2022/01/19 更新

写真a

クルミザカ ヒトシ
胡桃坂 仁志
所属
理工学術院
職名
名誉教授

学歴

  • 1992年04月
    -
    1995年03月

    埼玉大学大学院   理工学研究科博士後期課程  

  • 1991年04月
    -
    1992年03月

    東京薬科大学大学院   薬学研究科博士後期課程  

  • 1989年04月
    -
    1991年03月

    東京薬科大学大学院   薬学研究科博士前期課程  

  • 1985年04月
    -
    1989年03月

    東京薬科大学   薬学部   衛生薬学科  

学位

  • 埼玉大学   博士(学術)

経歴

  • 2018年04月
    -
    継続中

    東京大学   定量生命科学研究所   教授

  • 2008年04月
    -
    2018年03月

    早稲田大学   理工学術院 電気・情報生命工学科   教授

  • 2007年04月
    -
    2008年03月

    早稲田大学   理工学術院 電気・情報生命工学科   准教授

  • 2004年09月
    -
    2007年03月

    早稲田大学   理工学術院 電気・情報生命工学科   助教授

  • 2003年04月
    -
    2004年08月

    早稲田大学   理工学部 電気・情報生命工学科   助教授

  • 1997年06月
    -
    2003年03月

    理化学研究所   研究員

  • 1997年04月
    -
    1997年05月

    National Institutes of Health   National Institute of Child Health and Human Development   Visiting Fellow

  • 1996年04月
    -
    1997年03月

    National Institutes of Health   National Institute of Child Health and Human Development (日本学術振興会)   日本学術振興会海外特別研究員

  • 1995年06月
    -
    1996年03月

    National Institutes of Health   National Institute of Child Health and Human Development   Visiting Fellow

  • 1995年04月
    -
    1995年05月

    理化学研究所   奨励研究員

▼全件表示

所属学協会

  •  
     
     

    日本生物物理学会

  •  
     
     

    日本生化学会

  •  
     
     

    日本分子生物学会

  •  
     
     

    日本蛋白質科学会

 

研究分野

  • 分子生物学

  • 機能生物化学

  • 構造生物化学

研究キーワード

  • 遺伝子修復

  • 生体高分子構造・機能、遺伝的組換え

  • 遺伝情報複製・転写装置・再編・制御

  • 遺伝子の情報発現と複製

  • 遺伝子及び染色体

  • クライオ電子顕微鏡

  • X線結晶構造解析

  • ヌクレオソーム

  • ヒストン修飾

  • ヒストンバリアント

  • ヒストン

  • エピジェネティクス

  • 染色体

  • クロマチン

▼全件表示

論文

  • Unusual nucleosome formation and transcriptome influence by the histone H3mm18 variant

    Seiya Hirai, Kosuke Tomimatsu, Atsuko Miyawaki-Kuwakado, Yoshimasa Takizawa, Tetsuro Komatsu, Taro Tachibana, Yutaro Fukushima, Yasuko Takeda, Lumi Negishi, Tomoya Kujirai, Masako Koyama, Yasuyuki Ohkawa, Hitoshi Kurumizaka

    Nucleic Acids Research    2021年12月  [査読有り]

     概要を見る

    <title>Abstract</title>
    Histone H3mm18 is a non-allelic H3 variant expressed in skeletal muscle and brain in mice. However, its function has remained enigmatic. We found that H3mm18 is incorporated into chromatin in cells with low efficiency, as compared to H3.3. We determined the structures of the nucleosome core particle (NCP) containing H3mm18 by cryo-electron microscopy, which revealed that the entry/exit DNA regions are drastically disordered in the H3mm18 NCP. Consistently, the H3mm18 NCP is substantially unstable in vitro. The forced expression of H3mm18 in mouse myoblast C2C12 cells markedly suppressed muscle differentiation. A transcriptome analysis revealed that the forced expression of H3mm18 affected the expression of multiple genes, and suppressed a group of genes involved in muscle development. These results suggest a novel gene expression regulation system in which the chromatin landscape is altered by the formation of unusual nucleosomes with a histone variant, H3mm18, and provide important insight into understanding transcription regulation by chromatin.

    DOI

  • Structural basis for DNA sequence recognition by pioneer factors in nucleosomes

    Wataru Kagawa, Hitoshi Kurumizaka

    Current Opinion in Structural Biology   71   59 - 64  2021年12月

    DOI

  • Modeling population size independent tissue epigenomes by ChIL‐seq with single thin sections

    Kazumitsu Maehara, Kosuke Tomimatsu, Akihito Harada, Kaori Tanaka, Shoko Sato, Megumi Fukuoka, Seiji Okada, Tetsuya Handa, Hitoshi Kurumizaka, Noriko Saitoh, Hiroshi Kimura, Yasuyuki Ohkawa

    Molecular Systems Biology   17 ( 11 )  2021年11月  [査読有り]

    DOI

  • Cryo-EM structure of the nucleosome core particle containing Giardia lamblia histones

    Shoko Sato, Yoshimasa Takizawa, Fumika Hoshikawa, Mariko Dacher, Hiroki Tanaka, Hiroaki Tachiwana, Tomoya Kujirai, Yukari Iikura, Cheng-Han Ho, Naruhiko Adachi, Indu Patwal, Andrew Flaus, Hitoshi Kurumizaka

    Nucleic Acids Research   49 ( 15 ) 8934 - 8946  2021年08月  [査読有り]

     概要を見る

    <title>Abstract</title>
    Giardia lamblia is a pathogenic unicellular eukaryotic parasite that causes giardiasis. Its genome encodes the canonical histones H2A, H2B, H3, and H4, which share low amino acid sequence identity with their human orthologues. We determined the structure of the G. lamblia nucleosome core particle (NCP) at 3.6 Å resolution by cryo-electron microscopy. G. lamblia histones form a characteristic NCP, in which the visible 125 base-pair region of the DNA is wrapped in a left-handed supercoil. The acidic patch on the G. lamblia octamer is deeper, due to an insertion extending the H2B α1 helix and L1 loop, and thus cannot bind the LANA acidic patch binding peptide. The DNA and histone regions near the DNA entry-exit sites could not be assigned, suggesting that these regions are asymmetrically flexible in the G. lamblia NCP. Characterization by thermal unfolding in solution revealed that both the H2A–H2B and DNA association with the G. lamblia H3–H4 were weaker than those for human H3–H4. These results demonstrate the uniformity of the histone octamer as the organizing platform for eukaryotic chromatin, but also illustrate the unrecognized capability for large scale sequence variations that enable the adaptability of histone octamer surfaces and confer internal stability.

    DOI

  • The N-terminal Tails of Histones H2A and H2B Adopt Two Distinct Conformations in the Nucleosome with Contact and Reduced Contact to DNA

    Hideaki Ohtomo, Jun-ichi Kurita, Shun Sakuraba, Zhenhai Li, Yasuhiro Arimura, Masatoshi Wakamori, Yasuo Tsunaka, Takashi Umehara, Hitoshi Kurumizaka, Hidetoshi Kono, Yoshifumi Nishimura

    Journal of Molecular Biology   433 ( 15 ) 167110 - 167110  2021年07月  [査読有り]

    DOI

  • Sequence-dependent nucleosome formation in trinucleotide repeats evaluated by in vivo chemical mapping

    Koji Katsumata, Yuichi Ichikawa, Tomohiro Fuse, Hitoshi Kurumizaka, Akio Yanagida, Takeshi Urano, Hiroaki Kato, Mitsuhiro Shimizu

    Biochemical and Biophysical Research Communications   556   179 - 184  2021年06月  [査読有り]

    DOI

  • Chromatin structure-dependent histone incorporation revealed by a genome-wide deposition assay

    Hiroaki Tachiwana, Mariko Dacher, Kazumitsu Maehara, Akihito Harada, Yosuke Seto, Ryohei Katayama, Yasuyuki Ohkawa, Hiroshi Kimura, Hitoshi Kurumizaka, Noriko Saitoh

    eLife   10   e66290  2021年05月  [査読有り]

     概要を見る

    In eukaryotes, histone variant distribution within the genome is the key epigenetic feature. To understand how each histone variant is targeted to the genome, we developed a new method, the RhIP (<italic>R</italic>econstituted <italic>h</italic>istone complex <italic>I</italic>ncorporation into chromatin of <italic>P</italic>ermeabilized cell) assay, in which epitope-tagged histone complexes are introduced into permeabilized cells and incorporated into their chromatin. Using this method, we found that H3.1 and H3.3 were incorporated into chromatin in replication-dependent and -independent manners, respectively. We further found that the incorporation of histones H2A and H2A.Z mainly occurred at less condensed chromatin (open), suggesting that condensed chromatin (closed) is a barrier for histone incorporation. To overcome this barrier, H2A, but not H2A.Z, uses a replication-coupled deposition mechanism. Our study revealed that the combination of chromatin structure and DNA replication dictates the differential histone deposition to maintain the epigenetic chromatin states.

    DOI

  • Structural basis of nucleosomal histone H4 lysine 20 methylation by SET8 methyltransferase

    Cheng-Han Ho, Yoshimasa Takizawa, Wataru Kobayashi, Yasuhiro Arimura, Hiroshi Kimura, Hitoshi Kurumizaka

    Life Science Alliance   4 ( 4 ) e202000919 - e202000919  2021年04月  [査読有り]

     概要を見る

    SET8 is solely responsible for histone H4 lysine-20 (H4K20) monomethylation, which preferentially occurs in nucleosomal H4. However, the underlying mechanism by which SET8 specifically promotes the H4K20 monomethylation in the nucleosome has not been elucidated. Here, we report the cryo-EM structures of the human SET8–nucleosome complexes with histone H3 and the centromeric H3 variant, CENP-A. Surprisingly, we found that the overall cryo-EM structures of the SET8–nucleosome complexes are substantially different from the previous crystal structure models. In the complexes with H3 and CENP-A nucleosomes, SET8 specifically binds the nucleosomal acidic patch via an arginine anchor, composed of the Arg188 and Arg192 residues. Mutational analyses revealed that the interaction between the SET8 arginine anchor and the nucleosomal acidic patch plays an essential role in the H4K20 monomethylation activity. These results provide the groundwork for understanding the mechanism by which SET8 specifically accomplishes the H4K20 monomethylation in the nucleosome.

    DOI

  • Organoruthenium-catalyzed chemical protein synthesis to elucidate the functions of epigenetic modifications on heterochromatin factors

    Naoki Kamo, Tomoya Kujirai, Hitoshi Kurumizaka, Hiroshi Murakami, Gosuke Hayashi, Akimitsu Okamoto

    Chemical Science   12 ( 16 ) 5926 - 5937  2021年03月  [査読有り]

     概要を見る

    <p>Chemical protein synthesis assisted by an organoruthenium catalyst streamlined the production of heterochromatin factors bearing various patterns of epigenetic modifications, and their biological significance was elucidated.</p>

    DOI

  • H4K20me1 and H3K27me3 are concurrently loaded onto the inactive X chromosome but dispensable for inducing gene silencing

    Sjoerd J D Tjalsma, Mayako Hori, Yuko Sato, Aurelie Bousard, Akito Ohi, Ana Cláudia Raposo, Julia Roensch, Agnes Le Saux, Jumpei Nogami, Kazumitsu Maehara, Tomoya Kujirai, Tetsuya Handa, Sandra Bagés‐Arnal, Yasuyuki Ohkawa, Hitoshi Kurumizaka, Simão Teixeira da Rocha, Jan J Żylicz, Hiroshi Kimura, Edith Heard

    EMBO reports     e51989 - e51989  2021年02月  [査読有り]

    DOI

  • Histone variant H2A.B-H2B dimers are spontaneously exchanged with canonical H2A-H2B in the nucleosome

    Rina Hirano, Yasuhiro Arimura, Tomoya Kujirai, Mikihiro Shibata, Aya Okuda, Ken Morishima, Rintaro Inoue, Masaaki Sugiyama, Hitoshi Kurumizaka

    Communications Biology   4 ( 1 ) 191 - 191  2021年02月  [査読有り]

     概要を見る

    <title>Abstract</title>H2A.B is an evolutionarily distant histone H2A variant that accumulates on DNA repair sites, DNA replication sites, and actively transcribing regions in genomes. In cells, H2A.B exchanges rapidly in chromatin, but the mechanism has remained enigmatic. In the present study, we found that the H2A.B-H2B dimer incorporated within the nucleosome exchanges with the canonical H2A-H2B dimer without assistance from additional factors, such as histone chaperones and nucleosome remodelers. High-speed atomic force microscopy revealed that the H2A.B nucleosome, but not the canonical H2A nucleosome, transiently forms an intermediate “open conformation”, in which two H2A.B-H2B dimers may be detached from the H3-H4 tetramer and bind to the DNA regions near the entry/exit sites. Mutational analyses revealed that the H2A.B C-terminal region is responsible for the adoption of the open conformation and the H2A.B-H2B exchange in the nucleosome. These findings provide mechanistic insights into the histone exchange of the H2A.B nucleosome.

    DOI

  • Live-cell epigenome manipulation by synthetic histone acetylation catalyst system

    Yusuke Fujiwara, Yuki Yamanashi, Akiko Fujimura, Yuko Sato, Tomoya Kujirai, Hitoshi Kurumizaka, Hiroshi Kimura, Kenzo Yamatsugu, Shigehiro A. Kawashima, Motomu Kanai

    Proceedings of the National Academy of Sciences   118 ( 4 ) e2019554118 - e2019554118  2021年01月  [査読有り]

     概要を見る

    Chemical modifications of histones, such as lysine acetylation and ubiquitination, play pivotal roles in epigenetic regulation of gene expression. Methods to alter the epigenome thus hold promise as tools for elucidating epigenetic mechanisms and as therapeutics. However, an entirely chemical method to introduce histone modifications in living cells without genetic manipulation is unprecedented. Here, we developed a chemical catalyst, PEG-LANA-DSSMe 11, that binds with nucleosome’s acidic patch and promotes regioselective, synthetic histone acetylation at H2BK120 in living cells. The size of polyethylene glycol in the catalyst was a critical determinant for its in-cell metabolic stability, binding affinity to histones, and high activity. The synthetic acetylation promoted by 11 without genetic manipulation competed with and suppressed physiological H2B ubiquitination, a mark regulating chromatin functions, such as transcription and DNA damage response. Thus, the chemical catalyst will be a useful tool to manipulate epigenome for unraveling epigenetic mechanisms in living cells.

    DOI

  • Cryo‐EM structure of the CENP‐A nucleosome in complex with phosphorylated CENP‐C

    Mariko Ariyoshi, Fumiaki Makino, Reito Watanabe, Reiko Nakagawa, Takayuki Kato, Keiichi Namba, Yasuhiro Arimura, Risa Fujita, Hitoshi Kurumizaka, Ei‐ichi Okumura, Masatoshi Hara, Tatsuo Fukagawa

    The EMBO Journal     e105671 - e105671  2021年01月  [査読有り]

    DOI

  • Acetylation-modulated communication between the H3 N-terminal tail domain and the intrinsically disordered H1 C-terminal domain

    Fanfan Hao, Kevin J Murphy, Tomoya Kujirai, Naoki Kamo, Junko Kato, Masako Koyama, Akimitsu Okamato, Gosuke Hayashi, Hitoshi Kurumizaka, Jeffrey J Hayes

    Nucleic Acids Research   48 ( 20 ) 11510 - 11520  2020年11月  [査読有り]

     概要を見る

    <title>Abstract</title>
    Linker histones (H1s) are key structural components of the chromatin of higher eukaryotes. However, the mechanisms by which the intrinsically disordered linker histone carboxy-terminal domain (H1 CTD) influences chromatin structure and gene regulation remain unclear. We previously demonstrated that the CTD of H1.0 undergoes a significant condensation (reduction of end-to-end distance) upon binding to nucleosomes, consistent with a transition to an ordered structure or ensemble of structures. Here, we show that deletion of the H3 N-terminal tail or the installation of acetylation mimics or bona fide acetylation within H3 N-terminal tail alters the condensation of the nucleosome-bound H1 CTD. Additionally, we present evidence that the H3 N-tail influences H1 CTD condensation through direct protein-protein interaction, rather than alterations in linker DNA trajectory. These results support an emerging hypothesis wherein the H1 CTD serves as a nexus for signaling in the nucleosome.

    DOI

  • Essentiality of CENP-A Depends on Its Binding Mode to HJURP

    Tetsuya Hori, JingHui Cao, Kohei Nishimura, Mariko Ariyoshi, Yasuhiro Arimura, Hitoshi Kurumizaka, Tatsuo Fukagawa

    Cell Reports   33 ( 7 ) 108388 - 108388  2020年11月  [査読有り]

    DOI

  • Contributions of Histone Variants in Nucleosome Structure and Function

    Hitoshi Kurumizaka, Tomoya Kujirai, Yoshimasa Takizawa

    Journal of Molecular Biology   36 ( 20 ) 30586 - 30586  2020年10月

    DOI

  • Chromatin integration labeling for mapping DNA-binding proteins and modifications with low input.

    Tetsuya Handa, Akihito Harada, Kazumitsu Maehara, Shoko Sato, Masaru Nakao, Naoki Goto, Hitoshi Kurumizaka, Yasuyuki Ohkawa, Hiroshi Kimura

    Nature protocols   15 ( 10 ) 3334 - 3360  2020年10月  [査読有り]  [国際誌]

     概要を見る

    Cell identity is determined by the selective activation or silencing of specific genes via transcription factor binding and epigenetic modifications on the genome. Chromatin immunoprecipitation (ChIP) has been the standard technique for mapping the sites of transcription factor binding and histone modification. Recently, alternative methods to ChIP have been developed for addressing the increasing demands for low-input epigenomic profiling. Chromatin integration labeling (ChIL) followed by sequencing (ChIL-seq) has been demonstrated to be particularly useful for epigenomic profiling of low-input samples or even single cells because the technique amplifies the target genomic sequence before cell lysis. After labeling the target protein or modification in situ with an oligonucleotide-conjugated antibody (ChIL probe), the nearby genome sequence is amplified by Tn5 transposase-mediated transposition followed by T7 RNA polymerase-mediated transcription. ChIL-seq enables the detection of the antibody target localization under a fluorescence microscope and at the genomic level. Here we describe the detailed protocol of ChIL-seq with assessment methods for the key steps, including ChIL probe reaction, transposition, in situ transcription and sequencing library preparation. The protocol usually takes 3 d to prepare the sequencing library, including overnight incubations for the ChIL probe reaction and in situ transcription. The ChIL probe can be separately prepared and stored for several months, and its preparation and evaluation protocols are also documented in detail. An optional analysis for multiple targets (multitarget ChIL-seq) is also described. We anticipate that the protocol presented here will make the ChIL technique more widely accessible for analyzing precious samples and facilitate further applications.

    DOI PubMed

  • Structural basis for the inhibition of cGAS by nucleosomes.

    Tomoya Kujirai, Christian Zierhut, Yoshimasa Takizawa, Ryan Kim, Lumi Negishi, Nobuki Uruma, Seiya Hirai, Hironori Funabiki, Hitoshi Kurumizaka

    Science (New York, N.Y.)    2020年09月  [査読有り]  [国際誌]

     概要を見る

    The cyclic GMP-AMP synthase (cGAS) senses invasion of pathogenic DNA and stimulates inflammatory signaling, autophagy and apoptosis. Organization of host DNA into nucleosomes was proposed to limit cGAS autoinduction, but the underlying mechanism was unknown. Here, we report the structural basis for this inhibition. In the cryo-EM structure of the human cGAS-nucleosome core particle (NCP) complex, two cGAS monomers bridge two NCPs by binding the acidic patch of H2A-H2B and nucleosomal DNA. In this configuration, all three known cGAS DNA-binding sites, required for cGAS activation, are repurposed or become inaccessible, and cGAS dimerization, another prerequisite for activation, is inhibited. Mutating key residues linking cGAS and the acidic patch alleviates nucleosomal inhibition. This study establishes a structural framework for why cGAS is silenced on chromatinized self-DNA.

    DOI PubMed

  • Acetylated histone H4 tail enhances histone H3 tail acetylation by altering their mutual dynamics in the nucleosome.

    Ayako Furukawa, Masatoshi Wakamori, Yasuhiro Arimura, Hideaki Ohtomo, Yasuo Tsunaka, Hitoshi Kurumizaka, Takashi Umehara, Yoshifumi Nishimura

    Proceedings of the National Academy of Sciences of the United States of America   117 ( 33 ) 19661 - 19663  2020年08月  [査読有り]  [国際誌]

     概要を見る

    The structural unit of eukaryotic chromatin is a nucleosome, comprising two histone H2A-H2B heterodimers and one histone (H3-H4)2 tetramer, wrapped around by ∼146 bp of DNA. The N-terminal flexible histone tails stick out from the histone core and have extensive posttranslational modifications, causing epigenetic changes of chromatin. Although crystal and cryogenic electron microscopy structures of nucleosomes are available, the flexible tail structures remain elusive. Using NMR, we have examined the dynamics of histone H3 tails in nucleosomes containing unmodified and tetra-acetylated H4 tails. In unmodified nucleosome, the H3 tail adopts a dynamic equilibrium structure between DNA-contact and reduced-contact states. In acetylated H4 nucleosome, however, the H3 tail equilibrium shifts to a mainly DNA-contact state with a minor reduced-contact state. The acetylated H4 tail is dynamically released from its own DNA-contact state to a reduced-contact state, while the H3 tail DNA-contact state becomes major. Notably, H3 K14 in the acetylated H4 nucleosome is much more accessible to acetyltransferase Gcn5 relative to unmodified nucleosome, possibly due to the formation of a favorable H3 tail conformation for Gcn5. In summary, each histone tail adopts a characteristic dynamic state but regulates one other, probably creating a histone tail network even on a nucleosome.

    DOI PubMed

  • Interaction of the pioneer transcription factor GATA3 with nucleosomes.

    Hiroki Tanaka, Yoshimasa Takizawa, Motoki Takaku, Daiki Kato, Yusuke Kumagawa, Sara A Grimm, Paul A Wade, Hitoshi Kurumizaka

    Nature communications   11 ( 1 ) 4136 - 4136  2020年08月  [査読有り]  [国際誌]

     概要を見る

    During cellular reprogramming, the pioneer transcription factor GATA3 binds chromatin, and in a context-dependent manner directs local chromatin remodeling and enhancer formation. Here, we use high-resolution nucleosome mapping in human cells to explore the impact of the position of GATA motifs on the surface of nucleosomes on productive enhancer formation, finding productivity correlates with binding sites located near the nucleosomal dyad axis. Biochemical experiments with model nucleosomes demonstrate sufficiently stable transcription factor-nucleosome interaction to empower cryo-electron microscopy structure determination of the complex at 3.15 Å resolution. The GATA3 zinc fingers efficiently bind their target 5'-GAT-3' sequences in the nucleosome when they are located in solvent accessible, consecutive major grooves without significant changes in nucleosome structure. Analysis of genomic loci bound by GATA3 during reprogramming suggests a correlation of recognition motif sequence and spacing that may distinguish productivity of new enhancer formation.

    DOI PubMed

  • Linker DNA and histone contributions in nucleosome binding by p53

    Masahiro Nishimura, Yasuhiro Arimura, Kayo Nozawa, Hitoshi Kurumizaka

    The Journal of Biochemistry    2020年07月  [査読有り]

     概要を見る

    <title>Abstract</title>
    The tumor suppressor protein p53 regulates various genes involved in cell-cycle arrest, apoptosis, and DNA repair in response to cellular stress, and apparently functions as a pioneer transcription factor. The pioneer transcription factors can bind nucleosomal DNA, where many transcription factors are largely restricted. However, the mechanisms by which p53 recognizes the nucleosomal DNA are poorly understood. In the present study, we found that p53 requires linker DNAs for the efficient formation of p53-nucleosome complexes. p53 forms an additional specific complex with the nucleosome, when the p53 binding sequence is located around the entry/exit region of the nucleosomal DNA. We also showed that p53 directly binds to the histone H3-H4 complex via its N-terminal 1-93 amino acid region. These results shed light on the mechanism of nucleosome recognition by p53.

    DOI

  • Nucleosome binding by the pioneer transcription factor OCT4

    Kenta Echigoya, Masako Koyama, Lumi Negishi, Yoshimasa Takizawa, Yuka Mizukami, Hideki Shimabayashi, Akari Kuroda, Hitoshi Kurumizaka

    Scientific Reports   10 ( 1 )  2020年07月  [査読有り]

    DOI

  • Integral approach to biomacromolecular structure by analytical-ultracentrifugation and small-angle scattering

    Morishima K, Okuda A, Inoue R, Sato N, Miyamoto Y, Urade R, Yagi-Utsumi M, Kato K, Hirano R, Kujirai T, Kurumizaka H, Sugiyama M

    Communications Biology   3 ( 1 ) 294  2020年06月  [査読有り]

    DOI

  • The N‐terminal and C‐terminal halves of histone H2A.Z independently function in nucleosome positioning and stability

    Shoko Sato, Naoki Tanaka, Yasuhiro Arimura, Tomoya Kujirai, Hitoshi Kurumizaka

    Genes to Cells    2020年06月  [査読有り]

    DOI

  • Structural Studies of Overlapping Dinucleosomes in Solution

    Atsushi Matsumoto, Masaaki Sugiyama, Zhenhai Li, Anne Martel, Lionel Porcar, Rintaro Inoue, Daiki Kato, Akihisa Osakabe, Hitoshi Kurumizaka, Hidetoshi Kono

    Biophysical Journal   118 ( 9 ) 2209 - 2219  2020年05月  [査読有り]

    DOI PubMed

  • Improved Methods for Preparing the Telomere Tethering Complex Bqt1–Bqt2 for Structural Studies

    Yuzurihara H, Aizawa Y, Saotome M, Ichikawa Y, Yokoyama H, Chikashige Y, Haraguchi T, Hiraoka Y, Kurumizaka H, Kagawa W

    Protein J.   39   174 - 181  2020年04月  [査読有り]

    DOI

  • Nucleosome destabilization by nuclear non-coding RNAs.

    Risa Fujita, Tatsuro Yamamoto, Yasuhiro Arimura, Saori Fujiwara, Hiroaki Tachiwana, Yuichi Ichikawa, Yuka Sakata, Liying Yang, Reo Maruyama, Michiaki Hamada, Mitsuyoshi Nakao, Noriko Saitoh, Hitoshi Kurumizaka

    Communications biology   3 ( 1 ) 60 - 60  2020年02月  [査読有り]  [国際誌]

     概要を見る

    In the nucleus, genomic DNA is wrapped around histone octamers to form nucleosomes. In principle, nucleosomes are substantial barriers to transcriptional activities. Nuclear non-coding RNAs (ncRNAs) are proposed to function in chromatin conformation modulation and transcriptional regulation. However, it remains unclear how ncRNAs affect the nucleosome structure. Eleanors are clusters of ncRNAs that accumulate around the estrogen receptor-α (ESR1) gene locus in long-term estrogen deprivation (LTED) breast cancer cells, and markedly enhance the transcription of the ESR1 gene. Here we detected nucleosome depletion around the transcription site of Eleanor2, the most highly expressed Eleanor in the LTED cells. We found that the purified Eleanor2 RNA fragment drastically destabilized the nucleosome in vitro. This activity was also exerted by other ncRNAs, but not by poly(U) RNA or DNA. The RNA-mediated nucleosome destabilization may be a common feature among natural nuclear RNAs, and may function in transcription regulation in chromatin.

    DOI PubMed

  • Cryo-EM Structures of Centromeric Tri-nucleosomes Containing a Central CENP-A Nucleosome.

    Yoshimasa Takizawa, Cheng-Han Ho, Hiroaki Tachiwana, Hideyuki Matsunami, Wataru Kobayashi, Midori Suzuki, Yasuhiro Arimura, Tetsuya Hori, Tatsuo Fukagawa, Melanie D Ohi, Matthias Wolf, Hitoshi Kurumizaka

    Structure (London, England : 1993)   28 ( 1 ) 44 - 53  2020年01月  [査読有り]  [国際誌]

     概要を見る

    The histone H3 variant CENP-A is a crucial epigenetic marker for centromere specification. CENP-A forms a characteristic nucleosome and dictates the higher-order configuration of centromeric chromatin. However, little is known about how the CENP-A nucleosome affects the architecture of centromeric chromatin. In this study, we reconstituted tri-nucleosomes mimicking a centromeric nucleosome arrangement containing the CENP-A nucleosome, and determined their 3D structures by cryoelectron microscopy. The H3-CENP-A-H3 tri-nucleosomes adopt an untwisted architecture, with an outward-facing linker DNA path between nucleosomes. This is distinct from the H3-H3-H3 tri-nucleosome architecture, with an inward-facing DNA path. Intriguingly, the untwisted architecture may allow the CENP-A nucleosome to be exposed to the solvent in the condensed chromatin model. These results provide a structural basis for understanding the 3D configuration of CENP-A-containing chromatin, and may explain how centromeric proteins can specifically target the CENP-A nucleosomes buried in robust amounts of H3 nucleosomes in centromeres.

    DOI PubMed

  • Native Mass Spectrometry of Protein and DNA Complexes Prepared in Nonvolatile Buffers

    Kazumi Saikusa, Daiki Kato, Aritaka Nagadoi, Hitoshi Kurumizaka, Satoko Akashi

    Journal of the American Society for Mass Spectrometry   31 ( 3 ) 711 - 718  2020年01月  [査読有り]

    DOI

  • Incorporation and influence of Leishmania histone H3 in chromatin.

    Mariko Dacher, Hiroaki Tachiwana, Naoki Horikoshi, Tomoya Kujirai, Hiroyuki Taguchi, Hiroshi Kimura, Hitoshi Kurumizaka

    Nucleic acids research   47 ( 22 ) 11637 - 11648  2019年12月  [査読有り]  [国際誌]

     概要を見る

    Immunopathologies caused by Leishmania cause severe human morbidity and mortality. This protozoan parasite invades and persists inside host cells, resulting in disease development. Leishmania modifies the epigenomic status of the host cells, thus probably averting the host cell defense mechanism. To accomplish this, Leishmania may change the host cell chromatin structure. However, the mechanism by which the parasite changes the host cell chromatin has not been characterized. In the present study, we found that ectopically produced Leishmania histone H3, LmaH3, which mimics the secreted LmaH3 in infected cells, is incorporated into chromatin in human cells. A crystallographic analysis revealed that LmaH3 forms nucleosomes with human histones H2A, H2B and H4. We found that LmaH3 was less stably incorporated into the nucleosome, as compared to human H3.1. Consistently, we observed that LmaH3-H4 association was remarkably weakened. Mutational analyses revealed that the specific LmaH3 Trp35, Gln57 and Met98 residues, which correspond to the H3.1 Tyr41, Arg63 and Phe104 residues, might be responsible for the instability of the LmaH3 nucleosome. Nucleosomes containing LmaH3 resisted the Mg2+-mediated compaction of the chromatin fiber. These distinct physical characteristics of LmaH3 support the possibility that histones secreted by parasites during infection may modulate the host chromatin structure.

    DOI PubMed

  • The CENP-A centromere targeting domain facilitates H4K20 monomethylation in the nucleosome by structural polymorphism

    Yasuhiro Arimura, Hiroaki Tachiwana, Hiroki Takagi, Tetsuya Hori, Hiroshi Kimura, Tatsuo Fukagawa, Hitoshi Kurumizaka

    Nature Communications   10 ( 1 ) 576  2019年12月  [査読有り]

     概要を見る

    © 2019, The Author(s). Centromeric nucleosomes are composed of the centromere-specific histone H3 variant CENP-A and the core histones H2A, H2B, and H4. To establish a functional kinetochore, histone H4 lysine-20 (H4K20) must be monomethylated, but the underlying mechanism has remained enigmatic. To provide structural insights into H4K20 methylation, we here solve the crystal structure of a nucleosome containing an H3.1-CENP-A chimera, H3.1 CATD , which has a CENP-A centromere targeting domain and preserves essential CENP-A functions in vivo. Compared to the canonical H3.1 nucleosome, the H3.1 CATD nucleosome exhibits conformational changes in the H4 N-terminal tail leading to a relocation of H4K20. In particular, the H4 N-terminal tail interacts with glutamine-76 and aspartate-77 of canonical H3.1 while these interactions are cancelled in the presence of the CENP-A-specific residues valine-76 and lysine-77. Mutations of valine-76 and lysine-77 impair H4K20 monomethylation both in vitro and in vivo. These findings suggest that a CENP-A-mediated structural polymorphism may explain the preferential H4K20 monomethylation in centromeric nucleosomes.

    DOI PubMed

  • Structural transition of the nucleosome during chromatin remodeling and transcription.

    Kobayashi W, Kurumizaka H

    Current opinion in structural biology   59   107 - 114  2019年12月  [査読有り]

    DOI PubMed

  • A novel mode of DnaA-DnaA interaction promotes ADP dissociation for reactivation of replication initiation activity.

    Sugiyama R, Kasho K, Miyoshi K, Ozaki S, Kagawa W, Kurumizaka H, Katayama T

    Nucleic acids research   47 ( 21 ) 11209 - 11224  2019年12月  [査読有り]

    DOI PubMed

  • Biochemical and structural analyses of the nucleosome containing human histone H2A.J.

    Tanaka H, Sato S, Koyama M, Kujirai T, Kurumizaka H

    Journal of biochemistry    2019年12月  [査読有り]

    DOI PubMed

  • Transcription through the nucleosome.

    Kujirai T, Kurumizaka H

    Current opinion in structural biology   61   42 - 49  2019年11月  [査読有り]

    DOI PubMed

  • H3K14 ubiquitylation promotes H3K9 methylation for heterochromatin assembly.

    Eriko Oya, Reiko Nakagawa, Yuriko Yoshimura, Mayo Tanaka, Gohei Nishibuchi, Shinichi Machida, Atsuko Shirai, Karl Ekwall, Hitoshi Kurumizaka, Hideaki Tagami, Jun-Ichi Nakayama

    EMBO reports   20 ( 10 ) e48111  2019年10月  [査読有り]  [国際誌]

     概要を見る

    The methylation of histone H3 at lysine 9 (H3K9me), performed by the methyltransferase Clr4/SUV39H, is a key event in heterochromatin assembly. In fission yeast, Clr4, together with the ubiquitin E3 ligase Cul4, forms the Clr4 methyltransferase complex (CLRC), whose physiological targets and biological role are currently unclear. Here, we show that CLRC-dependent H3 ubiquitylation regulates Clr4's methyltransferase activity. Affinity-purified CLRC ubiquitylates histone H3, and mass spectrometric and mutation analyses reveal that H3 lysine 14 (H3K14) is the preferred target of the complex. Chromatin immunoprecipitation analysis shows that H3K14 ubiquitylation (H3K14ub) is closely associated with H3K9me-enriched chromatin. Notably, the CLRC-mediated H3 ubiquitylation promotes H3K9me by Clr4, suggesting that H3 ubiquitylation is intimately linked to the establishment and/or maintenance of H3K9me. These findings demonstrate a cross-talk mechanism between histone ubiquitylation and methylation that is involved in heterochromatin assembly.

    DOI PubMed

  • Structure determination of the nucleosome core particle by selenium SAD phasing.

    Saotome M, Horikoshi N, Urano K, Kujirai T, Yuzurihara H, Kurumizaka H, Kagawa W

    Acta crystallographica. Section D, Structural biology   75 ( Pt 10 ) 930 - 936  2019年10月  [査読有り]

    DOI PubMed

  • Biochemical analysis of nucleosome targeting by Tn5 transposase.

    Shoko Sato, Yasuhiro Arimura, Tomoya Kujirai, Akihito Harada, Kazumitsu Maehara, Jumpei Nogami, Yasuyuki Ohkawa, Hitoshi Kurumizaka

    Open biology   9 ( 8 ) 190116 - 190116  2019年08月  [査読有り]  [国際誌]

     概要を見る

    Tn5 transposase is a bacterial enzyme that integrates a DNA fragment into genomic DNA, and is used as a tool for detecting nucleosome-free regions of genomic DNA in eukaryotes. However, in chromatin, the DNA targeting by Tn5 transposase has remained unclear. In the present study, we reconstituted well-positioned 601 dinucleosomes, in which two nucleosomes are connected with a linker DNA, and studied the DNA integration sites in the dinucleosomes by Tn5 transposase in vitro. We found that Tn5 transposase preferentially targets near the entry-exit DNA regions within the nucleosome. Tn5 transposase minimally cleaved the dinucleosome without a linker DNA, indicating that the linker DNA between two nucleosomes is important for the Tn5 transposase activity. In the presence of a 30 base-pair linker DNA, Tn5 transposase targets the middle of the linker DNA, in addition to the entry-exit sites of the nucleosome. Intriguingly, this Tn5-targeting characteristic is conserved in a dinucleosome substrate with a different DNA sequence from the 601 sequence. Therefore, the Tn5-targeting preference in the nucleosomal templates reported here provides important information for the interpretation of Tn5 transposase-based genomics methods, such as ATAC-seq.

    DOI PubMed

  • Structure-based design of an H2A.Z.1 mutant stabilizing a nucleosome in vitro and in vivo.

    Horikoshi N, Kujirai T, Sato K, Kimura H, Kurumizaka H

    Biochemical and biophysical research communications   515 ( 4 ) 719 - 724  2019年08月  [査読有り]

    DOI PubMed

  • The CHD4-related syndrome: a comprehensive investigation of the clinical spectrum, genotype-phenotype correlations, and molecular basis.

    Genet Med.    2019年08月  [査読有り]

    DOI

  • Intrabody-based FRET probe to visualize endogenous histone acetylation.

    Chung CI, Sato Y, Ohmuro-Matsuyama Y, Machida S, Kurumizaka H, Kimura H, Ueda H

    Scientific reports   9 ( 1 ) 10188  2019年07月  [査読有り]

    DOI PubMed

  • Biochemical characterization of the placeholder nucleosome for DNA hypomethylation maintenance.

    Hirano R, Kujirai T, Negishi L, Kurumizaka H

    Biochemistry and biophysics reports   18   100634  2019年07月  [査読有り]

    DOI PubMed

  • DNA damage detection in nucleosomes involves DNA register shifting.

    Matsumoto S, Cavadini S, Bunker RD, Grand RS, Potenza A, Rabl J, Yamamoto J, Schenk AD, Schübeler D, Iwai S, Sugasawa K, Kurumizaka H, Thomä NH

    Nature    2019年05月  [査読有り]

    DOI PubMed

  • Structural and biochemical analyses of the nuclear pore complex component ELYS identify residues responsible for nucleosome binding.

    Kobayashi W, Takizawa Y, Aihara M, Negishi L, Ishii H, Kurumizaka H

    Communications biology   2   163  2019年03月  [査読有り]

    DOI PubMed

  • Structural insight into nucleosome transcription by RNA polymerase II with elongation factors.

    Ehara H, Kujirai T, Fujino Y, Shirouzu M, Kurumizaka H, Sekine SI

    Science (New York, N.Y.)   363 ( 6428 ) 744 - 747  2019年02月  [査読有り]

    DOI PubMed

  • Author Correction: CHD3 helicase domain mutations cause a neurodevelopmental syndrome with macrocephaly and impaired speech and language.

    Blok LS, Rousseau J, Twist J, Ehresmann S, Takaku M, Venselaar H, Rodan LH, Nowak CB, Douglas J, Swoboda KJ, Steeves MA, Sahai I, Stumpel CTRM, Stegmann APA, Wheeler P, Willing M, Fiala E, Kochhar A, Gibson WT, Cohen ASA, Agbahovbe R, Innes AM, Au PYB, Rankin J, Anderson IJ, Skinner SA, Louie RJ, Warren HE, Afenjar A, Keren B, Nava C, Buratti J, Isapof A, Rodriguez D, Lewandowski R, Propst J, van Essen T, Choi M, Lee S, Chae JH, Price S, Schnur RE, Douglas G, Wentzensen IM, Zweier C, Reis A, Bialer MG, Moore C, Koopmans M, Brilstra EH, Monroe GR, van Gassen KLI, van Binsbergen E, Newbury-Ecob R, Bownass L, Bader I, Mayr JA, Wortmann SB, Jakielski KJ, Strand EA, Kloth K, Bierhals T, DDD study, Roberts JD, Petrovich RM, Machida S, Kurumizaka H, Lelieveld S, Pfundt R, Jansen S, Deriziotis P, Faivre L, Thevenon J, Assoum M, Shriberg L, Kleefstra T, Brunner HG, Wade PA, Fisher SE, Campeau PM

    Nature communications   10 ( 1 ) 883  2019年02月  [査読有り]

    DOI PubMed

  • A chromatin integration labelling method enables epigenomic profiling with lower input.

    Akihito Harada, Kazumitsu Maehara, Tetsuya Handa, Yasuhiro Arimura, Jumpei Nogami, Yoko Hayashi-Takanaka, Katsuhiko Shirahige, Hitoshi Kurumizaka, Hiroshi Kimura, Yasuyuki Ohkawa

    Nature cell biology   21 ( 2 ) 287 - 296  2019年02月  [査読有り]  [国際誌]

     概要を見る

    Chromatin plays a crucial role in gene regulation, and chromatin immunoprecipitation followed by sequencing (ChIP-seq) has been the standard technique for examining protein-DNA interactions across the whole genome. However, it is difficult to obtain epigenomic information from limited numbers of cells by ChIP-seq because of sample loss during chromatin preparation and inefficient immunoprecipitation. In this study, we established an immunoprecipitation-free epigenomic profiling method named chromatin integration labelling (ChIL), which enables the amplification of genomic sequences closely associated with the target molecules before cell lysis. Using ChIL followed by sequencing (ChIL-seq), we reliably detected the distributions of histone modifications and DNA-binding factors in 100-1,000 cells. In addition, ChIL-seq successfully detected genomic regions associated with histone marks at the single-cell level. Thus, ChIL-seq offers an alternative method to ChIP-seq for epigenomic profiling using small numbers of cells, in particular, those attached to culture plates and after immunofluorescence.

    DOI PubMed

  • Histone H3.3 sub-variant H3mm7 is required for normal skeletal muscle regeneration

    Akihito Harada, Kazumitsu Maehara, Yusuke Ono, Hiroyuki Taguchi, Kiyoshi Yoshioka, Yasuo Kitajima, Yan Xie, Yuko Sato, Takeshi Iwasaki, Jumpei Nogami, Seiji Okada, Tetsuro Komatsu, Yuichiro Semba, Tatsuya Takemoto, Hiroshi Kimura, Hitoshi Kurumizaka, Yasuyuki Ohkawa

    Nature Communications   9 ( 1 ) 1400  2018年12月  [査読有り]

     概要を見る

    © 2018 The Author(s). Regulation of gene expression requires selective incorporation of histone H3 variant H3.3 into chromatin. Histone H3.3 has several subsidiary variants but their functions are unclear. Here we characterize the function of histone H3.3 sub-variant, H3mm7, which is expressed in skeletal muscle satellite cells. H3mm7 knockout mice demonstrate an essential role of H3mm7 in skeletal muscle regeneration. Chromatin analysis reveals that H3mm7 facilitates transcription by forming an open chromatin structure around promoter regions including those of myogenic genes. The crystal structure of the nucleosome containing H3mm7 reveals that, unlike the S57 residue of other H3 proteins, the H3mm7-specific A57 residue cannot form a hydrogen bond with the R40 residue of the cognate H4 molecule. Consequently, the H3mm7 nucleosome is unstable in vitro and exhibited higher mobility in vivo compared with the H3.3 nucleosome. We conclude that the unstable H3mm7 nucleosome may be required for proper skeletal muscle differentiation.

    DOI PubMed

  • Mitotic phosphorylation of HP1α regulates its cell cycle-dependent chromatin binding.

    Nishibuchi G, Machida S, Nakagawa R, Yoshimura Y, Hiragami-Hamada K, Abe Y, Kurumizaka H, Tagami H, Nakayama JI

    Journal of biochemistry    2018年12月  [査読有り]

    DOI PubMed

  • Crystallographic analysis of the overlapping dinucleosome as a novel chromatin unit.

    Nishimura M, Nozawa K, Kurumizaka H

    Biophysics and physicobiology   15   251 - 254  2018年12月  [査読有り]

    DOI PubMed

  • Homologous pairing activities of Arabidopsis thaliana RAD51 and DMC1.

    Kobiayashi W, Liu E, Ishii H, Matsunaga S, Schlögelhofer P, Kurumizaka H

    Journal of biochemistry   165 ( 3 ) 289 - 295  2018年12月  [査読有り]

    DOI PubMed

  • Structural basis of the nucleosome transition during RNA polymerase II passage.

    Kujirai T, Ehara H, Fujino Y, Shirouzu M, Sekine SI, Kurumizaka H

    Science (New York, N.Y.)   362 ( 6414 ) 595 - 598  2018年11月  [査読有り]

    DOI PubMed

  • CHD3 helicase domain mutations cause a neurodevelopmental syndrome with macrocephaly and impaired speech and language.

    Snijders Blok L, Rousseau J, Twist J, Ehresmann S, Takaku M, Venselaar H, Rodan LH, Nowak CB, Douglas J, Swoboda KJ, Steeves MA, Sahai I, Stumpel CTRM, Stegmann APA, Wheeler P, Willing M, Fiala E, Kochhar A, Gibson WT, Cohen ASA, Agbahovbe R, Innes AM, Au PYB, Rankin J, Anderson IJ, Skinner SA, Louie RJ, Warren HE, Afenjar A, Keren B, Nava C, Buratti J, Isapof A, Rodriguez D, Lewandowski R, Propst J, van Essen T, Choi M, Lee S, Chae JH, Price S, Schnur RE, Douglas G, Wentzensen IM, Zweier C, Reis A, Bialer MG, Moore C, Koopmans M, Brilstra EH, Monroe GR, van Gassen KLI, van Binsbergen E, Newbury-Ecob R, Bownass L, Bader I, Mayr JA, Wortmann SB, Jakielski KJ, Strand EA, Kloth K, Bierhals T, Roberts JD, Petrovich RM, Machida S, Kurumizaka H, Lelieveld S, Pfundt R, Jansen S, Deriziotis P, Faive L, Thevenon J, Assoum M, Shriberg L, Kleefstra T, Brunner HG, Wade PA, Fisher SE, Campeau PM

    Nature communications   9 ( 1 ) 4619  2018年11月  [査読有り]

    DOI PubMed

  • Histone H2A variants confer specific properties to nucleosomes and impact on chromatin accessibility

    Akihisa Osakabe, Zdravko J Lorković, Wataru Kobayashi, Hiroaki Tachiwana, Ramesh Yelagandula, Hitoshi Kurumizaka, Frédéric Berger

    Nucleic Acids Research   46 ( 15 ) 7675 - 7685  2018年09月  [査読有り]

    DOI PubMed

  • 遺伝情報の維持継承におけるミクロとマクロの統合的理解:生化学の新たな挑戦 RAD51-単鎖DNA-ヌクレオソーム複合体の構造生物学的解析

    小林 航, 石井 初芽, 滝沢 由政, 胡桃坂 仁志

    日本生化学会大会プログラム・講演要旨集   91回   [2S06m - 04]  2018年09月  [査読有り]

  • Corrigendum to: Contribution of histone N-terminal tails to the structure and stability of nucleosomes.

    Iwasaki W, Miya Y, Horikoshi N, Osakabe A, Taguchi H, Tachiwana H, Shibata T, Kagawa W, Kurumizaka H

    FEBS open bio   8 ( 9 ) 1567  2018年09月  [査読有り]

    DOI PubMed

  • Structural Diversity of Nucleosomes Characterized by Native Mass Spectrometry

    Kazumi Saikusa, Akihisa Osakabe, Daiki Kato, Sotaro Fuchigami, Aritaka Nagadoi, Yoshifumi Nishimura, Hitoshi Kurumizaka, Satoko Akashi

    Analytical Chemistry   90 ( 13 ) 8217 - 8226  2018年07月  [査読有り]

    DOI PubMed

  • Histone Methylation by SETD1A Protects Nascent DNA through the Nucleosome Chaperone Activity of FANCD2.

    Higgs MR, Sato K, Reynolds JJ, Begum S, Bayley R, Goula A, Vernet A, Paquin KL, Skalnik DG, Kobayashi W, Takata M, Howlett NG, Kurumizaka H, Kimura H, Stewart GS

    Molecular cell   71 ( 1 ) 25 - 41.e6  2018年07月  [査読有り]

    DOI PubMed

  • Structural polymorphism of the Escherichia coli poly-α-L-glutamate synthetase RimK.

    Arimura Y, Kono T, Kino K, Kurumizaka H

    Acta crystallographica. Section F, Structural biology communications   74 ( Pt 7 ) 385 - 390  2018年07月  [査読有り]

    DOI PubMed

  • Cancer-associated mutations of histones H2B, H3.1 and H2A.Z.1 affect the structure and stability of the nucleosome.

    Arimura Y, Ikura M, Fujita R, Noda M, Kobayashi W, Horikoshi N, Sun J, Shi L, Kusakabe M, Harata M, Ohkawa Y, Tashiro S, Kimura H, Ikura T, Kurumizaka H

    Nucleic acids research   46 ( 19 ) 10007 - 10018  2018年07月  [査読有り]

    DOI PubMed

  • MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast.

    Luo D, Kato D, Nogami J, Ohkawa Y, Kurumizaka H, Kono H

    Nucleic acids research    2018年06月  [査読有り]

    DOI PubMed

  • Structural Basis of Homology-Directed DNA Repair Mediated by RAD52.

    Saotome M, Saito K, Yasuda T, Ohtomo H, Sugiyama S, Nishimura Y, Kurumizaka H, Kagawa W

    iScience   3   50 - 62  2018年05月  [査読有り]

    DOI PubMed

  • Replication stress induces accumulation of FANCD2 at central region of large fragile genes.

    Okamoto Y, Iwasaki WM, Kugou K, Takahashi KK, Oda A, Sato K, Kobayashi W, Kawai H, Sakasai R, Takaori-Kondo A, Yamamoto T, Kanemaki MT, Taoka M, Isobe T, Kurumizaka H, Innan H, Ohta K, Ishiai M, Takata M

    Nucleic acids research   46 ( 6 ) 2932 - 2944  2018年04月  [査読有り]

    DOI PubMed

  • Cryo-EM structure of the nucleosome containing the ALB1 enhancer DNA sequence.

    Takizawa Y, Tanaka H, Machida S, Koyama M, Maehara K, Ohkawa Y, Wade PA, Wolf M, Kurumizaka H

    Open biology   8 ( 3 )  2018年03月  [査読有り]

    DOI PubMed

  • LC-MS/MS-based quantitative study of the acyl group- and site-selectivity of human sirtuins to acylated nucleosomes.

    Tanabe K, Liu J, Kato D, Kurumizaka H, Yamatsugu K, Kanai M, Kawashima S.A

    Scientific Reports   8 ( 1 ) 2656 - 2656  2018年02月  [査読有り]

    DOI PubMed

  • Structural Basis of Heterochromatin Formation by Human HP1.

    Machida S, Takizawa Y, Ishimaru M, Sugita Y, Sekine S, Nakayama J, Wolf M, Kurumizaka H

    Molecular Cell   69 ( 3 ) 385 - 397  2018年02月  [査読有り]

    DOI PubMed

  • Roles of histone H3.5 in human spermatogenesis and spermatogenic disorders.

    Shiraishi K, Shindo A, Harada A, Kurumizaka H, Kimura H, Ohkawa Y, Matsuyama H

    Andrology   6 ( 1 ) 158 - 165  2018年01月  [査読有り]

    DOI

  • Methods for Preparing Nucleosomes Containing Histone Variants.

    Kujirai T, Arimura Y, Fujita R, Horikoshi N, Machida S, Kurumizaka H

    Methods in molecular biology (Clifton, N.J.)   1832   3 - 20  2018年  [査読有り]

    DOI PubMed

  • SUMO modification system facilitates the exchange of histone variant H2A.Z-2 at DNA damage sites

    Atsuhiko Fukuto, Masae Ikura, Tsuyoshi Ikura, Jiying Sun, Yasunori Horikoshi, Hiroki Shima, Kazuhiko Igarashi, Masayuki Kusakabe, Masahiko Harata, Naoki Horikoshi, Hitoshi Kurumizaka, Yoshiaki Kiuchi, Satoshi Tashiro

    Nucleus   9 ( 1 ) 87 - 94  2018年01月  [査読有り]

    DOI PubMed

  • ヒストンH3バリアントCENP-AのCATDドメインはヌクレオソーム中のH4K20のモノメチル化を促進する

    高木 大輝, 立和名 博昭, 有村 泰宏, 堀 哲也, 深川 竜郎, 胡桃坂 仁志

    生命科学系学会合同年次大会   2017年度   [2P - 0575]  2017年12月  [査読有り]

  • Phosphorylation of CBX2 controls its nucleosome-binding specificity

    Takayuki Kawaguchi, Shinichi Machida, Hitoshi Kurumizaka, Hideaki Tagami, Jun-ichi Nakayama

    JOURNAL OF BIOCHEMISTRY   162 ( 5 ) 343 - 355  2017年11月  [査読有り]

     概要を見る

    Chromobox 2 (CBX2), a component of polycomb repressive complex 1 (PRC1), binds lysine 27-methylated histone H3 (H3K27me3) via its chromodomain (CD) and plays a critical role in repressing developmentally regulated genes. The phosphorylation of CBX2 has been described in several studies, but the biological implications of this modification remain largely elusive. Here, we show that CBX2's phosphorylation plays an important role in its nucleosome binding. CBX2 is stably phosphorylated in vivo, and domain analysis showed that residues in CBX2's serine-rich (SR) region are the predominant phosphorylation sites. The serine residues in an SR region followed by an acidic-residue (AR) cluster coincide with the consensus target of casein kinase II (CK2), and CK2 efficiently phosphorylated the SR region in vitro. A nucleosome pull-down assay revealed that CK2-phosphorylated CBX2 had a high specificity for H3K27me3-modified nucleosomes. An electrophoretic mobility-shift assay showed that CK2-mediated phosphorylation diminished CBX2's AT-hook-associated DNA-binding activity. Mutant CBX2 lacking the SR region or its neighboring AR cluster failed to repress the transcription of p21, a gene targeted by PRC1. These results suggest that CBX2's phosphorylation is critical for its transcriptional repression of target genes.

    DOI PubMed

  • Structural diversity of the nucleosome.

    Koyama M, Kurumizaka H

    Journal of biochemistry   163 ( 2 ) 85 - 95  2017年11月  [査読有り]

    DOI PubMed

  • Parallel mapping with site-directed hydroxyl radicals and micrococcal nuclease reveals structural features of positioned nucleosomes in vivo

    Tomohiro Fuse, Koji Katsumata, Koya Morohoshi, Yukio Mukai, Yuichi Ichikawa, Hitoshi Kurumizaka, Akio Yanagida, Takeshi Urano, Hiroaki Kato, Mitsuhiro Shimizu

    PLOS ONE   12 ( 10 ) e0186974  2017年10月  [査読有り]

     概要を見る

    Micrococcal nuclease (MNase) has been widely used for analyses of nucleosome locations in many organisms. However, due to its sequence preference, the interpretations of the positions and occupancies of nucleosomes using MNase have remained controversial. Next-generation sequencing (NGS) has also been utilized for analyses of MNase-digests, but some technical biases are commonly present in the NGS experiments. Here, we established a gel-based method to map nucleosome positions in Saccharomyces cerevisiae, using isolated nuclei as the substrate for the histone H4 S47C-site-directed chemical cleavage in parallel with MNase digestion. The parallel mapping allowed us to compare the chemically and enzymatically cleaved sites by indirect end-labeling and primer extension mapping, and thus we could determine the nucleosome positions and the sizes of the nucleosome- free regions (or nucleosome-depleted regions) more accurately, as compared to nucleosome mapping by MNase alone. The analysis also revealed that the structural features of the nucleosomes flanked by the nucleosome-free region were different from those within regularly arrayed nucleosomes, showing that the structures and dynamics of individual nucleosomes strongly depend on their locations. Moreover, we demonstrated that the parallel mapping results were generally consistent with the previous genome-wide chemical mapping and MNase-Seq results. Thus, the gel-based parallel mapping will be useful for the analysis of a specific locus under various conditions.

    DOI PubMed

  • Activation of the FA pathway mediated by phosphorylation and ubiquitination

    Masamichi Ishiai, Koichi Sato, Junya Tomida, Hiroyuki Kitao, Hitoshi Kurumizaka, Minoru Takata

    MUTATION RESEARCH-FUNDAMENTAL AND MOLECULAR MECHANISMS OF MUTAGENESIS   803   89 - 95  2017年10月  [査読有り]

     概要を見る

    Fanconi anemia (FA) is a devastating hereditary condition that impacts genome integrity, leading to clinical features such as skeletal and visceral organ malformations, attrition of bone marrow stem cells, and carcinogenesis. At least 21 proteins, when absent or defective, have been implicated in this disorder, and they together constitute the FA pathway, which functions in detection and repair of, and tolerance to, endogenous DNA damage. The damage primarily handled by the FA pathway has been assumed to be related to DNA interstrand crosslinks (ICLs). The FA pathway is activated upon ICL damage, and a hallmark of this activation is the mono-ubiquitination events of the key FANCD2-FANCI protein complex. Recent data have revealed unexpectedly complex details in the regulation of FA pathway activation by ICLs. In this short review, we summarize the knowledge accumulated over the years regarding how the FA pathway is activated via protein modifications.

    DOI PubMed

  • SYCP3 regulates strand invasion activities of RAD51 and DMC1

    Wataru Kobayashi, Noriko Hosoya, Shinichi Machida, Kiyoshi Miyagawa, Hitoshi Kurumizaka

    GENES TO CELLS   22 ( 9 ) 799 - 809  2017年09月  [査読有り]

     概要を見る

    The synaptonemal complex is a higher-ordered proteinaceous architecture formed between homologous chromosomes. SYCP3 is a major component of the lateral/axial elements in the synaptonemal complex and is essential for meiotic recombination. Previous genetic studies showed that SYCP3 functions in meiotic homologous recombination biased to interhomologous chromosomes, by regulating the strand invasion activities of the RAD51 and DMC1 recombinases. However, the mechanism by which SYCP3 regulates RAD51- and DMC1-mediated strand invasion remains elusive. In this study, we found that SYCP3 significantly suppresses the RAD51-mediated, but not the DMC1-mediated, strand invasion reaction by competing with HOP2-MND1, which is an activator for both RAD51 and DMC1. A SYCP3 mutant with defective RAD51 binding does not inhibit the RAD51-mediated homologous recombination in human cells. Therefore, SYCP3 may promote the DMC1-driven homologous recombination by attenuating the RAD51 activity during meiosis.

    DOI PubMed

  • Histone H3 Methylated at Arginine 17 Is Essential for Reprogramming the Paternal Genome in Zygotes

    Yuki Hatanaka, Takeshi Tsusaka, Natsumi Shimizu, Kohtaro Morita, Takehiro Suzuki, Shinichi Machida, Manabu Satoh, Arata Honda, Michiko Hirose, Satoshi Kamimura, Narumi Ogonuki, Toshinobu Nakamura, Kimiko Inoue, Yoshihiko Hosoi, Naoshi Dohmae, Toru Nakano, Hitoshi Kurumizaka, Kazuya Matsumoto, Yoichi Shinkai, Atsuo Ogura

    CELL REPORTS   20 ( 12 ) 2756 - 2765  2017年09月  [査読有り]

     概要を見る

    At fertilization, the paternal genome undergoes extensive reprogramming through protamine-histone exchange and active DNA demethylation, but only a few maternal factors have been defined in these processes. We identified maternal Mettl23 as a protein arginine methyltransferase (PRMT), which most likely catalyzes the asymmetric dimethylation of histone H3R17 (H3R17me2a), as indicated by in vitro assays and treatment with TBBD, an H3R17 PRMT inhibitor. Maternal histone H3.3, which is essential for paternal nucleosomal assembly, is unable to be incorporated into the male pronucleus when it lacks R17me2a. Mettl23 interacts with Tet3, a 5mC-oxidizing enzyme responsible for active DNA demethylation, by binding to another maternal factor, GSE (gonad-specific expression). Depletion of Mettl23 from oocytes resulted in impaired accumulation of GSE, Tet3, and 5hmC in the male pronucleus, suggesting that Mettl23 may recruit GSE-Tet3 to chromatin. Our findings establish H3R17me2a and its catalyzing enzyme Mettl23 as key regulators of paternal genome reprogramming.

    DOI PubMed

  • Association of M18BP1/KNL2 with CENP-A Nucleosome Is Essential for Centromere Formation in Non-mammalian Vertebrates

    Tetsuya Hori, Wei-Hao Shang, Masatoshi Hara, Mariko Ariyoshi, Yasuhiro Arimura, Risa Fujita, Hitoshi Kurumizaka, Tatsuo Fukagawa

    DEVELOPMENTAL CELL   42 ( 2 ) 181 - +  2017年07月  [査読有り]

     概要を見る

    Centromeres are specified and maintained by sequence-independent epigenetic mechanisms through the incorporation of CENP-A into centromeres. Given that CENP-A incorporation requires the Mis18 complex to be in the centromere region, it is necessary to precisely understand how the Mis18 complex localizes to the centromere region. Here, we showed that centromere localization of the Mis18 complex depends on CENP-A, but not CENP-C or CENP-T, in chicken DT40 cells. Furthermore, we demonstrated that M18BP1/KNL2, a member of the Mis18 complex, contained the CENP-C-like motif in chicken and other vertebrates, which is essential for centromere localization and M18BP1/KNL2 function in DT40 cells. We also showed that in vitro reconstituted CENP-A nucleosome, but not H3 nucleosome, bound to the CENP-C-like motif containing M18BP1/KNL2. Based on these results, we conclude thatM18BP1/KNL2 is essential for centromere formation through direct binding to CENP-A nucleosome in non-mammalian vertebrates. This explains how new CENP-A recognizes the centromere position.

    DOI PubMed

  • Synthetic Posttranslational Modifications: Chemical Catalyst-Driven Regioselective Histone Acylation of Native Chromatin

    Yoshifumi Amamoto, Yuki Aoi, Nozomu Nagashima, Hiroki Suto, Daisuke Yoshidome, Yasuhiro Arimura, Akihisa Osakabe, Daiki Kato, Hitoshi Kurumizaka, Shigehiro A. Kawashima, Kenzo Yamatsugu, Motomu Kanai

    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY   139 ( 22 ) 7568 - 7576  2017年06月  [査読有り]

     概要を見る

    Posttranslational modifications (PTMs) of histones play an important role in the complex regulatory mechanisms governing gene transcription, and their dysregulation can cause diseases such as cancer. The lack of methods for site-selectively modifying native chromatin, however, limits our understanding of the functional roles of a specific histone PTM, not as a single mark, but in the intertwined PTM network. Here, we report a synthetic catalyst DMAP-SH (DSH), which activates chemically stable thioesters (including acetyl-CoA) under physiological conditions and transfers various acyl groups to the proximate amino groups. Our data suggest that DSH, conjugated with a nucleosome ligand, such as pyrrole-imidazole-polyamide and LANA (latency-associated nuclear antigen)-peptide, promotes both natural (including acetylation, butyrylation, malonylation, and ubiquitination) and non-natural (azido- and phosphoryl labeling) PTMs on histones in recombinant nucleosomes and/or in native chromatin, at lysine residues close to the DSH moiety. To investigate the validity of our method, we used LANA-DSH to promote histone H2B lysine-120 (K120) acylation, the function of which is largely unknown. H2BK120 acetylation and malonylation modulated higher-order chromatin structures by reducing internucleosomal interactions, and this modulation was further enhanced by histone tail acetylation. This approach, therefore, may have versatile applications for dissecting the regulatory mechanisms underlying chromatin function.

    DOI

  • Synthetic Chromatin Acylation by an Artificial Catalyst System

    Tadashi Ishiguro, Yoshifumi Amamoto, Kana Tanabe, Jiaan Liu, Hidetoshi Kajino, Akiko Fujimura, Yuki Aoi, Akihisa Osakabe, Naoki Horikoshi, Hitoshi Kurumizaka, Kenzo Yamatsugu, Shigehiro A. Kawashima, Motomu Kanai

    CHEM   2 ( 6 ) 840 - 859  2017年06月  [査読有り]

     概要を見る

    Histone acetylation is physiologically regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs) and constitutes a fundamental regulatory element in gene expression. New types of lysine acylation on histones have recently been identified, but it remains unclear how chromatin function is regulated by divergent types of histone acylation and various enzymes. Here, we report on an approach to modulating histone acylation states synthetically without relying on enzymes. We have developed an artificial catalyst system composed of nucleosome-binding catalysts and acyl donors, which preferentially acetylated or malonylated lysines on histone tails and suppressed intraand inter-nucleosome interactions similarly to HATs. We demonstrate the utility of our approach by identifying a site-selectivity difference between two HDAC isoforms, Sirt1 and Sirt6, and comparing the functions of histone malonylation and acetylation. Our system is applicable to endogenous chromatin without genetic manipulation; thus, it can be used to dissect the complex regulation of chromatin.

    DOI

  • RFWD3-Mediated Ubiquitination Promotes Timely Removal of Both RPA and RAD51 from DNA Damage Sites to Facilitate Homologous Recombination

    Shojiro Inano, Koichi Sato, Yoko Katsuki, Wataru Kobayashi, Hiroki Tanaka, Kazuhiro Nakajima, Shinichiro Nakada, Hiroyuki Miyoshi, Kerstin Knies, Akifumi Takaori-Kondo, Detlev Schindler, Masamichi Ishiai, Hitoshi Kurumizaka, Minoru Takata

    MOLECULAR CELL   66 ( 5 ) 622 - +  2017年06月  [査読有り]

     概要を見る

    RFWD3 is a recently identified Fanconi anemia protein FANCW whose E3 ligase activity toward RPA is essential in homologous recombination (HR) repair. However, how RPA ubiquitination promotes HR remained unknown. Here, we identified RAD51, the central HR protein, as another target of RFWD3. We show that RFWD3 polyubiquitinates both RPA and RAD51 in vitro and in vivo. Phosphorylation by ATR and ATM kinases is required for this activity in vivo. RFWD3 inhibits persistent mitomycin C (MMC)-induced RAD51 and RPA foci by promoting VCP/p97-mediated protein dynamics and subsequent degradation. Furthermore, MMC-induced chromatin loading of MCM8 and RAD54 is defective in cells with inactivated RFWD3 or expressing a ubiquitination-deficient mutant RAD51. Collectively, our data reveal a mechanism that facilitates timely removal of RPA and RAD51 from DNA damage sites, which is crucial for progression to the late-phase HR and suppression of the FA phenotype.

    DOI PubMed

  • Erratum: Structure and Dynamics of a 197 bp Nucleosome in Complex with Linker Histone H1 (Molecular Cell (2017) 66 (384–397)(S109727651730268X)(10.1016/j.molcel.2017.04.012)

    Bednar J, Garcia-Saez I, Boopathi R, Cutter A.R, Papai G, Reymer A, Syed S.H, Lone I.N, Tonchev O, Crucifix C, Menoni H, Papin C, Skoufias D.A, Kurumizaka H, Lavery R, Hamiche A, Hayes J.J, Schultz P, Angelov D, Petosa C, Dimitrov S

    Molecular Cell   66 ( 5 ) 729  2017年06月  [査読有り]

    DOI PubMed

  • Structure and Dynamics of a 197 bp Nucleosome in Complex with Linker Histone H1

    Bednar J, Garcia-Saez I, Boopathi R, Cutter A.R, Papai G, Reymer A, Syed S.H, Lone I.N, Tonchev O, Crucifix C, Menoni H, Papin C, Skoufias D.A, Kurumizaka H, Lavery R, Hamiche A, Hayes J.J, Schultz P, Angelov D, Petosa C, Dimitrov S

    Molecular Cell   66 ( 3 ) 384 - +  2017年05月  [査読有り]

    DOI

  • Structure and function of human histone H3.Y nucleosome.

    Kujirai T, Horikoshi N, Sato K, Maehara K, Machida S, Osakabe A, Kimura H, Ohkawa Y, Kurumizaka H

    Nucleic acids research   45 ( 6 ) 3612  2017年04月  [査読有り]

    DOI PubMed

  • Influence of polynucleosome preparation methods on sedimentation velocity analysis of chromatin

    Tomoya Kujirai, Shinichi Machida, Akihisa Osakabe, Hitoshi Kurumizaka

    JOURNAL OF BIOCHEMISTRY   161 ( 4 ) 381 - 388  2017年04月  [査読有り]

     概要を見る

    Chromatin dynamics and higher order structures play essential roles in genomic DNA functions. Histone variants and histone post-translational modifications are involved in the regulation of chromatin structure and dynamics, cooperatively with DNA methylation and chromatin binding proteins. Therefore, studies of higher-order chromatin conformations have become important to reveal how genomic DNA is regulated during DNA transcription, replication, recombination and repair. The sedimentation velocity analysis by analytical ultracentrifugation has been commonly used to evaluate the higher-order conformation of in vitro reconstituted polynucleosomes, as model chromatin. Three major preparation methods for the unpurified, purified, and partially purified polynucleosomes have been reported so far. It is important to clarify the effects of the different polynucleosome preparation methods on the sedimentation profiles. To accomplish this, in the present study, we prepared unpurified, purified and partially purified polynucleosomes, and compared their sedimentation velocity profiles by analytical ultracentrifugation. In addition, we tested how the histone occupancy affects the sedimentation velocities of polynucleosomes. Our results revealed how free histones and polynucleosome aggregates affect the sedimentation velocity profiles of the polynucleosomes, in the absence and presence of Mg2+ ions.

    DOI

  • Histone Variant H2AL2 Guides Transition Protein-Dependent Protamine Assembly in Male Germ Cells

    Sophie Barral, Yuichi Morozumi, Hiroki Tanaka, Emilie Montellier, Jerome Govin, Maud de Dieuleveult, Guillaume Charbonnier, Yohann Coute, Denis Puthier, Thierry Buchou, Faycal Boussouar, Takashi Urahama, Francois Fenaille, Sandrine Curtet, Patrick Hery, Nicolas Fernandez-Nunez, Hitoshi Shiota, Matthieu Gerard, Sophie Rousseaux, Hitoshi Kurumizaka, Saadi Khochbin

    MOLECULAR CELL   66 ( 1 ) 89 - +  2017年04月  [査読有り]

     概要を見る

    Histone replacement by transition proteins (TPs) and protamines (Prms) constitutes an essential step for the successful production of functional male gametes, yet nothing is known on the underlying functional interplay between histones, TPs, and Prms. Here, by studying spermatogenesis in the absence of a spermatid-specific histone variant, H2A.L.2, we discover a fundamental mechanism involved in the transformation of nucleosomes into nucleoprotamines. H2A.L.2 is synthesized at the same time as TPs and enables their loading onto the nucleosomes. TPs do not displace histones but rather drive the recruitment and processing of Prms, which are themselves responsible for histone eviction. Altogether, the incorporation of H2A.L.2 initiates and orchestrates a series of successive transitional states that ultimately shift to the fully compacted genome of the mature spermatozoa. Hence, the current view of histone-to-nucleoprotamine transition should be revisited and include an additional step with H2A.L.2 assembly prior to the action of TPs and Prms.

    DOI

  • Crystal structure of the overlapping dinucleosome composed of hexasome and octasome

    Daiki Kato, Akihisa Osakabe, Yasuhiro Arimura, Yuka Mizukami, Naoki Horikoshi, Kazumi Saikusa, Satoko Akashi, Yoshifumi Nishimura, Sam-Yong Park, Jumpei Nogami, Kazumitsu Maehara, Yasuyuki Ohkawa, Atsushi Matsumoto, Hidetoshi Kono, Rintaro Inoue, Masaaki Sugiyama, Hitoshi Kurumizaka

    SCIENCE   356 ( 6334 ) 205 - 208  2017年04月  [査読有り]

     概要を見る

    Nucleosomes are dynamic entities that are repositioned along DNA by chromatin remodeling processes. A nucleosome repositioned by the switch-sucrose nonfermentable (SWI/SNF) remodeler collides with a neighbor and forms the intermediate" overlapping dinucleosome." Here, we report the crystal structure of the overlapping dinucleosome, in which two nucleosomes are associated, at 3.14-angstrom resolution. In the overlapping dinucleosome structure, the unusual "hexasome" nucleosome, composed of the histone hexamer lacking one H2A-H2B dimer from the conventional histone octamer, contacts the canonical "octasome" nucleosome, and they intimately associate. Consequently, about 250 base pairs of DNA are left-handedly wrapped in three turns, without a linker DNA segment between the hexasome and octasome moieties. The overlapping dinucleosome structure may provide important information to understand how nucleosome repositioning occurs during the chromatin remodeling process.

    DOI

  • Crystal Structure and Characterization of Novel Human Histone H3 Variants, H3.6, H3.7, and H3.8

    Hiroyuki Taguchi, Yan Xie, Naoki Horikoshi, Kazumitsu Maehara, Akihito Harada, Jumpei Nogami, Koichi Sato, Yasuhiro Arimura, Akihisa Osakabe, Tomoya Kujirai, Takeshi Iwasaki, Yuichiro Semba, Taro Tachibana, Hiroshi Kimura, Yasuyuki Ohkawa, Hitoshi Kurumizaka

    BIOCHEMISTRY   56 ( 16 ) 2184 - 2196  2017年04月  [査読有り]

     概要を見る

    Non-allelic histone variants are considered as epigenetic factors that regulate genomic DNA functions in eukaryotic chromosomes. In this study, we identified three new human histone H3 variants (named H3.6, H3.7, and H3.8), which were previously annotated as pseudogenes. H3.6 and H3.8 conserve the H3.3-specific amino acid residues, but H3.7 shares the specific amino acid residues with H3.1. We successfully reconstituted the nucleosome containing H3.6 in vitro and determined its crystal structure. In the H3.6 nucleosome, the H3.6-specific Val62 residue hydrophobically contacts the cognate H4 molecule, but its contact area is smaller than that of the corresponding H3.3 Ile62 residue. The thermal stability assay revealed that the H3.6 nucleosome is substantially unstable, as compared to the, H3.3 nucleosome. Interestingly, mutational analysis demonstrated that the H3.6 Val62 residue is fully responsible for the H3.6 nucleosome instability, probably because of the weakened hydrophobic interaction with H4. We also reconstituted the nucleosome containing H3.8, but its thermal stability was quite low. In contrast, purified H3.7 failed to form nucleosomes in vitro. The identification and characterization of these novel human histone H3 variants provide important new insights into understanding the epigenetic regulation of the human genome.

    DOI

  • Xeroderma pigmentosum group C protein interacts with histones: regulation by acetylated states of histone H3

    Erina Kakumu, Seiya Nakanishi, Hiromi M. Shiratori, Akari Kato, Wataru Kobayashi, Shinichi Machida, Takeshi Yasuda, Naoko Adachi, Naoaki Saito, Tsuyoshi Ikura, Hitoshi Kurumizaka, Hiroshi Kimura, Masayuki Yokoi, Wataru Sakai, Kaoru Sugasawa

    GENES TO CELLS   22 ( 3 ) 310 - 327  2017年03月  [査読有り]

     概要を見る

    In the mammalian global genome nucleotide excision repair pathway, two damage recognition factors, XPC and UV-DDB, play pivotal roles in the initiation of the repair reaction. However, the molecular mechanisms underlying regulation of the lesion recognition process in the context of chromatin structures remain to be understood. Here, we show evidence that damage recognition factors tend to associate with chromatin regions devoid of certain types of acetylated histones. Treatment of cells with histone deacetylase inhibitors retarded recruitment of XPC to sites of UV-induced DNA damage and the subsequent repair process. Biochemical studies showed novel multifaceted interactions of XPC with histone H3, which were profoundly impaired by deletion of the N-terminal tail of histone H3. In addition, histone H1 also interacted with XPC. Importantly, acetylation of histone H3 markedly attenuated the interaction with XPC invitro, and local UV irradiation of cells decreased the level of H3K27ac in the damaged areas. Our results suggest that histone deacetylation plays a significant role in the process of DNA damage recognition for nucleotide excision repair and that the localization and functions of XPC can be regulated by acetylated states of histones.

    DOI

  • Testis-Specific Histone Variant H3t Gene Is Essential for Entry into Spermatogenesis

    Jun Ueda, Akihito Harada, Takashi Urahama, Shinichi Machida, Kazumitsu Maehara, Masashi Hada, Yoshinori Makino, Jumpei Nogami, Naoki Horikoshi, Akihisa Osakabe, Hiroyuki Taguchi, Hiroki Tanaka, Hiroaki Tachiwana, Tatsuma Yao, Minami Yamada, Takashi Iwamoto, Ayako Isotani, Masahito Ikawa, Taro Tachibana, Yuki Okada, Hiroshi Kimura, Yasuyuki Ohkawa, Hitoshi Kurumizaka, Kazuo Yamagata

    CELL REPORTS   18 ( 3 ) 593 - 600  2017年01月  [査読有り]

     概要を見る

    Cellular differentiation is associated with dynamic chromatin remodeling in establishing a cell-typespecific epigenomic landscape. Here, we find that mouse testis-specific and replication-dependent histone H3 variant H3t is essential for very early stages of spermatogenesis. H3t gene deficiency leads to azoospermia because of the loss of haploid germ cells. When differentiating spermatogonia emerge in normal spermatogenesis, H3t appears and replaces the canonical H3 proteins. Structural and biochemical analyses reveal that H3t-containing nucleosomes are more flexible than the canonical nucleosomes. Thus, by incorporating H3t into the genome during spermatogonial differentiation, male germ cells are able to enter meiosis and beyond.

    DOI

  • Polymorphism of apyrimidinic DNA structures in the nucleosome

    Akihisa Osakabe, Yasuhiro Arimura, Syota Matsumoto, Naoki Horikoshi, Kaoru Sugasawa, Hitoshi Kurumizaka

    SCIENTIFIC REPORTS   7  2017年01月  [査読有り]

     概要を見る

    Huge amounts (&gt;10,000/day) of apurinic/apyrimidinic (AP) sites are produced in genomes, but their structures in chromatin remain undetermined. We determined the crystal structure of the nucleosome containing AP-site analogs at two symmetric sites, which revealed structural polymorphism: one forms an inchworm configuration without an empty space at the AP site, and the other forms a B-form-like structure with an empty space and the orphan base. This unexpected inchworm configuration of the AP site is important to understand the AP DNA repair mechanism, because it may not be recognized by the major AP-binding protein, APE1, during the base excision repair process.

    DOI

  • In vitro reconstitution and biochemical analyses of the Schizosaccharomyces pombe nucleosome

    Masako Koyama, Wataru Nagakura, Hiroki Tanaka, Tomoya Kujirai, Yuji Chikashige, Tokuko Haraguchi, Yasushi Hiraoka, Hitoshi Kurumizaka

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   482 ( 4 ) 896 - 901  2017年01月  [査読有り]

     概要を見る

    Schizosaccharomyces pombe, which has a small genome but shares many physiological functions with higher eulcaryotes, is a useful single-cell, model eukaryotic organism. In particular, many features concerning chromatin structure and dynamics, including heterochromatin, centromeres, telomeres, and DNA replication origins, are well conserved between S. pombe and higher eulcaryotes. However, the S. pombe nucleosome, the fundamental structural unit of chromatin, has not been reconstituted in vitro. In the present study, we established the method to purify S. pombe histones H2A, H2B, H3, and H4, and successfully reconstituted the S. pombe nucleosome in vitro. Our thermal stability assay and micrococcal nuclease treatment assay revealed that the S. pombe nucleosome is markedly unstable and its DNA ends are quite accessible, as compared to the canonical human nucleosome. These findings are important to understand the mechanisms of epigenetic genomic DNA regulation in fission yeast. (C) 2016 Elsevier Inc. All rights reserved.

    DOI

  • Identification of the amino acid residues responsible for stable nucleosome formation by histone H3.Y

    Kujirai T, Horikoshi N, Xie Y, Taguchi H, Kurumizaka H

    Nucleus     1 - 10  2017年  [査読有り]

    DOI

  • FANCI-FANCD2 stabilizes the RAD51-DNA complex by binding RAD51 and protects the 5 '-DNA end

    Koichi Sato, Mayo Shimomuki, Yoko Katsuki, Daisuke Takahashi, Wataru Kobayashi, Masamichi Ishiai, Hiroyuki Miyoshi, Minoru Takata, Hitoshi Kurumizaka

    NUCLEIC ACIDS RESEARCH   44 ( 22 ) 10758 - 10771  2016年12月  [査読有り]

     概要を見る

    The FANCI-FANCD2 (I-D) complex is considered to work with RAD51 to protect the damaged DNA in the stalled replication fork. However, the means by which this DNA protection is accomplished have remained elusive. In the present study, we found that the I-D complex directly binds to RAD51, and stabilizes the RAD51-DNA filament. Unexpectedly, the DNA binding activity of FANCI, but not FANCD2, is explicitly required for the I-D complex-mediated RAD51-DNA filament stabilization. The RAD51 filament stabilized by the I-D complex actually protects the DNA end from nucleolytic degradation by an FA-associated nuclease, FAN1. This DNA end protection is not observed with the RAD51 mutant from FANCR patient cells. These results clearly answer the currently enigmatic question of how RAD51 functions with the I-D complex to prevent genomic instability at the stalled replication fork.

    DOI

  • The Flexible Ends of CENP-A Nucleosome Are Required for Mitotic Fidelity

    Yohan Roulland, Khalid Ouararhni, Mladen Naidenov, Lorrie Ramos, Muhammad Shuaib, Sajad Hussain Syed, Imtiaz Nizar Lone, Ramachandran Boopathi, Emeline Fontaine, Gabor Papai, Hiroaki Tachiwana, Thierry Gautier, Dimitrios Skoufias, Kiran Padmanabhan, Jan Bednar, Hitoshi Kurumizaka, Patrick Schultz, Dimitar Angelov, Ali Hamiche, Stefan Dimitrov

    MOLECULAR CELL   63 ( 4 ) 674 - 685  2016年08月  [査読有り]

     概要を見る

    CENP-A is a histone variant, which replaces histone H3 at centromeres and confers unique properties to centromeric chromatin. The crystal structure of CENP-A nucleosome suggests flexible nucleosomal DNA ends, but their dynamics in solution remains elusive and their implication in centromere function is unknown. Using electron cryo-microscopy, we determined the dynamic solution properties of the CENP-A nucleosome. Our biochemical, proteomic, and genetic data reveal that higher flexibility of DNA ends impairs histone H1 binding to the CENP-A nucleosome. Substituting the 2-turn alpha N-helix of CENP-A with the 3-turn alpha N-helix of H3 results in compact particles with rigidified DNA ends, able to bind histone H1. In vivo replacement of CENP-A with H3-CENP-A hybrid nucleosomes leads to H1 recruitment, delocalization of kinetochore proteins, and significant mitotic and cytokinesis defects. Our data reveal that the evolutionarily conserved flexible ends of the CENP-A nucleosomes are essential to ensure the fidelity of the mitotic pathway.

    DOI

  • Structure of the human DNA-repair protein RAD52 containing surface mutations

    Mika Saotome, Kengo Saito, Keiichi Onodera, Hitoshi Kurumizaka, Wataru Kagawa

    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS   72   598 - 603  2016年08月  [査読有り]

     概要を見る

    The Rad52 protein is a eukaryotic single-strand DNA-annealing protein that is involved in the homologous recombinational repair of DNA double-strand breaks. The isolated N-terminal half of the human RAD52 protein (RAD52(1-212)) forms an undecameric ring structure with a surface that is mostly positively charged. In the present study, it was found that RAD52(1-212) containing alanine mutations of the charged surface residues (Lys102, Lys133 and Glu202) is highly amenable to crystallization. The structure of the mutant RAD52(1-212) was solved at 2.4 angstrom resolution. The structure revealed an association between the symmetry-related RAD52(1-212) rings, in which a partially unfolded, C-terminal region of RAD52 extended into the DNA-binding groove of the neighbouring ring in the crystal. The alanine mutations probably reduced the surface entropy of the RAD52(1-212) ring and stabilized the ring-ring association observed in the crystal.

    DOI

  • Structure and function of human histone H3.Y nucleosome

    Tomoya Kujirai, Naoki Horikoshi, Koichi Sato, Kazumitsu Maehara, Shinichi Machida, Akihisa Osakabe, Hiroshi Kimura, Yasuyuki Ohkawa, Hitoshi Kurumizaka

    NUCLEIC ACIDS RESEARCH   44 ( 13 ) 6127 - 6141  2016年07月  [査読有り]

     概要を見る

    Histone H3.Y is a primate-specific, distant H3 variant. It is evolutionarily derived from H3.3, and may function in transcription regulation. However, the mechanism by which H3.Y regulates transcription has not been elucidated. In the present study, we determined the crystal structure of the H3. Y nucleosome, and found that many H3.Y-specific residues are located on the entry/exit sites of the nucleosome. Biochemical analyses revealed that the DNA ends of the H3.Y nucleosome were more flexible than those of the H3.3 nucleosome, although the H3. Y nucleosome was stable in vitro and in vivo. Interestingly, the linker histone H1, which compacts nucleosomal DNA, appears to bind to the H3. Y nucleosome less efficiently, as compared to the H3.3 nucleosome. These characteristics of the H3. Y nucleosome are also conserved in the H3. Y/H3.3 heterotypic nucleosome, which may be the predominant form in cells. In human cells, H3. Y preferentially accumulated around transcription start sites (TSSs). Taken together, H3.Y-containing nucleosomes around transcription start sites may form relaxed chromatin that allows transcription factor access, to regulate the transcription status of specific genes.

    DOI

  • Sequence-directed nucleosome-depletion is sufficient to activate transcription from a yeast core promoter in vivo

    Yuichi Ichikawa, Nobuyuki Morohashi, Nobuyuki Tomita, Aaron P. Mitchell, Hitoshi Kurumizaka, Mitsuhiro Shimizu

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   476 ( 2 ) 57 - 62  2016年07月  [査読有り]

     概要を見る

    Nucleosome-depleted regions (NDRs) (also called nucleosome-free regions or NFRs) are often found in the promoter regions of many yeast genes, and are formed by multiple mechanisms, including the binding of activators and enhancers, the actions of chromatin remodeling complexes, and the specific DNA sequences themselves. However, it remains unclear whether NDR formation per se is essential for transcriptional activation. Here, we examined the relationship between nucleosome organization and gene expression using a defined yeast reporter system, consisting of the CYC1 minimal core promoter and the lacZ gene. We introduced simple repeated sequences that should be either incorporated in nucleosomes or excluded from nucleosomes in the site upstream of the TATA boxes. The (CTG)(12), (GAA)(12) and (TGTAGG)(6) inserts were incorporated into a positioned nucleosome in the core promoter region, and did not affect the reporter gene expression. In contrast, the insertion of (CGG)(12), (TTAGGG)(6), (A)(34) or (CG)(8) induced lacZ expression by 10-20 fold. Nucleosome mapping analyses revealed that the inserts that induced the reporter gene expression prevented nucleosome formation, and created an NDR upstream of the TATA boxes. Thus, our results demonstrated that NDR formation dictated by DNA sequences is sufficient for transcriptional activation from the core promoter in vivo. (C) 2016 Elsevier Inc. All rights reserved.

    DOI

  • Structural and biochemical analyses of monoubiquitinated human histones H2B and H4

    Shinichi Machida, Satoshi Sekine, Yuuki Nishiyama, Naoki Horikoshi, Hitoshi Kurumizaka

    OPEN BIOLOGY   6 ( 6 )  2016年06月  [査読有り]

     概要を見る

    Monoubiquitination is a major histone post-translational modification. In humans, the histone H2B K120 and histone H4 K31 residues are monoubiquitinated and may form transcriptionally active chromatin. In this study, we reconstituted nucleosomes containing H2B monoubiquitinated at position 120 (H2Bub(120)) and/or H4 monoubiquitinated at position 31 (H4ub(31)). We found that the H2Bub(120) and H4ub(31) monoubiquitinations differently affect nucleosome stability: the H2Bub(120) monoubiquitination enhances the H2A-H2B association with the nucleosome, while the H4ub(31) monoubiquitination decreases the H3-H4 stability in the nucleosome, when compared with the unmodified nucleosome. The H2Bub(120) and H4ub(31) monoubiquitinations both antagonize the Mg2+-dependent compaction of a poly-nucleosome, suggesting that these monoubiquitinations maintain more relaxed conformations of chromatin. In the crystal structure, the H2Bub(120) and H4ub(31) monoubiquitinations do not change the structure of the nucleosome core particle and the ubiquitin molecules were flexibly disordered in the H2Bub(120)/H4ub(31) nucleosome structure. These results revealed the differences and similarities of the H2Bub(120) and H4ub(31) monoubiquitinations at the mono- and poly-nucleosome levels and provide novel information to clarify the roles of monoubiquitination in chromatin.

    DOI

  • Crystal structures of heterotypic nucleosomes containing histones H2A.Z and H2A

    Naoki Horikoshi, Yasuhiro Arimura, Hiroyuki Taguchi, Hitoshi Kurumizaka

    OPEN BIOLOGY   6 ( 6 )  2016年06月  [査読有り]

     概要を見る

    H2A.Z is incorporated into nucleosomes located around transcription start sites and functions as an epigenetic regulator for the transcription of certain genes. During transcriptional regulation, the heterotypic H2A.Z/H2A nucleosome containing one each of H2A.Z and H2A is formed. However, previous homotypic H2A.Z nucleosome structures suggested that the L1 loop region of H2A.Z would sterically clash with the corresponding region of canonical H2A in the heterotypic nucleosome. To resolve this issue, we determined the crystal structures of heterotypic H2A.Z/H2A nucleosomes. In the H2A.Z/H2A nucleosome structure, the H2A.Z L1 loop structure was drastically altered without any structural changes of the canonical H2A L1 loop, thus avoiding the steric clash. Unexpectedly, the heterotypic H2A.Z/H2A nucleosome is more stable than the homotypic H2A.Z nucleosome. These data suggested that the flexible character of the H2A.Z L1 loop plays an essential role in forming the stable heterotypic H2A.Z/H2A nucleosome.

    DOI

  • Chromatin architecture may dictate the target site for DMC1, but not for RAD51, during homologous pairing

    Wataru Kobayashi, Motoki Takaku, Shinichi Machida, Hiroaki Tachiwana, Kazumitsu Maehara, Yasuyuki Ohkawa, Hitoshi Kurumizaka

    SCIENTIFIC REPORTS   6  2016年04月  [査読有り]

     概要を見る

    In eukaryotes, genomic DNA is compacted as chromatin, in which histones and DNA form the nucleosome as the basic unit. DMC1 and RAD51 are essential eukaryotic recombinases that mediate homologous chromosome pairing during homologous recombination. However, the means by which these two recombinases distinctly function in chromatin have remained elusive. Here we found that, in chromatin, the human DMC1-single-stranded DNA complex bypasses binding to the nucleosome, and preferentially promotes homologous pairing at the nucleosome-depleted regions. Consistently, DMC1 forms ternary complex recombination intermediates with the nucleosome-free DNA or the nucleosome-depleted DNA region. Surprisingly, removal of the histone tails improperly enhances the nucleosome binding by DMC1. In contrast, RAD51 does not specifically target the nucleosome-depleted region in chromatin. These are the first demonstrations that the chromatin architecture specifies the sites to promote the homologous recombination reaction by DMC1, but not by RAD51.

    DOI

  • C-terminal acidic domain of histone chaperone human NAP1 is an efficient binding assistant for histone H2A-H2B, but not H3-H4

    Hideaki Ohtomo, Satoko Akashi, Yoshihito Moriwaki, Mitsuru Okuwaki, Akihisa Osakabe, Kyosuke Nagata, Hitoshi Kurumizaka, Yoshifumi Nishimura

    GENES TO CELLS   21 ( 3 ) 252 - 263  2016年03月  [査読有り]

     概要を見る

    Nucleosome assembly protein 1 (NAP1) binds both the (H3-H4)(2) tetramer and two H2A-H2B dimers, mediating their sequential deposition on DNA. NAP1 contains a C-terminal acidic domain (CTAD) and a core domain that promotes dimer formation. Here, we have investigated the roles of the core domain and CTAD of human NAP1 in binding to H2A-H2B and H3-H4 by isothermal calorimetry and native mass spectrometry and compared them with the roles of yeast NAP1. We show that the hNAP1 and yNAP1 dimers bind H2A-H2B by two different modes: a strong endothermic interaction and a weak exothermic interaction. A mutant hNAP1, but not yNAP1, dimer lacking CTAD loses the exothermic interaction and shows greatly reduced H2A-H2B binding activity. The isolated CTAD of hNAP1 binds H2A-H2B only exothermically with relatively stronger binding as compared with the exothermic interaction observed for the full-length hNAP1 dimer. Thus, the two CTADs in the hNAP1 dimer seem to provide binding assistance for the strong endothermic interaction of the core domain with H2A-H2B. By contrast, in the relatively weaker binding of hNAP1 to H3-H4 as compared with yNAP1, CTAD of hNAP1 has no significant role. To our knowledge, this is the first distinct role identified for the hNAP1 CTAD.

    DOI J-GLOBAL

  • Crystal structure of the nucleosome containing ultraviolet light-induced cyclobutane pyrimidine dimer

    Naoki Horikoshi, Hiroaki Tachiwana, Wataru Kagawa, Akihisa Osakabe, Syota Matsumoto, Shigenori Iwai, Kaoru Sugasawa, Hitoshi Kurumizaka

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   471 ( 1 ) 117 - 122  2016年02月  [査読有り]

     概要を見る

    The cyclobutane pyrimidine dimer (CPD) is induced in genomic DNA by ultraviolet (UV) light. In mammals, this photolesion is primarily induced within nucleosomal DNA, and repaired exclusively by the nucleotide excision repair (NER) pathway. However, the mechanism by which the CPD is accommodated within the nucleosome has remained unknown. We now report the crystal structure of a nucleosome containing CPDs. In the nucleosome, the CPD induces only limited local backbone distortion, and the affected bases are accommodated within the duplex. Interestingly, one of the affected thymine bases is located within 3.0 angstrom from the undamaged complementary adenine base, suggesting the formation of complementary hydrogen bonds in the nucleosome. We also found that UV-DDB, which binds the CPD at the initial stage of the NER pathway, also efficiently binds to the nucleosomal CPD. These results provide important structural and biochemical information for understanding how the CPD is accommodated and recognized in chromatin. (C) 2016 Elsevier Inc. All rights reserved.

    DOI

  • GATA3-dependent cellular reprogramming requires activation-domain dependent recruitment of a chromatin remodeler

    Motoki Takaku, Sara A. Grimm, Takashi Shimbo, Lalith Perera, Roberta Menafra, Hendrik G. Stunnenberg, Trevor K. Archer, Shinichi Machida, Hitoshi Kurumizaka, Paul A. Wade

    GENOME BIOLOGY   17 ( 1 ) 36  2016年02月  [査読有り]

     概要を見る

    Background: Transcription factor-dependent cellular reprogramming is integral to normal development and is central to production of induced pluripotent stem cells. This process typically requires pioneer transcription factors (TFs) to induce de novo formation of enhancers at previously closed chromatin. Mechanistic information on this process is currently sparse.
    Results: Here we explore the mechanistic basis by which GATA3 functions as a pioneer TF in a cellular reprogramming event relevant to breast cancer, the mesenchymal to epithelial transition (MET). In some instances, GATA3 binds previously inaccessible chromatin, characterized by stable, positioned nucleosomes where it induces nucleosome eviction, alters local histone modifications, and remodels local chromatin architecture. At other loci, GATA3 binding induces nucleosome sliding without concomitant generation of accessible chromatin. Deletion of the transactivation domain retains the chromatin binding ability of GATA3 but cripples chromatin reprogramming ability, resulting in failure to induce MET.
    Conclusions: These data provide mechanistic insights into GATA3-mediated chromatin reprogramming during MET, and suggest unexpected complexity to TF pioneering. Successful reprogramming requires stable binding to a nucleosomal site; activation domain-dependent recruitment of co-factors including BRG1, the ATPase subunit of the SWI/SNF chromatin remodeling complex; and appropriate genomic context. The resulting model provides a new conceptual framework for de novo enhancer establishment by a pioneer TF.

    DOI J-GLOBAL

  • Relaxed Chromatin Formation and Weak Suppression of Homologous Pairing by the Testis-Specific Linker Histone H1T

    Shinichi Machida, Ryota Hayashida, Motoki Takaku, Atsuhiko Fukuto, Jiying Sun, Aiko Kinomura, Satoshi Tashiro, Hitoshi Kurumizaka

    BIOCHEMISTRY   55 ( 4 ) 637 - 646  2016年02月  [査読有り]

     概要を見る

    Linker histones bind to nucleosomes and compact polynucleosomes into a higher-order: chromatin configuration. Somatic and germ cell-specific linker histone subtypes have been identified and may have distinct functions. In this study; we reconstituted polynucleosomes containing human histones H1.2 and H1T, as representative somatic and germ cell-specific linker histones, respectively, and found that HIT forms less compacted chromatin, as compared to H1.2. An in vitro homologous pairing assay revealed that H1T weakly inhibited RAD51/RAD54-mediated homologous pairing in chromatin, although the somatic H1 subtypes, H1.0, H1.1, H1.2, H1.3, H1.4, and H1.53 substantially suppressed it. An in vivo recombination assay revealed that H1T overproduction minimally affected the recombination frequency, but significant suppression was observed When H1.2 was overproduced in human cells. These results suggested that the testis-specific linker histone, H1T, possesses a Specific function to produce the chromatin architecture required for proper chromosome regulation, such as homologous recombination.

    DOI J-GLOBAL

  • Bivalent interaction of the PZP domain of BRPF1 with the nucleosome impacts chromatin dynamics and acetylation

    Brianna J. Klein, Uma M. Muthurajan, Marie-Eve Lalonde, Matthew D. Gibson, Forest H. Andrews, Maggie Hepler, Shinichi Machida, Kezhi Yan, Hitoshi Kurumizaka, Michael G. Poirier, Jacques Cote, Karolin Luger, Tatiana G. Kutateladze

    NUCLEIC ACIDS RESEARCH   44 ( 1 ) 472 - 484  2016年01月  [査読有り]

     概要を見る

    BRPF1 (bromodomain PHD finger 1) is a core subunit of the MOZ histone acetyltransferase (HAT) complex, critical for normal developmental programs and implicated in acute leukemias. BRPF1 contains a unique assembly of zinc fingers, termed a PZP domain, the physiological role of which remains unclear. Here, we elucidate the structure-function relationship of this novel epigenetic reader and detail the biological and mechanistic consequences of its interaction with nucleosomes. PZP has a globular architecture and forms a 2: 1 stoichiometry complex with the nucleosome, bivalently interacting with histone H3 and DNA. This binding impacts the nucleosome dynamics, shifting the DNA unwrapping/rewrapping equilibrium toward the unwrapped state and increasing DNA accessibility. We demonstrate that the DNA-binding function of the BRPF1 PZP domain is required for the MOZ-BRPF1-ING5-hEaf6 HAT complex to be recruited to chromatin and to acetylate nucleosomal histones. Our findings reveal a novel link between chromatin dynamics and MOZ-mediated acetylation.

    DOI

  • A Genetically Encoded Probe for Live-Cell Imaging of H4K20 Monomethylation

    Sato Y, Kujirai T, Arai R, Asakawa H, Ohtsuki C, Horikoshi N, Yamagata K, Ueda J, Nagase T, Haraguchi T, Hiraoka Y, Kimura A, Kurumizaka H, Kimura H

    Journal of Molecular Biology   428 ( 20 ) 3885 - 3902  2016年  [査読有り]

    DOI

  • Histone H3.5 forms an unstable nucleosome and accumulates around transcription start sites in human testis

    Takashi Urahama, Akihito Harada, Kazumitsu Maehara, Naoki Horikoshi, Koichi Sato, Yuko Sato, Koji Shiraishi, Norihiro Sugino, Akihisa Osakabe, Hiroaki Tachiwana, Wataru Kagawa, Hiroshi Kimura, Yasuyuki Ohkawa, Hitoshi Kurumizaka

    EPIGENETICS & CHROMATIN   9 ( Jan ) 2  2016年01月  [査読有り]

     概要を見る

    Background: Human histone H3.5 is a non-allelic H3 variant evolutionally derived from H3.3. The H3.5 mRNA is highly expressed in human testis. However, the function of H3.5 has remained poorly understood.
    Results: We found that the H3.5 nucleosome is less stable than the H3.3 nucleosome. The crystal structure of the H3.5 nucleosome showed that the H3.5-specific Leu103 residue, which corresponds to the H3.3 Phe104 residue, reduces the hydrophobic interaction with histone H4. Mutational analyses revealed that the H3.5-specific Leu103 residue is responsible for the instability of the H3.5 nucleosome, both in vitro and in living cells. The H3.5 protein was present in human seminiferous tubules, but little to none was found in mature sperm. A chromatin immunoprecipitation coupled with sequencing analysis revealed that H3.5 accumulated around transcription start sites (TSSs) in testicular cells.
    Conclusions: We performed comprehensive studies of H3.5, and found the instability of the H3.5 nucleosome and the accumulation of H3.5 protein around TSSs in human testis. The unstable H3.5 nucleosome may function in the chromatin dynamics around the TSSs, during spermatogenesis.

    DOI J-GLOBAL

  • Crystal structure of the nucleosome containing histone H3 with crotonylated lysine 122

    Yuya Suzuki, Naoki Horikoshi, Daiki Kato, Hitoshi Kurumizaka

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   469 ( 3 ) 483 - 489  2016年01月  [査読有り]

     概要を見る

    The crotonylation of histones is an important post-translational modification, and epigenetically functions in the regulation of genomic DNA activity. The histone modifications in the structured "histone-fold" domains are considered to have an especially important impact on the nucleosome structure and dynamics. In the present study, we reconstituted the human nucleosome containing histone H3.2 crotonylated at the Lys122 residue, and determined its crystal structure at 2.56 angstrom resolution. We found that the crotonylation of the H3 Lys122 residue does not affect the overall nucleosome structure, but locally impedes the formation of the water-mediated hydrogen bond with the DNA backbone. Consistently, thermal stability assays revealed that the H3 Lys122 crotonylation, as well as the H3 Lys122 acetylation, clearly reduced the histone-DNA association. (C) 2015 Elsevier Inc. All rights reserved.

    DOI J-GLOBAL

  • Structural basis of pyrimidine-pyrimidone (6-4) photoproduct recognition by UV-DDB in the nucleosome.

    Akihisa Osakabe, Hiroaki Tachiwana, Wataru Kagawa, Naoki Horikoshi, Syota Matsumoto, Mayu Hasegawa, Naoyuki Matsumoto, Tatsuya Toga, Junpei Yamamoto, Fumio Hanaoka, Nicolas H Thomä, Kaoru Sugasawa, Shigenori Iwai, Hitoshi Kurumizaka

    Scientific reports   5   16330 - 16330  2015年11月  [査読有り]  [国際誌]

     概要を見る

    UV-DDB, an initiation factor for the nucleotide excision repair pathway, recognizes 6-4PP lesions through a base flipping mechanism. As genomic DNA is almost entirely accommodated within nucleosomes, the flipping of the 6-4PP bases is supposed to be extremely difficult if the lesion occurs in a nucleosome, especially on the strand directly contacting the histone surface. Here we report that UV-DDB binds efficiently to nucleosomal 6-4PPs that are rotationally positioned on the solvent accessible or occluded surface. We determined the crystal structures of nucleosomes containing 6-4PPs in these rotational positions, and found that the 6-4PP DNA regions were flexibly disordered, especially in the strand exposed to the solvent. This characteristic of 6-4PP may facilitate UV-DDB binding to the damaged nucleosome. We present the first atomic-resolution pictures of the detrimental DNA cross-links of neighboring pyrimidine bases within the nucleosome, and provide the mechanistic framework for lesion recognition by UV-DDB in chromatin.

    DOI PubMed J-GLOBAL

  • Dynamic changes in CCAN organization through CENP-C during cell-cycle progression

    Harsh Nagpal, Tetsuya Hori, Ayako Furukawa, Kenji Sugase, Akihisa Osakabe, Hitoshi Kurumizaka, Tatsuo Fukagawa

    Molecular Biology of the Cell   26 ( 21 ) 3768 - 3776  2015年11月  [査読有り]

     概要を見る

    The kinetochore is a crucial structure for faithful chromosome segregation during mitosis and is formed in the centromeric region of each chromosome. The 16-subunit protein complex known as the constitutive centromere-associated network (CCAN) forms the foundation for kinetochore assembly on the centromeric chromatin. Although the CCAN can be divided into several subcomplexes, it remains unclear how CCAN proteins are organized to form the functional kinetochore. In particular, this organization may vary as the cell cycle progresses. To address this, we analyzed the relationship of centromeric protein (CENP)-C with the CENP-H complex during progression of the cell cycle. We find that the middle portion of chicken CENP-C (CENP-C166–324) is sufficient for centromere localization during interphase, potentially through association with the CENP-L-N complex. The C-terminus of CENP-C (CENP-C601–864) is essential for centromere localization during mitosis, through binding to CENP-A nucleosomes, independent of the CENP-H complex. On the basis of these results, we propose that CCAN organization changes dynamically during progression of the cell cycle.

    DOI

  • Influence of DNA methylation on positioning and DNA flexibility of nucleosomes with pericentric satellite DNA

    Akihisa Osakabe, Fumiya Adachi, Yasuhiro Arimura, Kazumitsu Maehara, Yasuyuki Ohkawa, Hitoshi Kurumizaka

    OPEN BIOLOGY   5 ( 10 ) 150128  2015年10月  [査読有り]

     概要を見る

    DNA methylation occurs on CpG sites and is important to form pericentric heterochromatin domains. The satellite 2 sequence, containing seven CpG sites, is located in the pericentric region of human chromosome 1 and is highly methylated in normal cells. In contrast, the satellite 2 region is reportedly hypomethylated in cancer cells, suggesting that the methylation status may affect the chromatin structure around the pericentric regions in tumours. In this study, we mapped the nucleosome positioning on the satellite 2 sequence in vitro and found that DNA methylation modestly affects the distribution of the nucleosome positioning. The micrococcal nuclease assay revealed that the DNA end flexibility of the nucleosomes changes, depending on the DNA methylation status. However, the structures and thermal stabilities of the nucleosomes are unaffected by DNA methylation. These findings provide new information to understand how DNA methylation functions in regulating pericentric heterochromatin formation and maintenance in normal and malignant cells.

    DOI J-GLOBAL

  • hCAS/CSE1L regulates RAD51 distribution and focus formation for homologous recombinational repair

    Satoshi Okimoto, Jiying Sun, Atsuhiko Fukuto, Yasunori Horikoshi, Shun Matsuda, Tomonari Matsuda, Masae Ikura, Tsuyoshi Ikura, Shinichi Machida, Hitoshi Kurumizaka, Yoichi Miyamoto, Masahiro Oka, Yoshihiro Yoneda, Yoshiaki Kiuchi, Satoshi Tashiro

    GENES TO CELLS   20 ( 9 ) 681 - 694  2015年09月  [査読有り]

     概要を見る

    Homologous recombinational repair (HR) is one of the major repair systems for DNA double-strand breaks. RAD51 is a key molecule in HR, and the RAD51 concentration in the cell nucleus increases after DNA damage induction. However, the mechanism that regulates the intracellular distribution of RAD51 is still unclear. Here, we show that hCAS/CSE1L associates with RAD51 in human cells. We found that hCAS/CSE1L negatively regulates the nuclear protein level of RAD51 under normal conditions. hCAS/CSE1L is also required to repress the DNA damage-induced focus formation of RAD51. Moreover, we show that hCAS/CSE1L plays roles in the regulation of the HR activity and in chromosome stability. These findings suggest that hCAS/CSE1L is responsible for controlling the HR activity by directly interacting with RAD51.

    DOI

  • Human FAN1 promotes strand incision in 5 '-flapped DNA complexed with RPA

    Daisuke Takahashi, Koichi Sato, Emiko Hirayama, Minoru Takata, Hitoshi Kurumizaka

    JOURNAL OF BIOCHEMISTRY   158 ( 3 ) 263 - 270  2015年09月  [査読有り]

     概要を見る

    Fanconi anaemia (FA) is a human infantile recessive disorder. Seventeen FA causal proteins cooperatively function in the DNA interstrand crosslink (ICL) repair pathway. Dual DNA strand incisions around the crosslink are critical steps in ICL repair. FA-associated nuclease 1 (FAN1) is a DNA structure-specific endonuclease that is considered to be involved in DNA incision at the stalled replication fork. Replication protein A (RPA) rapidly assembles on the single-stranded DNA region of the stalled fork. However, the effect of RPA on the FAN1-mediated DNA incision has not been determined. In this study, we purified human FAN1, as a bacterially expressed recombinant protein. FAN1 exhibited robust endonuclease activity with 5'-flapped DNA, which is formed at the stalled replication fork. We found that FAN1 efficiently promoted DNA incision at the proper site of RPA-coated 5'-flapped DNA. Therefore, FAN1 possesses the ability to promote the ICL repair of 5'-flapped DNA covered by RPA.

    DOI J-GLOBAL

  • Structure of RizA, an L-amino-acid ligase from Bacillus subtilis

    Wataru Kagawa, Toshinobu Arai, Shun Ishikura, Kuniki Kino, Hitoshi Kurumizaka

    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS   71 ( Pt 9 ) 1125 - 1130  2015年09月  [査読有り]

     概要を見る

    RizA is an L-amino-acid ligase from Bacillus subtilis that participates in the biosynthesis of rhizocticin, an oligopeptide antibiotic. The substrate-free form of RizA has been crystallized and the structure was solved at 2.8 angstrom resolution. The amino-acid-binding site appears to be capable of accommodating multiple amino acids, consistent with previous biochemical studies.

    DOI J-GLOBAL

  • Cryogenic coherent x-ray diffraction imaging for biological non-crystalline particles using the KOTOBUKI-1 diffraction apparatus at SACLA

    Tomotaka Oroguchi, Yuki Sekiguchi, Amane Kobayashi, Yu Masaki, Asahi Fukuda, Saki Hashimoto, Masayoshi Nakasako, Yuichi Ichikawa, Hitoshi Kurumizaka, Mitsuhiro Shimizu, Yayoi Inui, Sachihiro Matsunaga, Takayuki Kato, Keiichi Namba, Keiichi Yamaguchi, Kazuo Kuwata, Hiroshi Kameda, Naoya Fukui, Yasushi Kawata, Takashi Kameshima, Yuki Takayama, Koji Yonekura, Masaki Yamamoto

    JOURNAL OF PHYSICS B-ATOMIC MOLECULAR AND OPTICAL PHYSICS   48 ( 18 )  2015年09月  [査読有り]

     概要を見る

    We have developed an experimental apparatus named KOTOBUKI-1 for use in the coherent x-ray diffraction imaging experiments of frozen-hydrated non-crystalline particles at cryogenic temperature. The apparatus allows us to collect diffraction data for frozen-hydrated specimens at 66 K and provides an experimental environment to easily transfer frozen-hydrated specimens from liquid nitrogen storage to the specimen stage for x-ray exposure. Since 2012, the apparatus has been used in the single-shot diffraction data collection of non-crystalline biological cells and cellular components with dimensions from micrometer to submicrometer using x-ray free electron lasers at SACLA. Here we report on the performance of the KOTOBUKI-1 diffraction apparatus and some structure analyses of biological cells and cellular components. Based on the present results, we also discuss the future developments of diffraction apparatus for more efficient data collection.

    DOI J-GLOBAL

  • Charge-neutralization effect of the tail regions on the histone H2A/H2B dimer structure

    Kazumi Saikusa, Singo Shimoyama, Yuuki Asano, Aritaka Nagadoi, Mamoru Sato, Hitoshi Kurumizaka, Yoshifumi Nishimura, Satoko Akashi

    PROTEIN SCIENCE   24 ( 8 ) 1224 - 1231  2015年08月  [査読有り]

     概要を見る

    It is well known that various modifications of histone tails play important roles in the regulation of transcription initiation. In this study, some lysine (Lys) and arginine (Arg) residues were acetylated and deiminated, respectively, in the histone H2A/H2B dimer, and charge-neutralization effects on the dimer structure were studied by native mass spectrometry. Given that both acetylation and deimination neutralize the positive charges of basic amino acid residues, it had been expected that these modifications would correspondingly affect the gas-phase behavior of the histone H2A/H2B dimer. Contrary to this expectation, it was found that Arg deimination led to greater difficulty of dissociation of the dimer by gas-phase collision, whereas acetylation of Lys residues did not cause such a drastic change in the dimer stability. In contrast, ion mobility-mass spectrometry (IM-MS) experiments showed that arrival times in the mobility cell both of acetylated and of deiminated dimer ions changed little from those of the unmodified dimer ions, indicating that the sizes of the dimer ions did not change by modification. Charge neutralization of Arg, basicity of which is higher than Lys, might have triggered some alteration of the dimer structure that cannot be found in IM-MS but can be detected by collision in the gas phase.

    DOI J-GLOBAL

  • Structural basis of a nucleosome containing histone H(2)A.B/H(2)A.Bbd that transiently associates with reorganized chromatin (vol 3, 3510, 2013)

    Yasuhiro Arimura, Hiroshi Kimura, Takashi Oda, Koichi Sato, Akihisa Osakabe, Hiroaki Tachiwana, Yuko Sato, Yasuha Kinugasa, Tsuyoshi Ikura, Masaaki Sugiyama, Mamoru Sato, Hitoshi Kurumizaka

    SCIENTIFIC REPORTS   5   9628  2015年07月  [査読有り]

    DOI PubMed

  • Cdt1-binding protein GRWD1 is a novel histone-binding protein that facilitates MCM loading through its influence on chromatin architecture

    Nozomi Sugimoto, Kazumitsu Maehara, Kazumasa Yoshida, Shuhei Yasukouchi, Satoko Osano, Shinya Watanabe, Masahiro Aizawa, Takashi Yugawa, Tohru Kiyono, Hitoshi Kurumizaka, Yasuyuki Ohkawa, Masatoshi Fujita

    NUCLEIC ACIDS RESEARCH   43 ( 12 ) 5898 - 5911  2015年07月  [査読有り]

     概要を見る

    Efficient pre-replication complex (pre-RC) formation on chromatin templates is crucial for the maintenance of genome integrity. However, the regulation of chromatin dynamics during this process has remained elusive. We found that a conserved protein, GRWD1 (glutamate-rich WD40 repeat containing 1), binds to two representative replication origins specifically during G1 phase in a CDC6- and Cdt1-dependent manner, and that depletion of GRWD1 reduces loading of MCM but not CDC6 and Cdt1. Furthermore, chromatin immunoprecipitation coupled with high-throughput sequencing (Seq) revealed significant genome-wide co-localization of GRWD1 with CDC6. We found that GRWD1 has histone-binding activity. To investigate the effect of GRWD1 on chromatin architecture, we used formaldehyde-assisted isolation of regulatory elements (FAIRE)-seq or FAIRE-quantitative PCR analyses, and the results suggest that GRWD1 regulates chromatin openness at specific chromatin locations. Taken together, these findings suggest that GRWD1 may be a novel histone-binding protein that regulates chromatin dynamics and MCM loading at replication origins.

    DOI J-GLOBAL

  • Mutations in the Gene Encoding the E2 Conjugating Enzyme UBE2T Cause Fanconi Anemia

    Asuka Hira, Kenichi Yoshida, Koichi Sato, Yusuke Okuno, Yuichi Shiraishi, Kenichi Chiba, Hiroko Tanaka, Satoru Miyano, Akira Shimamoto, Hidetoshi Tahara, Etsuro Ito, Seiji Kojima, Hitoshi Kurumizaka, Seishi Ogawa, Minoru Takata, Hiromasa Yabe, Miharu Yabe

    AMERICAN JOURNAL OF HUMAN GENETICS   96 ( 6 ) 1001 - 1007  2015年06月  [査読有り]

     概要を見る

    Fanconi anemia (FA) is a rare genetic disorder characterized by genome instability, increased cancer susceptibility, progressive bone marrow failure (BMF), and various developmental abnormalities resulting from the defective FA pathway. FA is caused by mutations in genes that mediate repair processes of interstrand crosslinks and/or DNA adducts generated by endogenous aldehydes. The UBE2T E2 ubiquitin conjugating enzyme acts in FANCD2/FANCI monoubiquitination, a critical event in the pathway. Here we identified two unrelated FA-affected individuals, each harboring biallelic mutations in UBE2T. They both produced a defective UBE2T protein with the same missense alteration (p.Gln2Glu) that abolished FANCD2 monoubiquitination and interaction with FANCL. We suggest this FA complementation group be named FA-T.

    DOI J-GLOBAL

  • Stable complex formation of CENP-B with the CENP-A nucleosome

    Risa Fujita, Koichiro Otake, Yasuhiro Arimura, Naoki Horikoshi, Yuta Miya, Tatsuya Shiga, Akihisa Osakabe, Hiroaki Tachiwana, Jun-ichirou Ohzeki, Vladimir Larionov, Hiroshi Masumoto, Hitoshi Kurumizaka

    NUCLEIC ACIDS RESEARCH   43 ( 10 ) 4909 - 4922  2015年05月  [査読有り]

     概要を見る

    CENP-A and CENP-B are major components of centromeric chromatin. CENP-A is the histone H3 variant, which forms the centromere-specific nucleosome. CENP-B specifically binds to the CENP-B box DNA sequence on the centromere-specific repetitive DNA. In the present study, we found that the CENP-A nucleosome more stably retains human CENP-B than the H3.1 nucleosome in vitro. Specifically, CENP-B forms a stable complex with the CENP-A nucleosome, when the CENP-B box sequence is located at the proximal edge of the nucleosome. Surprisingly, the CENP-B binding was weaker when the CENP-B box sequence was located in the distal linker region of the nucleosome. This difference in CENP-B binding, depending on the CENP-B box location, was not observed with the H3.1 nucleosome. Consistently, we found that the DNA-binding domain of CENP-B specifically interacted with the CENP-A-H4 complex, but not with the H3.1-H4 complex, in vitro. These results suggested that CENP-B forms a more stable complex with the CENP-A nucleosome through specific interactions with CENP-A, if the CENP-B box is located proximal to the CENP-A nucleosome. Our in vivo assay also revealed that CENP-B binding in the vicinity of the CENP-A nucleosome substantially stabilizes the CENP-A nucleosome on alphoid DNA in human cells.

    DOI J-GLOBAL

  • Comprehensive analysis of Japanese Fanconi anemia (FA) patients has led to the identification of an E2 enzyme UBE2T as a novel FA gene

    Minoru Takata, Asuka Hira, Kenichi Yoshida, Koichi Sato, Akira Shimamoto, Hidetoshi Tahara, Hitoshi Kurumizaka, Seishi Ogawa, Hiromasa Yabe, Miharu Yabe

    DNA REPAIR   28   140 - 141  2015年04月

  • Structures of the nucleosomes containing UV-damaged DNA bases and the damaged base recognition mechanism by DNA repair proteins

    Akihisa Osakabe, Hiroaki Tachiwana, Naoki Horikoshi, Wataru Kagawa, Junpei Yamamoto, Takeshi Yasuda, Fumio Hanaoka, Kaoru Sugasawa, Shigenori Iwai, Hitoshi Kurumizaka

    DNA REPAIR   28   139 - 140  2015年04月

  • Two Arginine Residues Suppress the Flexibility of Nucleosomal DNA in the Canonical Nucleosome Core

    Hidetoshi Kono, Kazuyoshi Shirayama, Yasuhiro Arimura, Hiroaki Tachiwana, Hitoshi Kurumizaka

    PLOS ONE   10 ( 3 ) e0120635  2015年03月  [査読有り]

     概要を見る

    The dynamics of nucleosomes containing either canonical H3 or its centromere-specific variant CENP-A were investigated using molecular dynamics simulations. The simulations showed that the histone cores were structurally stable during simulation periods of 100 ns and 50 ns, while DNA was highly flexible at the entry and exit regions and partially dissociated from the histone core. In particular, approximately 20-25 bp of DNA at the entry and exit regions of the CENP-A nucleosome exhibited larger fluctuations than DNA at the entry and exit regions of the H3 nucleosome. Our detailed analysis clarified that this difference in dynamics was attributable to a difference in two basic amino acids in the aN helix; two arginine (Arg) residues in H3 were substituted by lysine (Lys) residues at the corresponding sites in CENP-A. The difference in the ability to form hydrogen bonds with DNA of these two residues regulated the flexibility of nucleosomal DNA at the entry and exit regions. Our exonuclease III assay consistently revealed that replacement of these two Arg residues in the H3 nucleosome by Lys enhanced endonuclease susceptibility, suggesting that the DNA ends of the CENP-A nucleosome are more flexible than those of the H3 nucleosome. This difference in the dynamics between the two types of nucleosomes may be important for forming higher order structures in different phases.

    DOI J-GLOBAL

  • Mass Spectrometric Approach for Characterizing the Disordered Tail Regions of the Histone H2A/H2B Dimer

    Kazumi Saikusa, Aritaka Nagadoi, Kana Hara, Sotaro Fuchigami, Hitoshi Kurumizaka, Yoshifumi Nishimura, Satoko Akashi

    ANALYTICAL CHEMISTRY   87 ( 4 ) 2220 - 2227  2015年02月  [査読有り]

     概要を見る

    The histone H2A/H2B dimer is a component of nucleosome core particles (NCPs). The structure of the dimer at the atomic level has not yet been revealed. A possible reason for this is that the dimer has three intrinsically disordered tail regions: the N- and C-termini of H2A and the N-terminus of H2B. To investigate the role of the tail regions of the H2A/H2B dimer structure, we characterized behaviors of the H2A/H2B mutant dimers, in which these functionally important disordered regions were depleted, using mass spectrometry (MS). After verifying that the acetylation of Lys residues in the tail regions had little effect on the gas-phase conformations of the wild-type dimer, we prepared two histone H2A/H2B dimer mutants: an H2A/H2B dimer depleted of both N-termini (dN-H2A/dN-H2B) and a dimer with the N- and C-termini of H2A and the N-terminus of H2B depleted (dNC-H2A/dN-H2B). We analyzed these mutants using ion mobility-mass spectrometry (IM-MS) and hydrogen/deuterium exchange mass spectrometry (HDX-MS). With IM-MS, reduced structural diversity was observed for each of the tail-truncated H2A/H2B mutants. In addition, global HDX-MS proved that the dimer mutant dNC-H2A/dN-H2B was susceptible to deuteration, suggesting that its structure in solution was somewhat loosened. A partial relaxation of the mutants structure was demonstrated also by IM-MS. In this study, we characterized the relationship between the tail lengths and the conformations of the H2A/H2B dimer in solution and gas phases, and demonstrated, using mass spectrometry, that disordered tail regions play an important role in stabilizing the conformation of the core region of the dimer in both phases.

    DOI J-GLOBAL

  • Human tNASP Promotes in Vitro Nucleosome Assembly with Histone H3.3

    Daiki Kato, Akihisa Osakabe, Hiroaki Tachiwana, Hiroki Tanaka, Hitoshi Kurumizaka

    BIOCHEMISTRY   54 ( 5 ) 1171 - 1179  2015年02月  [査読有り]

     概要を見る

    Nuclear autoantigenic sperm proteins (NASPs) are members of the acidic histone chaperones, which promote nucleosome assembly. In humans, two splicing variants proposed for the somatic and testicular isoforms, sNASP and tNASP, respectively, have been found, and the shorter form, sNASP, reportedly promotes nucleosome assembly with the histone H3 isoforms, H3.1, H3.2, and H3.3. However, the biochemical properties of the longer form, tNASP, have not been reported. tNASP is considered to exist specifically in the testis. Our present results revealed that the tNASP protein is ubiquitously produced in various human tissues, in addition to testis. Unexpectedly, we found that the nucleosome assembly activity of purified tNASP was extremely low with the canonical histone H3.1 or H3.2, but was substantially detected with the replacement histone H3.3 variant. A mutational analysis revealed that the H3.3 Ile89 residue, corresponding to the H3.1 Val89 residue, is responsible for the tNASP-mediated nucleosome assembly with H3.3. A histone deposition assay showed that the H3.3-H4 complex is more efficiently deposited onto DNA by tNASP than the H3.1-H4 complex. These results provide evidence that tNASP is ubiquitously produced in various types of human tissues and promotes in vitro nucleosome assembly with H3 variant specificity.

    DOI J-GLOBAL

  • Solution structure of variant H2A.Z.1 nucleosome investigated by small-angle X-ray and neutron scatterings

    Sugiyama M, Horikoshi N, Suzuki Y, Taguchi H, Kujirai T, Inoue R, Oba Y, Sato N, Martel A, Porcar L, Kurumizaka H

    Biochemistry and Biophysics Reports   4   28 - 32  2015年  [査読有り]

    DOI

  • hCAS/CSE1L regulates RAD51 distribution and focus formation for homologous recombinational repair.

    Okimoto S, Sun J, Fukuto A, Horikoshi Y, Matsuda S, Matsuda T, Ikura M, Ikura T, Machida S, Kurumizaka H, Miyamoto Y, Oka M, Yoneda Y, Kiuchi Y, Tashiro S

    hCAS/CSE1L regulates RAD51 distribution and focus formation for homologous recombinational repair.   20 ( 9 ) 681 - 694  2015年  [査読有り]

     概要を見る

    :Homologous recombinational repair (HR) is one of the major repair systems for DNA double-strand breaks. RAD51 is a key molecule in HR, and the RAD51 concentration in the cell nucleus increases after DNA damage induction. However, the mechanism that regulates the intracellular distribution of RAD51 is still unclear. Here, we show that hCAS/CSE1L associates with RAD51 in human cells. We found that hCAS/CSE1L negatively regulates the nuclear protein level of RAD51 under normal conditions. hCAS/CSE1L is also required to repress the DNA damage-induced focus formation of RAD51. Moreover, we show that hCAS/CSE1L plays roles in the regulation of the HR activity and in chromosome stability. These findings suggest that hCAS/CSE1L is responsible for controlling the HR activity by directly interacting with RAD51.

    DOI

  • Nucleosome organization and chromatin dynamics in telomeres.

    Ichikawa, Y, Nishimura, Y, Kurumizaka, H, Shimizu, M

    Biomol Concepts.   6 ( 1 ) 67 - 75  2015年  [査読有り]

    DOI J-GLOBAL

  • A method for evaluating nucleosome stability with a protein-binding fluorescent dye

    Hiroyuki Taguchi, Naoki Horikoshi, Yasuhiro Arimura, Hitoshi Kurumizaka

    METHODS   70 ( 2-3 ) 119 - 126  2014年12月  [査読有り]

     概要を見る

    Nucleosomes are extremely stable histone-DNA complexes that form the building blocks of chromatin, which accommodates genomic DNA within the nucleus. The dynamic properties of chromatin play essential roles in regulating genomic DNA functions, such as DNA replication, recombination, repair, and transcription. Histones are the protein components of nucleosomes, and their diverse modifications and variants increase the versatility of nucleosome structures and their dynamics in chromatin. Therefore, a technique to evaluate the physical properties of nucleosomes would facilitate functional studies of the various nucleosomes. In this report, we describe a convenient assay for evaluating the thermal stability of nucleosomes in vitro. (C) 2014 Elsevier Inc. All rights reserved.

    DOI J-GLOBAL

  • Defective FANCI Binding by a Fanconi Anemia-Related FANCD2 Mutant

    Koichi Sato, Masamichi Ishiai, Minoru Takata, Hitoshi Kurumizaka

    PLOS ONE   9 ( 12 ) e114752  2014年12月  [査読有り]

     概要を見る

    FANCD2 is a product of one of the genes associated with Fanconi anemia (FA), a rare recessive disease characterized by bone marrow failure, skeletal malformations, developmental defects, and cancer predisposition. FANCD2 forms a complex with FANCI (ID complex) and is monoubiquitinated, which facilitates the downstream interstrand crosslink (ICL) repair steps, such as ICL unhooking and nucleolytic end resection. In the present study, we focused on the chicken FANCD2 (cFANCD2) mutant harboring the Leu234 to Arg (L234R) substitution. cFANCD2 L234R corresponds to the human FANCD2 L231R mutation identified in an FA patient. We found that cFANCD2 L234R did not complement the defective ICL repair in FANCD2(-/-) DT40 cells. Purified cFANCD2 L234R did not bind to chicken FANCI, and its monoubiquitination was significantly deficient, probably due to the abnormal ID complex formation. In addition, the histone chaperone activity of cFANCD2 L234R was also defective. These findings may explain some aspects of Fanconi anemia pathogenesis by a FANCD2 missense mutation.

    DOI J-GLOBAL

  • Expression and purification of human FANCI and FANCD2 using Escherichia coli cells

    Daisuke Takahashi, Koichi Sato, Mayo Shimomuki, Minoru Takata, Hitoshi Kurumizaka

    PROTEIN EXPRESSION AND PURIFICATION   103   8 - 15  2014年11月  [査読有り]

     概要を見る

    The DNA interstrand crosslink (ICL) is an extremely deleterious DNA lesion that covalently crosslinks complementary strands and prevents the strand-separation reaction. In higher eukaryotes, the Fanconi anemia proteins, FANCI and FANCD2, form a heterodimer and play essential roles in ICL repair. Human FANCI and FANCD2 are large proteins with molecular masses of 149 kDa and 164 kDa, respectively, and were reportedly purified using a baculovirus expression system with insect cells. We have established a novel expression and purification procedure for human FANCD2 and FANCI, using Escherichia coli cells. The human FANCD2 and FANCI proteins purified by this bacterial expression method formed a stable heterodimer, and exhibited DNA binding and histone chaperone activities, as previously reported for the proteins purified by the baculovirus system. Therefore, these purification methods for human FANCI and FANCD2 provide novel procedures to facilitate structural and biochemical studies of human FANCI and FANCD2. (C) 2014 Elsevier Inc. All rights reserved.

    DOI J-GLOBAL

  • N-terminal phosphorylation of HP1α increases its nucleosome-binding specificity.

    Nishibuchi, G, Machida, S, Osakabe, A, Murakoshi, H, Hiragami-Hamada, K, Nakagawa, R, Fischle, W, Nishimura, Y, Kurumizaka, H, Tagami, H, Nakayama, J

    Nucleic Acids Res.   42 ( 20 ) 12498 - 12511  2014年11月  [査読有り]

    DOI J-GLOBAL

  • Crystal structure and stable property of the cancer-associated heterotypic nucleosome containing CENP-A and H3.3

    Yasuhiro Arimura, Kazuyoshi Shirayama, Naoki Horikoshi, Risa Fujita, Hiroyuki Taguchi, Wataru Kagawa, Tatsuo Fukagawa, Genevieve Almouzni, Hitoshi Kurumizaka

    SCIENTIFIC REPORTS   4   7115  2014年11月  [査読有り]

     概要を見る

    The centromere-specific histone H3 variant, CENP-A, is overexpressed in particular aggressive cancer cells, where it can be mislocalized ectopically in the form of heterotypic nucleosomes containing H3.3. In the present study, we report the crystal structure of the heterotypic CENP-A/H3.3 particle and reveal its "hybrid structure", in which the physical characteristics of CENP-A and H3.3 are conserved independently within the same particle. The CENP-A/H3.3 nucleosome forms an unexpectedly stable structure as compared to the CENP-A nucleosome, and allows the binding of the essential centromeric protein, CENP-C, which is ectopically mislocalized in the chromosomes of CENP-A overexpressing cells.

    DOI J-GLOBAL

  • DNA Binding Properties of the Actin-Related Protein Arp8 and Its Role in DNA Repair

    Akihisa Osakabe, Yuichiro Takahashi, Hirokazu Murakami, Kenji Otawa, Hiroaki Tachiwana, Yukako Oma, Hitoshi Nishijima, Kei-ich Shibahara, Hitoshi Kurumizaka, Masahiko Harata

    PLOS ONE   9 ( 10 ) e108354  2014年10月  [査読有り]

     概要を見る

    Actin and actin-related proteins (Arps), which are members of the actin family, are essential components of many of these remodeling complexes. Actin, Arp4, Arp5, and Arp8 are found to be evolutionarily conserved components of the INO80 chromatin remodeling complex, which is involved in transcriptional regulation, DNA replication, and DNA repair. A recent report showed that Arp8 forms a module in the INO80 complex and this module can directly capture a nucleosome. In the present study, we showed that recombinant human Arp8 binds to DNAs, and preferentially binds to single-stranded DNA. Analysis of the binding of adenine nucleotides to Arp8 mutants suggested that the ATP-binding pocket, located in the evolutionarily conserved actin fold, plays a regulatory role in the binding of Arp8 to DNA. To determine the cellular function of Arp8, we derived tetracycline-inducible Arp8 knockout cells from a cultured human cell line. Analysis of results obtained after treating these cells with aphidicolin and camptothecin revealed that Arp8 is involved in DNA repair. Together with the previous observation that Arp8, but not gamma-H2AX, is indispensable for recruiting INO80 complex to DSB in human, results of our study suggest an individual role for Arp8 in DNA repair.

    DOI J-GLOBAL

  • Evaluation of Chemical Fluorescent Dyes as a Protein Conjugation Partner for Live Cell Imaging

    Yoko Hayashi-Takanaka, Timothy J. Stasevich, Hitoshi Kurumizaka, Naohito Nozaki, Hiroshi Kimura

    PLOS ONE   9 ( 9 ) e106271  2014年09月  [査読有り]

     概要を見る

    To optimize live cell fluorescence imaging, the choice of fluorescent substrate is a critical factor. Although genetically encoded fluorescent proteins have been used widely, chemical fluorescent dyes are still useful when conjugated to proteins or ligands. However, little information is available for the suitability of different fluorescent dyes for live imaging. We here systematically analyzed the property of a number of commercial fluorescent dyes when conjugated with antigen-binding (Fab) fragments directed against specific histone modifications, in particular, phosphorylated H3S28 (H3S28ph) and acetylated H3K9 (H3K9ac). These Fab fragments were conjugated with a fluorescent dye and loaded into living HeLa cells. H3S28ph-specific Fab fragments were expected to be enriched in condensed chromosomes, as H3S28 is phosphorylated during mitosis. However, the degree of Fab fragment enrichment on mitotic chromosomes varied depending on the conjugated dye. In general, green fluorescent dyes showed higher enrichment, compared to red and far-red fluorescent dyes, even when dye: protein conjugation ratios were similar. These differences are partly explained by an altered affinity of Fab fragment after dye-conjugation; some dyes have less effect on the affinity, while others can affect it more. Moreover, red and far-red fluorescent dyes tended to form aggregates in the cytoplasm. Similar results were observed when H3K9ac-specific Fab fragments were used, suggesting that the properties of each dye affect different Fab fragments similarly. According to our analysis, conjugation with green fluorescent dyes, like Alexa Fluor 488 and Dylight 488, has the least effect on Fab affinity and is the best for live cell imaging, although these dyes are less photostable than red fluorescent dyes. When multicolor imaging is required, we recommend the following dye combinations for optimal results: Alexa Fluor 488 (green), Cy3 (red), and Cy5 or CF640 (far-red).

    DOI J-GLOBAL

  • Green fluorescent protein fused to the C terminus of RAD51 specifically interferes with secondary DNA binding by the RAD51-ssDNA complex

    Wataru Kobayashi, Satoshi Sekine, Shinichi Machida, Hitoshi Kurumizaka

    GENES & GENETIC SYSTEMS   89 ( 4 ) 169 - 179  2014年08月  [査読有り]

     概要を見る

    Green fluorescent protein (GFP), fused to the N or C terminus of a protein of interest, is widely used to monitor the localization and mobility of proteins in cells. RAD51 is an essential protein that functions in mitotic DNA repair and meiotic chromosome segregation by promoting the homologous recombination reaction. A previous genetic study with Arabidopsis thaliana revealed that GFP fused to the C terminus of RAD51 (RAD51-GFP) inhibits mitotic DNA repair, but meiotic homologous recombination remained unaffected. To determine how the C-terminal GFP specifically inhibits mitotic DNA repair by RAD51, we purified rice RAD51A1-GFP and RAD51A2-GFP, and performed biochemical analyses. Interestingly, purified RAD51A1-GFP and RAD51A2-GFP are proficient in DNA binding and ATP hydrolysis. However, nucleoprotein complexes containing single-stranded DNA and RAD51A1-GFP or RAD51A2-GFP are significantly defective in binding to the second DNA molecule (secondary DNA binding), and consequently fail to catalyze homologous pairing. In contrast, RAD51A1-GFP and RAD51A2-GFP efficiently stimulated homologous pairing promoted by the meiosis-specific RAD51 isoform DMC1. These biochemical characteristics are well conserved in human RAD51-GFP. Therefore, GFP fused to the C terminus of RAD51 abolishes the homologous pairing activity of RAD51 by disrupting secondary DNA binding, but does not affect its DMC1-stimulating activity.

    DOI J-GLOBAL

  • FANCD2 Binds CtIP and Regulates DNA-End Resection during DNA Interstrand Crosslink Repair

    Junya Unno, Akiko Itaya, Masato Taoka, Koichi Sato, Junya Tomida, Wataru Sakai, Kaoru Sugasawa, Masamichi Ishiai, Tsuyoshi Ikura, Toshiaki Isobe, Hitoshi Kurumizaka, Minoru Takata

    CELL REPORTS   7 ( 4 ) 1039 - 1047  2014年05月  [査読有り]

     概要を見る

    The Fanconi anemia (FA) pathway is critically involved in the maintenance of hematopoietic stem cells and the suppression of carcinogenesis. A key FA protein, FANCD2, is monoubiquitinated and accumulates in chromatin in response to DNA interstrand crosslinks (ICLs), where it coordinates DNA repair through mechanisms that are still poorly understood. Here, we report that CtIP protein directly interacts with FANCD2. A region spanning amino acids 166 to 273 of CtIP and monoubiquitination of FANCD2 are both essential for the FANCD2-CtIP interaction and mitomycin C (MMC)-induced CtIP foci. Remarkably, both FANCD2 and CtIP are critical for MMC-induced RPA2 hyperphosphorylation, an event that accompanies end resection of double-strand breaks. Collectively, our results reveal a role of monoubiquitinated FANCD2 in end resection that depends on its binding to CtIP during ICL repair.

    DOI J-GLOBAL

  • Distinct Features of the Histone Core Structure in Nucleosomes Containing the Histone H2A.B Variant

    Masaaki Sugiyama, Yasuhiro Arimura, Kazuyoshi Shirayama, Risa Fujita, Yojiro Oba, Nobuhiro Sato, Rintaro Inoue, Takashi Oda, Mamoru Sato, Richard K. Heenan, Hitoshi Kurumizaka

    BIOPHYSICAL JOURNAL   106 ( 10 ) 2206 - 2213  2014年05月  [査読有り]

     概要を見る

    Nucleosomes containing a human histone variant, H2A.B, in an aqueous solution were analyzed by small-angle neutron scattering utilizing a contrast variation technique. Comparisons with the canonical H2A nucleosome structure revealed that the DNA termini of the H2A.B nucleosome are detached from the histone core surface, and flexibly expanded toward the solvent. In contrast, the histone tails are compacted in H2A.B nucleosomes compared to those in canonical H2A nucleosomes, suggesting that they bind to the surface of the histone core and/or DNA. Therefore, the histone tail dynamics may function to regulate the flexibility of the DNA termini in the nucleosomes.

    DOI J-GLOBAL

  • Nap1 stimulates homologous recombination by RAD51 and RAD54 in higher-ordered chromatin containing histone H1

    Shinichi Machida, Motoki Takaku, Masae Ikura, Jiying Sun, Hidekazu Suzuki, Wataru Kobayashi, Aiko Kinomura, Akihisa Osakabe, Hiroaki Tachiwana, Yasunori Horikoshi, Atsuhiko Fukuto, Ryo Matsuda, Kiyoe Ura, Satoshi Tashiro, Tsuyoshi Ikura, Hitoshi Kurumizaka

    SCIENTIFIC REPORTS   4 ( 4863 )  2014年05月  [査読有り]

     概要を見る

    Homologous recombination plays essential roles in mitotic DNA double strand break (DSB) repair and meiotic genetic recombination. In eukaryotes, RAD51 promotes the central homologous-pairing step during homologous recombination, but is not sufficient to overcome the reaction barrier imposed by nucleosomes. RAD54, a member of the ATP-dependent nucleosome remodeling factor family, is required to promote the RAD51-mediated homologous pairing in nucleosomal DNA. In higher eukaryotes, most nucleosomes form higher-ordered chromatin containing the linker histone H1. However, the mechanism by which RAD51/RAD54-mediated homologous pairing occurs in higher-ordered chromatin has not been elucidated. In this study, we found that a histone chaperone, Nap1, accumulates on DSB sites in human cells, and DSB repair is substantially decreased in Nap1-knockdown cells. We determined that Nap1 binds to RAD54, enhances the RAD54-mediated nucleosome remodeling by evicting histone H1, and eventually stimulates the RAD51-mediated homologous pairing in higher-ordered chromatin containing histone H1.

    DOI J-GLOBAL

  • Structure of human nucleosome containing the testis-specific histone variant TSH2B

    Takashi Urahama, Naoki Horikoshi, Akihisa Osakabe, Hiroaki Tachiwana, Hitoshi Kurumizaka

    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS   70 ( F70 ) 444 - 449  2014年04月

     概要を見る

    The human histone H2B variant TSH2B is highly expressed in testis and may function in the chromatin transition during spermatogenesis. In the present study, the crystal structure of the human testis-specific nucleosome containing TSH2B was determined at 2.8 angstrom resolution. A local structural difference between TSH2B and canonical H2B in nucleosomes was detected around the TSH2B-specific amino-acid residue Ser85. The TSH2B Ser85 residue does not interact with H4 in the nucleosome, but in the canonical nucleosome the H2B Asn84 residue (corresponding to the TSH2B Ser85 residue) forms water-mediated hydrogen bonds with the H4 Arg78 residue. In contrast, the other TSH2B-specific amino-acid residues did not induce any significant local structural changes in the TSH2B nucleosome. These findings may provide important information for understanding how testis-specific histone variants form nucleosomes during spermatogenesis.

    DOI

  • Structure of human nucleosome containing the testis-specific histone variant TSH2B

    Takashi Urahama, Naoki Horikoshi, Akihisa Osakabe, Hiroaki Tachiwana, Hitoshi Kurumizaka

    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS   70 ( 4 ) 444 - 449  2014年04月  [査読有り]

     概要を見る

    The human histone H2B variant TSH2B is highly expressed in testis and may function in the chromatin transition during spermatogenesis. In the present study, the crystal structure of the human testis-specific nucleosome containing TSH2B was determined at 2.8 angstrom resolution. A local structural difference between TSH2B and canonical H2B in nucleosomes was detected around the TSH2B-specific amino-acid residue Ser85. The TSH2B Ser85 residue does not interact with H4 in the nucleosome, but in the canonical nucleosome the H2B Asn84 residue (corresponding to the TSH2B Ser85 residue) forms water-mediated hydrogen bonds with the H4 Arg78 residue. In contrast, the other TSH2B-specific amino-acid residues did not induce any significant local structural changes in the TSH2B nucleosome. These findings may provide important information for understanding how testis-specific histone variants form nucleosomes during spermatogenesis.

    DOI

  • Telomeric repeats act as nucleosome-disfavouring sequences in vivo

    Yuichi Ichikawa, Nobuyuki Morohashi, Yoshifumi Nishimura, Hitoshi Kurumizaka, Mitsuhiro Shimizu

    NUCLEIC ACIDS RESEARCH   42 ( 3 ) 1541 - 1552  2014年02月  [査読有り]

     概要を見る

    Telomeric DNAs consist of tandem repeats of G-clusters such as TTAGGG and TG(1-3), which are the human and yeast repeat sequences, respectively. In the yeast Saccharomyces cerevisiae, the telomeric repeats are non-nucleosomal, whereas in humans, they are organized in tightly packaged nucleosomes. However, previous in vitro studies revealed that the binding affinities of human and yeast telomeric repeat sequences to histone octamers in vitro were similar, which is apparently inconsistent with the differences in the human and yeast telomeric chromatin structures. To further investigate the relationship between telomeric sequences and chromatin structure, we examined the effect of telomeric repeats on the formation of positioned nucleosomes in vivo by indirect end-label mapping, primer extension mapping and nucleosome repeat analyses, using a defined minichromosome in yeast cells. We found that the human and yeast telomeric repeat sequences both disfavour nucleosome assembly and alter nucleosome positioning in the yeast minichromosome. We further demonstrated that the G-clusters in the telomeric repeats are required for the nucleosome-disfavouring properties. Thus, our results suggest that this inherent structural feature of the telomeric repeat sequences is involved in the functional dynamics of the telomeric chromatin structure.

    DOI J-GLOBAL

  • The centromeric nucleosome-like CENP-T-W-S-X complex induces positive supercoils into DNA

    Kozo Takeuchi, Tatsuya Nishino, Kouta Mayanagi, Naoki Horikoshi, Akihisa Osakabe, Hiroaki Tachiwana, Tetsuya Hori, Hitoshi Kurumizaka, Tatsuo Fukagawa

    Nucleic Acids Research   42 ( 3 ) 1644 - 1655  2014年02月  [査読有り]

     概要を見る

    The centromere is a specific genomic region upon which the kinetochore is formed to attach to spindle microtubules for faithful chromosome segregation. To distinguish this chromosomal region from other genomic loci, the centromere contains a specific chromatin structure including specialized nucleosomes containing the histone H3 variant CENP-A. In addition to CENP-A nucleosomes, we have found that centromeres contain a nucleosome-like structure comprised of the histone-fold CENP-T-W-S-X complex. However, it is unclear how the CENP-T-W-S-X complex associates with centromere chromatin. Here, we demonstrate that the CENP-T-W-S-X complex binds preferentially to ∼100 bp of linker DNA rather than nucleosome-bound DNA. In addition, we find that the CENP-T-W-S-X complex primarily binds to DNA as a (CENP-T-W-S-X)2 structure. Interestingly, in contrast to canonical nucleosomes that negatively supercoil DNA, the CENP-T-W-S-X complex induces positive DNA supercoils. We found that the DNA-binding regions in CENP-T or CENP-W, but not CENP-S or CENP-X, are required for this positive supercoiling activity and the kinetochore targeting of the CENP-T-W-S-X complex. In summary, our work reveals the structural features and properties of the CENP-T-W-S-X complex for its localization to centromeres. © 2013 The Author(s) 2013. Published by Oxford University Press.

    DOI PubMed J-GLOBAL

  • Compensatory Functions and Interdependency of the DNA-Binding Domain of BRCA2 with the BRCA1-PALB2-BRCA2 Complex

    Muthana Al Abo, Donniphat Dejsuphong, Kouji Hirota, Yasukazu Yonetani, Mitsuyoshi Yamazoe, Hitoshi Kurumizaka, Shunichi Takeda

    CANCER RESEARCH   74 ( 3 ) 797 - 807  2014年02月  [査読有り]

     概要を見る

    BRCA1, BRCA2, and PALB2 are key players in cellular tolerance to chemotherapeutic agents, including camptothecin, cisplatin, and PARP inhibitor. The N-terminal segment of BRCA2 interacts with PALB2, thus contributing to the formation of the BRCA1-PALB2-BRCA2 complex. To understand the role played by BRCA2 in this complex, we deleted its N-terminal segment and generated BRCA2(Delta N) mutant cells. Although previous studies have suggested that BRCA1-PALB2 plays a role in the recruitment of BRCA2 to DNA-damage sites, BRCA2(Delta N) mutant cells displayed a considerably milder phenotype than did BRCA2(-/-) null-deficient cells. We hypothesized that the DNA-binding domain (DBD) of BRCA2 might compensate for a defect in BRCA2(Delta N) that prevented stable interaction with PALB2. To test this hypothesis, we disrupted the DBD of BRCA2 in wild-type and BRCA2(Delta N) cells. Remarkably, although the resulting BRCA2(Delta DBD) cells displayed a moderate phenotype, the BRCA2(Delta N+DBD) cells displayed a very severe phenotype, as did the BRCA2(-/-) cells, suggesting that the N-terminal segment and the DBD play a substantially overlapping role in the functionality of BRCA2. We also showed that the formation of both the BRCA1-PALB2-BRCA2 complex and the DBD is required for efficient recruitment of BRCA2 to DNA-damage sites. Our study revealed the essential role played by both the BRCA1-PALB2-BRCA2 complex and the DBD in the functionality of BRCA2, as each can compensate for the other in the recruitment of BRCA2 to DNA-damage sites. This knowledge adds to our ability to accurately predict the efficacy of antimalignant therapies for patients carrying mutations in the BRCA2 gene. (C) 2013 AACR.

    DOI J-GLOBAL

  • Mislocalization of the Centromeric Histone Variant CenH3/CENP-A in Human Cells Depends on the Chaperone DAXX

    Nicolas Lacoste, Adam Woolfe, Hiroaki Tachiwana, Ana Villar Garea, Teresa Barth, Sylvain Cantaloube, Hitoshi Kurumizaka, Axel Imhof, Genevieve Almouzni

    MOLECULAR CELL   53 ( 4 ) 631 - 644  2014年02月  [査読有り]

     概要を見る

    Centromeres are essential for ensuring proper chromosome segregation in eukaryotes. Their definition relies on the presence of a centromere-specific H3 histone variant CenH3, known as CENP-A in mammals. Its overexpression in aggressive cancers raises questions concerning its effect on chromatin dynamics and contribution to tumorigenesis. We find that CenH3 overexpression in human cells leads to ectopic enrichment at sites of active histone turnover involving a heterotypic tetramer containing CenH3-H4 with H3.3-H4. Ectopic localization of this particle depends on the H3.3 chaperone DAXX rather than the dedicated CenH3 chaperone HJURP. This aberrant nucleosome occludes CTCF binding and has a minor effect on gene expression. Cells overexpressing CenH3 are more tolerant of DNA damage. Both the survival advantage and CTCF occlusion in these cells are dependent on DAXX. Our findings illustrate how changes in histone variant levels can disrupt chromatin dynamics and suggests a possible mechanism for cell resistance to anticancer treatments.

    DOI J-GLOBAL

  • Functional analyses of the C-terminal half of the Saccharomyces cerevisiae Rad52 protein

    Wataru Kagawa, Naoto Arai, Yuichi Ichikawa, Kengo Saito, Shusei Sugiyama, Mika Saotome, Takehiko Shibata, Hitoshi Kurumizaka

    NUCLEIC ACIDS RESEARCH   42 ( 2 ) 941 - 951  2014年01月

     概要を見る

    The Saccharomyces cerevisiae Rad52 protein is essential for efficient homologous recombination (HR). An important role of Rad52 in HR is the loading of Rad51 onto replication protein A-coated single-stranded DNA (ssDNA), which is referred to as the recombination mediator activity. In vitro, Rad52 displays additional activities, including self-association, DNA binding and ssDNA annealing. Although Rad52 has been a subject of extensive genetic, biochemical and structural studies, the mechanisms by which these activities are coordinated in the various roles of Rad52 in HR remain largely unknown. In the present study, we found that an isolated C-terminal half of Rad52 disrupted the Rad51 oligomer and formed a heterodimeric complex with Rad51. The Rad52 fragment inhibited the binding of Rad51 to double-stranded DNA, but not to ssDNA. The phenylalanine-349 and tyrosine-409 residues present in the C-terminal half of Rad52 were critical for the interaction with Rad51, the disruption of Rad51 oligomers, the mediator activity of the full-length protein and for DNA repair in vivo in the presence of methyl methanesulfonate. Our studies suggested that phenylalanine-349 and tyrosine-409 are key residues in the C-terminal half of Rad52 and probably play an important role in the mediator activity.

    DOI

  • Functional analyses of the C-terminal half of the Saccharomyces cerevisiae Rad52 protein

    Wataru Kagawa, Naoto Arai, Yuichi Ichikawa, Kengo Saito, Shusei Sugiyama, Mika Saotome, Takehiko Shibata, Hitoshi Kurumizaka

    NUCLEIC ACIDS RESEARCH   42 ( 2 ) 941 - 951  2014年01月  [査読有り]

     概要を見る

    The Saccharomyces cerevisiae Rad52 protein is essential for efficient homologous recombination (HR). An important role of Rad52 in HR is the loading of Rad51 onto replication protein A-coated single-stranded DNA (ssDNA), which is referred to as the recombination mediator activity. In vitro, Rad52 displays additional activities, including self-association, DNA binding and ssDNA annealing. Although Rad52 has been a subject of extensive genetic, biochemical and structural studies, the mechanisms by which these activities are coordinated in the various roles of Rad52 in HR remain largely unknown. In the present study, we found that an isolated C-terminal half of Rad52 disrupted the Rad51 oligomer and formed a heterodimeric complex with Rad51. The Rad52 fragment inhibited the binding of Rad51 to double-stranded DNA, but not to ssDNA. The phenylalanine-349 and tyrosine-409 residues present in the C-terminal half of Rad52 were critical for the interaction with Rad51, the disruption of Rad51 oligomers, the mediator activity of the full-length protein and for DNA repair in vivo in the presence of methyl methanesulfonate. Our studies suggested that phenylalanine-349 and tyrosine-409 are key residues in the C-terminal half of Rad52 and probably play an important role in the mediator activity.

    DOI J-GLOBAL

  • Structural polymorphism in the L1 loop regions of human H2A.Z.1 and H2A.Z.2

    Horikoshi, Naoki, Sato, Koichi, Shimada, Keisuke, Arimura, Yasuhiro, Osakabe, Akihisa, Tachiwana, Hiroaki, Hayashi-Takanaka, Yoko, Iwasaki, Wakana, Kagawa, Wataru, Harata, Masahiko, Kimura, Hiroshi, Kurumizaka, Hitoshi

    Acta crystallographica. Section D, Biological crystallography   69 ( Pt 12 ) 2431 - 2439  2013年12月  [査読有り]

     概要を見る

    The histone H2A.Z variant is widely conserved among eukaryotes. Two isoforms, H2A.Z.1 and H2A.Z.2, have been identified in vertebrates and may have distinct functions in cell growth and gene expression. However, no structural differences between H2A.Z.1 and H2A.Z.2 have been reported. In the present study, the crystal structures of nucleosomes containing human H2A.Z.1 and H2A.Z.2 were determined. The structures of the L1 loop regions were found to clearly differ between H2A.Z.1 and H2A.Z.2, although their amino-acid sequences in this region are identical. This structural polymorphism may have been induced by a substitution that evolutionally occurred at the position of amino acid 38 and by the flexible nature of the L1 loops of H2A.Z.1 and H2A.Z.2. It was also found that in living cells nucleosomal H2A.Z.1 exchanges more rapidly than H2A.Z.2. A mutational analysis revealed that the amino-acid difference at position 38 is at least partially responsible for the distinctive dynamics of H2A.Z.1 and H2A.Z.2. These findings provide important new information for understanding the differences in the regulation and functions of H2A.Z.1 and H2A.Z.2 in cells.

    DOI

  • Structural polymorphism in the L1 loop regions of human H2AZ1 and H2AZ2

    Naoki Horikoshi, Koichi Sato, Keisuke Shimada, Yasuhiro Arimura, Akihisa Osakabe, Hiroaki Tachiwana, Yoko Hayashi-Takanaka, Wakana Iwasaki, Wataru Kagawa, Masahiko Harata, Hiroshi Kimura, Hitoshi Kurumizaka

    ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY   69 ( 12 ) 2431 - 2439  2013年12月  [査読有り]

     概要を見る

    The histone H2A.Z variant is widely conserved among eukaryotes. Two isoforms, H2A.Z.1 and H2A.Z.2, have been identified in vertebrates and may have distinct functions in cell growth and gene expression. However, no structural differences between H2A.Z.1 and H2A.Z.2 have been reported. In the present study, the crystal structures of nucleosomes containing human H2A.Z.1 and H2A.Z.2 were determined. The structures of the L1 loop regions were found to clearly differ between H2A.Z.1 and H2A.Z.2, although their amino-acid sequences in this region are identical. This structural polymorphism may have been induced by a substitution that evolutionally occurred at the position of amino acid 38 and by the flexible nature of the L1 loops of H2A.Z.1 and H2A.Z.2. It was also found that in living cells nucleosomal H2A.Z.1 exchanges more rapidly than H2A.Z.2. A mutational analysis revealed that the amino-acid difference at position 38 is at least partially responsible for the distinctive dynamics of H2A.Z.1 and H2A.Z.2. These findings provide important new information for understanding the differences in the regulation and functions of H2A.Z.1 and H2A.Z.2 in cells.

    DOI J-GLOBAL

  • Structural basis of a nucleosome containing histone H2A.B/H2A.Bbd that transiently associates with reorganized chromatin

    Yasuhiro Arimura, Hiroshi Kimura, Takashi Oda, Koichi Sato, Akihisa Osakabe, Hiroaki Tachiwana, Yuko Sato, Yasuha Kinugasa, Tsuyoshi Ikura, Masaaki Sugiyama, Mamoru Sato, Hitoshi Kurumizaka

    SCIENTIFIC REPORTS   3 ( 3 )  2013年12月  [査読有り]

     概要を見る

    Human histone H2A.B (formerly H2A.Bbd), a non-allelic H2A variant, exchanges rapidly as compared to canonical H2A, and preferentially associates with actively transcribed genes. We found that H2A. B transiently accumulated at DNA replication and repair foci in living cells. To explore the biochemical function of H2A. B, we performed nucleosome reconstitution analyses using various lengths of DNA. Two types of H2A.B nucleosomes, octasome and hexasome, were formed with 116, 124, or 130 base pairs (bp) of DNA, and only the octasome was formed with 136 or 146 bp DNA. In contrast, only hexasome formation was observed by canonical H2A with 116 or 124 bp DNA. A small-angle X-ray scattering analysis revealed that the H2A.B octasome is more extended, due to the flexible detachment of the DNA regions at the entry/exit sites from the histone surface. These results suggested that H2A. B rapidly and transiently forms nucleosomes with short DNA segments during chromatin reorganization.

    DOI J-GLOBAL

  • Sufficient Amounts of Functional HOP2/MND1 Complex Promote Interhomolog DNA Repair but Are Dispensable for Intersister DNA Repair during Meiosis in Arabidopsis

    Clemens Uanschou, Arnaud Ronceret, Mona Von Harder, Arnaud De Muyt, Daniel Vezon, Lucie Pereira, Liudmila Chelysheva, Wataru Kobayashi, Hitoshi Kurumizaka, Peter Schloegelhofer, Mathilde Grelon

    PLANT CELL   25 ( 12 ) 4924 - 4940  2013年12月  [査読有り]

     概要を見る

    During meiosis, homologous recombination (HR) is essential to repair programmed DNA double-strand breaks (DSBs), and a dedicated protein machinery ensures that the homologous chromosome is favored over the nearby sister chromatid as a repair template. The HOMOLOGOUS-PAIRING PROTEIN2/MEIOTIC NUCLEAR DIVISION PROTEIN1 (HOP2/MND1) protein complex has been identified as a crucial factor of meiotic HR in Arabidopsis thaliana, since loss of either MND1 or HOP2 results in failure of DNA repair. We isolated two mutant alleles of HOP2 (hop2-2 and hop2-3) that retained the capacity to repair meiotic DSBs via the sister chromatid but failed to use the homologous chromosome. We show that in these alleles, the recombinases RADIATION SENSITIVE51 (RAD51) and DISRUPTED MEIOTIC cDNA1 (DMC1) are loaded, but only the intersister DNA repair pathway is activated. The hop2-2 phenotype is correlated with a decrease in HOP2/MND1 complex abundance. In hop2-3, a truncated HOP2 protein is produced that retains its ability to bind to DMC1 and DNA but forms less stable complexes with MND1 and fails to efficiently stimulate DMC1-driven D-loop formation. Genetic analyses demonstrated that in the absence of DMC1, HOP2/MND1 is dispensable for RAD51-mediated intersister DNA repair, while in the presence of DMC1, a minimal amount of functional HOP2/MND1 is essential to drive intersister DNA repair.

    DOI J-GLOBAL

  • Activation of the SUMO modification system is required for the accumulation of RAD51 at sites of DNA damage

    Hiroki Shima, Hidekazu Suzuki, Jiying Sun, Kazuteru Kono, Lin Shi, Aiko Kinomura, Yasunori Horikoshi, Tsuyoshi Ikura, Masae Ikura, Roland Kanaar, Kazuhiko Igarashi, Hisato Saitoh, Hitoshi Kurumizaka, Satoshi Tashiro

    JOURNAL OF CELL SCIENCE   126 ( 22 ) 5284 - 5292  2013年11月  [査読有り]

     概要を見る

    Genetic information encoded in chromosomal DNA is challenged by intrinsic and exogenous sources of DNA damage. DNA double-strand breaks (DSBs) are extremely dangerous DNA lesions. RAD51 plays a central role in homologous DSB repair, by facilitating the recombination of damaged DNA with intact DNA in eukaryotes. RAD51 accumulates at sites containing DNA damage to form nuclear foci. However, the mechanism of RAD51 accumulation at sites of DNA damage is still unclear. Post-translational modifications of proteins, such as phosphorylation, acetylation and ubiquitylation play a role in the regulation of protein localization and dynamics. Recently, the covalent binding of small ubiquitin-like modifier (SUMO) proteins to target proteins, termed SUMOylation, at sites containing DNA damage has been shown to play a role in the regulation of the DNA-damage response. Here, we show that the SUMOylation E2 ligase UBC9, and E3 ligases PIAS1 and PIAS4, are required for RAD51 accretion at sites containing DNA damage in human cells. Moreover, we identified a SUMO-interacting motif (SIM) in RAD51, which is necessary for accumulation of RAD51 at sites of DNA damage. These findings suggest that the SUMO-SIM system plays an important role in DNA repair, through the regulation of RAD51 dynamics.

    DOI J-GLOBAL

  • Homologous Pairing Activities of Two Rice RAD51 Proteins, RAD51A1 and RAD51A2

    Yuichi Morozumi, Ryohei Ino, Shukuko Ikawa, Naozumi Mimida, Takeshi Shimizu, Seiichi Toki, Hiroaki Ichikawa, Takehiko Shibata, Hitoshi Kurumizaka

    PLOS ONE   8 ( 10 ) e75451  2013年10月  [査読有り]

     概要を見る

    In higher eukaryotes, RAD51 functions as an essential protein in homologous recombination and recombinational repair of DNA double strand breaks. During these processes, RAD51 catalyzes homologous pairing between single-stranded DNA and double-stranded DNA. Japonica cultivars of rice (Oryza sativa) encode two RAD51 proteins, RAD51A1 and RAD51A2, whereas only one RAD51 exists in yeast and mammals. However, the functional differences between RAD51A1 and RAD51A2 have not been elucidated, because their biochemical properties have not been characterized. In the present study, we purified RAD51A1 and RAD51A2, and found that RAD51A2 robustly promotes homologous pairing in vitro. RAD51A1 also possesses homologous-pairing activity, but it is only about 10% of the RAD51A2 activity. Both RAD51A1 and RAD51A2 bind to ssDNA and dsDNA, and their DNA binding strictly requires ATP, which modulates the polymer formation activities of RAD51A1 and RAD51A2. These findings suggest that although both RAD51A1 and RAD51A2 have the potential to catalyze homologous pairing, RAD51A2 may be the major recombinase in rice.

    DOI J-GLOBAL

  • Conclusive Evidence of the Reconstituted Hexasome Proven by Native Mass Spectrometry

    Nanako Azegami, Kazumi Saikusa, Yasuto Todokoro, Aritaka Nagadoi, Hitoshi Kurumizaka, Yoshifumi Nishimura, Satoko Akashi

    BIOCHEMISTRY   52 ( 31 ) 5155 - 5157  2013年08月  [査読有り]

     概要を見る

    It has been suggested that the hexasome, in which one of the H2A/H2B dimers is depleted from the canonical nucleosome core particle (NCP), is an essential intermediate during NCP assembly and disassembly, but little structural evidence of this exists. In this study, reconstituted products in a conventional NCP preparation were analyzed by native electrospray ionization mass spectrometry, and it was found that the hexasome, which migrated in a manner almost identical to that of the octasome NCP in native polyacrylamide gel electrophoresis, was produced simultaneously with the octasome NCP. This result might contribute to understanding the assembly and disassembly mechanism of NCPs.

    DOI J-GLOBAL

  • Genetically encoded system to track histone modification in vivo.

    Sato Y, Mukai M, Ueda J, Muraki M, Stasevich TJ, Horikoshi N, Kujirai T, Kita H, Kimura T, Hira S, Okada Y, Hayashi-Takanaka Y, Obuse C, Kurumizaka H, Kawahara A, Yamagata K, Nozaki N, Kimura H

    Scientific reports   3 ( 10 ) 1038  2013年08月  [査読有り]

    DOI J-GLOBAL

  • Gas-Phase Structure of the Histone Multimers Characterized by Ion Mobility Mass Spectrometry and Molecular Dynamics Simulation

    Kazumi Saikusa, Sotaro Fuchigami, Kyohei Takahashi, Yuuki Asano, Aritaka Nagadoi, Hiroaki Tachiwana, Hitoshi Kurumizaka, Mitsunori Ikeguchi, Yoshifumi Nishimura, Satoko Akashi

    ANALYTICAL CHEMISTRY   85 ( 8 ) 4165 - 4171  2013年04月  [査読有り]

     概要を見る

    The minimum structural unit of chromatin is the nucleosome core particle (NCP), consisting of 146 bp of DNA wrapped around a histone octamer, which itself contains two H2A/H2B dimers and one (H3/H4)(2) tetramer. These multimers possess functionally important tail regions that are intrinsically disordered. In order to elucidate the mechanisms behind NCP assembly and disassembly processes, which are highly related to gene expression, structural characterization of the H2A/H2B dimer and (H3/H4)(2) tetramer will be of importance. In the present study, human histone multimers with disordered tail regions were characterized by electrospray ionization (ESI) ion mobility-mass spectrometry (IM-MS) and molecular dynamics (MD) simulation. Experimentally obtained arrival times of these histone multimer ions showed rather wide distributions, implying that multiple conformers exist for each histone multimer in the gas phase. To examine their structures, MD simulations of the histone multimers were performed first in solution and then in vacuo at four temperatures, resulting in a variety of histone multimer structures. Theoretical collision cross-section (CCS) values calculated for the simulated structures revealed that structural models with smaller CCS values had more compact tail regions than those with larger CCS values. This implied that variation of the CCS values of the histone multimers were primarily due to the random behaviors of the tail regions in the gas phase. The combination of IM-MS and MD simulation enabled clear and comprehensive characterization of the gas-phase structures of histone multimers containing disordered tails.

    DOI J-GLOBAL

  • Purification and characterization of the fission yeast telomere clustering factors, Bqt1 and Bqt2

    Yuichi Ichikawa, Wataru Kagawa, Kengo Saito, Yuji Chikashige, Tokuko Haraguchi, Yasushi Hiraoka, Hitoshi Kurumizaka

    PROTEIN EXPRESSION AND PURIFICATION   88 ( 2 ) 207 - 213  2013年04月  [査読有り]

     概要を見る

    During meiosis, chromosomes adopt a bouquet arrangement, which is widely conserved among eukaryotes. This arrangement is assumed to play an important role in the normal progression of meiosis, by mediating the proper pairing of homologous chromosomes. In Schizosaccharomyces pombe, the complex of Bqt1 and Bqt2 plays a key role in telomere clustering and the subsequent bouquet arrangement of chromosomes during early meiotic prophase. Bqt1 and Bqt2 are part of a multi-protein complex that mediates the attachment of the telomere to the nuclear membrane. However, the structural details of the complex are needed to clarify the mechanism of telomere clustering. To enable biophysical studies of Bqt1 and Bqt2, we established a purification procedure for the Schizosaccharomyces japonicus Bqt1-Bqt2 complex, which is closely related to the S. pombe Bqt1-Bqt2 complex. A co-expression vector, in which one of the expressed subunits is fused to a removable SUMO tag, yielded high amounts of the proteins in the soluble fraction. The solubility of the Bqt1-Bqt2 complex after the removal of the SUMO tag was maintained by including CHAPS, a nondenaturing, zwitterionic detergent, in the purification buffers. These procedures enabled us to rapidly purify the stable Bqt1-Bqt2 complex. The co-purified Bqt1 and Bqt2 proteins formed a stable heterodimer, consistent with results from in vivo studies showing the requirement of both proteins for the bouquet arrangement The expression and purification procedures established here will facilitate further biophysical studies of the Bqt1-Bqt2 complex. (C) 2013 Elsevier Ltd. All rights reserved.

    DOI J-GLOBAL

  • Interaction between basic residues of Epstein-Barr virus EBNA1 protein and cellular chromatin mediates viral plasmid maintenance.

    Kanda T, Horikoshi N, Murata T, Kawashima D, Sugimoto A, Narita Y, Kurumizaka H, Tsurumi T

    The Journal of biological chemistry   288 ( 33 ) 24189 - 24199  2013年04月  [査読有り]

    DOI J-GLOBAL

  • Vertebrate Spt2 is a novel nucleolar histone chaperone that assists in ribosomal DNA transcription

    Akihisa Osakabe, Hiroaki Tachiwana, Motoki Takaku, Tetsuya Hori, Chikashi Obuse, Hiroshi Kimura, Tatsuo Fukagawa, Hitoshi Kurumizaka

    JOURNAL OF CELL SCIENCE   126 ( 6 ) 1323 - 1332  2013年03月  [査読有り]

     概要を見る

    In eukaryotes, transcription occurs in the chromatin context with the assistance of histone-binding proteins, such as chromatin/nucleosome remodeling factors and histone chaperones. However, it is unclear how each remodeling factor or histone chaperone functions in transcription. Here, we identify a novel histone-binding protein, Spt2, in higher eukaryotes. Recombinant human Spt2 binds to histones and DNA, and promotes nucleosome assembly in vitro. Spt2 accumulates in nucleoli and interacts with RNA polymerase I in chicken DT40 cells, suggesting its involvement in ribosomal RNA transcription. Consistently, Spt2-deficient chicken DT40 cells are sensitive to RNA polymerase I inhibitors and exhibit decreased transcription activity, as shown by a transcription run-on assay. Domain analyses of Spt2 revealed that the C-terminal region, containing the region homologous to yeast Spt2, is responsible for histone binding, while the central region is essential for nucleolar localization and DNA binding. Based on these results, we conclude that vertebrate Spt2 is a novel histone chaperone with a separate DNA-binding domain that facilitates ribosomal DNA transcription through chromatin remodeling during transcription.

    DOI

  • Nap1 regulates proper CENP-B binding to nucleosomes

    Hiroaki Tachiwana, Yuta Miya, Nobuaki Shono, Jun-ichirou Ohzeki, Akihisa Osakabe, Koichiro Otake, Vladimir Larionov, William C. Earnshaw, Hiroshi Kimura, Hiroshi Masumoto, Hitoshi Kurumizaka

    NUCLEIC ACIDS RESEARCH   41 ( 5 ) 2869 - 2880  2013年03月  [査読有り]

     概要を見る

    CENP-B is a widely conserved centromeric satellite DNA-binding protein, which specifically binds to a 17-bp DNA sequence known as the CENP-B box. CENP-B functions positively in the de novo assembly of centromeric nucleosomes, containing the centromere-specific histone H3 variant, CENP-A. At the same time, CENP-B also prevents undesired assembly of the CENP-A nucleosome through heterochromatin formation on satellite DNA integrated into ectopic sites. Therefore, improper CENP-B binding to chromosomes could be harmful. However, no CENP-B eviction mechanism has yet been reported. In the present study, we found that human Nap1, an acidic histone chaperone, inhibited the non-specific binding of CENP-B to nucleosomes and apparently stimulated CENP-B binding to its cognate CENP-B box DNA in nucleosomes. In human cells, the CENP-B eviction activity of Nap1 was confirmed in model experiments, in which the CENP-B binding to a human artificial chromosome or an ectopic chromosome locus bearing CENP-B boxes was significantly decreased when Nap1 was tethered near the CENP-B box sequence. In contrast, another acidic histone chaperone, sNASP, did not promote CENP-B eviction in vitro and in vivo and did not stimulate specific CENP-B binding to CENP-A nucleosomes in vitro. We therefore propose a novel mechanism of CENP-B regulation by Nap1.

    DOI J-GLOBAL

  • Vertebrate Spt2 is a novel nucleolar histone chaperone that assists in rebosomal DNA transcription.

    Osakabe,A, Tachiwana, H, Takaku, M, Hori,T, Obuse, C, Kimura, H, Fukagawa, T, Kurumizaka, H

    Journal of Cell Science   126 ( Pt6 ) 1232 - 1323  2013年02月

    DOI

  • Current progress on structural studies of nucleosomes containing histone H3 variants.

    Kurumizaka H, Horikoshi N, Tachiwana H, Kagawa W

    Current Opinion in Structural Biology   23 ( 1 ) 109 - 115  2013年02月  [査読有り]

    DOI J-GLOBAL

  • Activation of the SUMO modification system is required for the accumulation of RAD51 at sites of DNA damage.

    Shima Hiroki, Suzuki Hidekazu, Sun Jiying, Kono Kazuteru, Shi Lin, Kinomura Aiko, Horikoshi Yasunori, Ikura Tsuyoshi, Ikura Masae, Kanaar Roland, Igarashi Kazuhiko, Saitoh Hisato, Kurumizaka Hitoshi, Tashiro Satoshi

    Journal of cell science   126 ( Pt 22 )  2013年

     概要を見る

    :Genetic information encoded in chromosomal DNA is challenged by intrinsic and exogenous sources of DNA damage. DNA double-strand breaks (DSBs) are extremely dangerous DNA lesions. RAD51 plays a central role in homologous DSB repair, by facilitating the recombination of damaged DNA with intact DNA in eukaryotes. RAD51 accumulates at sites containing DNA damage to form nuclear foci. However, the mechanism of RAD51 accumulation at sites of DNA damage is still unclear. Post-translational modifications of proteins, such as phosphorylation, acetylation and ubiquitylation play a role in the regulation of protein localization and dynamics. Recently, the covalent binding of small ubiquitin-like modifier (SUMO) proteins to target proteins, termed SUMOylation, at sites containing DNA damage has been shown to play a role in the regulation of the DNA-damage response. Here, we show that the SUMOylation E2 ligase UBC9, and E3 ligases PIAS1 and PIAS4, are required for RAD51 accretion at sites containing DNA damage in human cells. Moreover, we identified a SUMO-interacting motif (SIM) in RAD51, which is necessary for accumulation of RAD51 at sites of DNA damage. These findings suggest that the SUM

    DOI

  • Crystallization and preliminary X-ray diffraction analysis of the secreted protein Athe_0614 from Caldicellulosiruptor bescii

    Yokoyama H, Yamashita T, Horikoshi N, Kurumizaka H, Kagawa W

    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS   69 ( 4 ) 438 - 440  2013年  [査読有り]

    DOI J-GLOBAL

  • Contribution of histone N-terminal tails to the structure and stability of nucleosomes

    Wakana Iwasaki, Yuta Miya, Naoki Horikoshi, Akihisa Osakabe, Hiroyuki Taguchi, Hiroaki Tachiwana, Takehiko Shibata, Wataru Kagawa, Hitoshi Kurumizaka

    FEBS OPEN BIO   3 ( 3 ) 363 - 369  2013年  [査読有り]

     概要を見る

    Histones are the protein components of the nucleosome, which forms the basic architecture of eukaryotic chromatin. Histones H2A, H2B, H3, and H4 are composed of two common regions, the "histone fold" and the "histone tail". Many efforts have been focused on the mechanisms by which the post-translational modifications of histone tails regulate the higher-order chromatin architecture. On the other hand, previous biochemical studies have suggested that histone tails also affect the structure and stability of the nucleosome core particle itself. However, the precise contributions of each histone tail are unclear. In the present study, we determined the crystal structures of four mutant nucleosomes, in which one of the four histones, H2A, H2B, H3, or H4, lacked the N-terminal tail. We found that the deletion of the H2B or H3 N-terminal tail affected histone-DNA interactions and substantially decreased nucleosome stability. These findings provide important information for understanding the complex roles of histone tails in regulating chromatin structure. (C) 2013 The Authors. Published by Elsevier B.V. on behalf of Federation of European Biochemical Societies. All rights reserved.

    DOI J-GLOBAL

  • 新規ヒストン相互作用因子hsSpt2の核小体クロマチンダイナミクスにおける機能

    越阪部 晃永, 立和名 博昭, 高久 誉大, 堀 哲也, 小布施 力史, 木村 宏, 深川 竜郎, 胡桃坂 仁志

    日本生化学会大会プログラム・講演要旨集   85回   3T22 - 01  2012年12月  [査読有り]

  • Identification of new acetylated proteins involved in DNA damage response

    Takeshi Yasuda, Kengo Saito, Wataru Kagawa, Tomoo Ogi, Takehiro Suzuki, Naoshi Dohmae, Takuya Hino, Yuka Nakazawa, Ai Nakamura, Masaharu Hazawa, Fumio Hanaoka, Kaoru Sugasawa, Ryuichi Okayasu, Hitoshi Kurumizaka, Katsushi Tajima

    GENES & GENETIC SYSTEMS   87 ( 6 ) 411 - 411  2012年12月

  • Histone chaperone activity of Fanconi anemia proteins, FANCD2 and FANCI, is required for DNA crosslink repair

    Koichi Sato, Masamichi Ishiai, Kazue Toda, Satoshi Furukoshi, Akihisa Osakabe, Hiroaki Tachiwana, Yoshimasa Takizawa, Wataru Kagawa, Hiroyuki Kitao, Naoshi Dohmae, Chikashi Obuse, Hiroshi Kimura, Minoru Takata, Hitoshi Kurumizaka

    EMBO JOURNAL   31 ( 17 ) 3524 - 3536  2012年08月  [査読有り]

     概要を見る

    Fanconi anaemia (FA) is a rare hereditary disorder characterized by genomic instability and cancer susceptibility. A key FA protein, FANCD2, is targeted to chromatin with its partner, FANCI, and plays a critical role in DNA crosslink repair. However, the molecular function of chromatin-bound FANCD2-FANCI is still poorly understood. In the present study, we found that FANCD2 possesses nucleosome-assembly activity in vitro. The mobility of histone H3 was reduced in FANCD2-knockdown cells following treatment with an interstrand DNA crosslinker, mitomycin C. Furthermore, cells harbouring FANCD2 mutations that were defective in nucleosome assembly displayed impaired survival upon cisplatin treatment. Although FANCI by itself lacked nucleosome-assembly activity, it significantly stimulated FANCD2-mediated nucleosome assembly. These observations suggest that FANCD2-FANCI may regulate chromatin dynamics during DNA repair. The EMBO Journal (2012) 31, 3524-3536. doi: 10.1038/emboj.2012.197; Published online 24 July 2012 Subject Categories: genome stability & dynamics; chromatin & transcription

    DOI J-GLOBAL

  • Chd2 interacts with H3.3 to determine myogenic cell fate

    Akihito Harada, Seiji Okada, Daijiro Konno, Jun Odawara, Tomohiko Yoshimi, Saori Yoshimura, Hiromi Kumamaru, Hirokazu Saiwai, Toshiaki Tsubota, Hitoshi Kurumizaka, Koichi Akashi, Taro Tachibana, Anthony N. Imbalzano, Yasuyuki Ohkawa

    EMBO JOURNAL   31 ( 13 ) 2994 - 3007  2012年06月  [査読有り]

     概要を見る

    Cell differentiation is mediated by lineage-determining transcription factors. We show that chromodomain helicase DNA-binding domain 2 (Chd2), a SNF2 chromatin remodelling enzyme family member, interacts with MyoD and myogenic gene regulatory sequences to specifically mark these loci via deposition of the histone variant H3.3 prior to cell differentiation. Directed and genome-wide analysis of endogenous H3.3 incorporation demonstrates that knockdown of Chd2 prevents H3.3 deposition at differentiation-dependent, but not housekeeping, genes and inhibits myogenic gene activation. The data indicate that MyoD determines cell fate and facilitates differentiation-dependent gene expression through Chd2-dependent deposition of H3.3 at myogenic loci prior to differentiation. The EMBO Journal (2012) 31, 2994-3007. doi: 10.1038/emboj.2012.136; Published online 8 May 2012

    DOI J-GLOBAL

  • DNA robustly stimulates FANCD2 monoubiquitylation in the complex with FANCI

    Koichi Sato, Kazue Toda, Masamichi Ishiai, Minoru Takata, Hitoshi Kurumizaka

    NUCLEIC ACIDS RESEARCH   40 ( 10 ) 4553 - 4561  2012年05月  [査読有り]

     概要を見る

    FANCI and FANCD2 form a complex, and play essential roles in the repair of interstrand DNA crosslinks (ICLs) by the Fanconi anemia (FA) pathway. FANCD2 is monoubiquitylated by the FA core complex, composed of 10 FA proteins including FANCL as the catalytic E3 subunit. FANCD2 monoubiquitylation can be reconstituted with purified minimal components, such as FANCI, E1, UBE2T (E2) and FANCL (E3) in vitro; however, its efficiency is quite low as compared to the in vivo monoubiquitylation of FANCD2. In this study, we found that various forms of DNA, such as single-stranded, double-stranded and branched DNA, robustly stimulated the FANCD2 monoubiquitylation in vitro up to a level comparable to its in vivo monoubiquitylation. This stimulation of the FANCD2 monoubiquitylation strictly required FANCI, suggesting that FANCD2 monoubiquitylation may occur in the FANCI-FANCD2 complex. A FANCI mutant that was defective in DNA binding was also significantly defective in FANCD2 monoubiquitylation in vitro. In the presence of 5' flapped DNA, a DNA substrate mimicking the arrested replication fork, about 70% of the input FANCD2 was monoubiquitylated, while less than 1% FANCD2 monoubiquitylation was observed in the absence of the DNA. Therefore, DNA may be the unidentified factor required for proper FANCD2 monoubiquitylation.

    DOI J-GLOBAL

  • Human PSF concentrates DNA and stimulates duplex capture in DMC1-mediated homologous pairing

    Yuichi Morozumi, Ryohei Ino, Motoki Takaku, Mihoko Hosokawa, Shinichiro Chuma, Hitoshi Kurumizaka

    NUCLEIC ACIDS RESEARCH   40 ( 7 ) 3031 - 3041  2012年04月

     概要を見る

    PSF is considered to have multiple functions in RNA processing, transcription and DNA repair by mitotic recombination. In the present study, we found that PSF is produced in spermatogonia, spermatocytes and spermatids, suggesting that PSF may also function in meiotic recombination. We tested the effect of PSF on homologous pairing by the meiosis-specific recombinase DMC1, and found that human PSF robustly stimulated it. PSF synergistically enhanced the formation of a synaptic complex containing DMC1, ssDNA and dsDNA during homologous pairing. The PSF-mediated DMC1 stimulation may be promoted by its DNA aggregation activity, which increases the local concentrations of ssDNA and dsDNA for homologous pairing by DMC1. These results suggested that PSF may function as an activator for the meiosis-specific recombinase DMC1 in higher eukaryotes.

    DOI

  • Human PSF concentrates DNA and stimulates duplex capture in DMC1-mediated homologous pairing

    Yuichi Morozumi, Ryohei Ino, Motoki Takaku, Mihoko Hosokawa, Shinichiro Chuma, Hitoshi Kurumizaka

    NUCLEIC ACIDS RESEARCH   40 ( 7 ) 3031 - 3041  2012年04月  [査読有り]

     概要を見る

    PSF is considered to have multiple functions in RNA processing, transcription and DNA repair by mitotic recombination. In the present study, we found that PSF is produced in spermatogonia, spermatocytes and spermatids, suggesting that PSF may also function in meiotic recombination. We tested the effect of PSF on homologous pairing by the meiosis-specific recombinase DMC1, and found that human PSF robustly stimulated it. PSF synergistically enhanced the formation of a synaptic complex containing DMC1, ssDNA and dsDNA during homologous pairing. The PSF-mediated DMC1 stimulation may be promoted by its DNA aggregation activity, which increases the local concentrations of ssDNA and dsDNA for homologous pairing by DMC1. These results suggested that PSF may function as an activator for the meiosis-specific recombinase DMC1 in higher eukaryotes.

    DOI

  • Structural Analysis of the Hexasome, Lacking One Histone H2A/H2B Dimer from the Conventional Nucleosome

    Yasuhiro Arimura, Hiroaki Tachiwana, Takashi Oda, Mamoru Sato, Hitoshi Kurumizaka

    BIOCHEMISTRY   51 ( 15 ) 3302 - 3309  2012年04月  [査読有り]

     概要を見る

    Genomic DNA is packaged into chromatin in eukaryotes, and the nucleosome is the fundamental unit of chromatin. The canonical nucleosome is the octasome, which is composed of two H2A/H2B dimers and two H3/H4 dimers. During transcription elongation, one of the H2A/H2B dimers is removed from the octasome. The depletion of the H2A/H2B dimer is also suggested to occur during DNA replication and repair. The remaining histone components are believed to maintain a nucleosomal structure called a "hexasome", which is probably important for the regulation of gene expression, DNA replication, and repair in chromatin. However, hexasomes are currently poorly understood, due to the lack of in vivo and in vitro studies. Biochemical and structural studies of hexasomes have been hampered by the difficulty of preparing purified hexasomes. In the present study, we successfully reconstituted hexasomes, using recombinant human histones. A micrococcal nuclease treatment and in vitro reconstitution assays revealed that the hexasome tightly wraps approximately 110 base-pairs of DNA, about 40 base-pairs shorter than the length of the DNA wrapped within the canonical nucleosome. A small-angle X-ray scattering analysis revealed that the global structure of the hexasome is similar to that of the canonical nucleosome. Our studies suggest that octasomes can be converted into hexasomes by the eviction of one of the H2A/H2B dimers, and the release of about 40 base-pairs of DNA, without involving large structural changes in the nucleosome core particle.

    DOI J-GLOBAL

  • ATR-ATRIP Kinase Complex Triggers Activation of the Fanconi Anemia DNA Repair Pathway

    Tomoko Shigechi, Junya Tomida, Koichi Sato, Masahiko Kobayashi, John K. Eykelenboom, Fabio Pessina, Yanbin Zhang, Emi Uchida, Masamichi Ishiai, Noel F. Lowndes, Kenichi Yamamoto, Hitoshi Kurumizaka, Yoshihiko Maehara, Minoru Takata

    CANCER RESEARCH   72 ( 5 ) 1149 - 1156  2012年03月  [査読有り]

     概要を見る

    ATR kinase activates the S-phase checkpoint when replication forks stall at sites of DNA damage. This event also causes phosphorylation of the Fanconi anemia (FA) protein FANCI, triggering its monoubiquitination of the key DNA repair factor FANCD2 by the FA core E3 ligase complex, thereby promoting this central pathway of DNA repair which permits replication to be restarted. However, the interplay between ATR and the FA pathway has been unclear. In this study, we present evidence that their action is directly linked, gaining insights into this relationship in a DT40 mutant cell line that is conditionally deficient in the critical ATR-binding partner protein ATRIP. Using this system, we showed that ATRIP was crucial for DNA damage-induced FANCD2 monoubiquitination and FANCI phosphorylation. ATR kinase phosphorylated recombinant FANCI protein in vitro, which was facilitated by the presence of FANCD2. Mechanistic investigations revealed that the RPA region but not the TopBP1 region of ATRIP was required for FANCD2 monoubiquitination, whereas Chk1 phosphorylation relied upon both domains. Together, our findings identify ATR as the kinase responsible for activating the FA pathway of DNA repair. Cancer Res; 72(5); 1149-56. (C) 2012 AACR.

    DOI J-GLOBAL

  • RAD51 Plays a Crucial Role in Halting Cell Death Program Induced by Ionizing Radiation in Bovine Oocytes

    Loro L. Kujjo, Reg Ronningen, Pablo Ross, Ricardo J. G. Pereira, Ramon Rodriguez, Zeki Beyhan, Marcelo D. Goissis, Thomas Baumann, Wataru Kagawa, Cagri Camsari, George W. Smith, Hitoshi Kurumizaka, Shigeyuki Yokoyama, Jose B. Cibelli, Gloria I. Perez

    BIOLOGY OF REPRODUCTION   86 ( 3 ) 1 - 11  2012年03月  [査読有り]

     概要を見る

    Reproductive health of humans and animals exposed to daily irradiants from solar/cosmic particles remains largely understudied. We evaluated the sensitivities of bovine and mouse oocytes to bombardment by krypton-78 (1 Gy) or ultraviolet B (UV-B; 100 microjoules). Mouse oocytes responded to irradiation by undergoing massive activation of caspases, rapid loss of energy without cytochrome-c release, and subsequent necrotic death. In contrast, bovine oocytes became positive for annexin-V, exhibited cytochrome-c release, and displayed mild activation of caspases and downstream DNAses but with the absence of a complete cell death program; therefore, cytoplasmic fragmentation was never observed. However, massive cytoplasmic fragmentation and increased DNA damage were induced experimentally by both inhibiting RAD51 and increasing caspase 3 activity before irradiation. Microinjection of recombinant human RAD51 prior to irradiation markedly decreased both cytoplasmic fragmentation and DNA damage in both bovine and mouse oocytes. RAD51 response to damaged DNA occurred faster in bovine oocytes than in mouse oocytes. Therefore, we conclude that upon exposure to irradiation, bovine oocytes create a physiologically indeterminate state of partial cell death, attributed to rapid induction of DNA repair and low activation of caspases. The persistence of these damaged cells may represent an adaptive mechanism with potential implications for livestock productivity and long-term health risks associated with human activity in space.

    DOI J-GLOBAL

  • Comparison between the CENP-A and histone H3 structures in nucleosomes

    Hiroaki Tachiwana, Wataru Kagawa, Hitoshi Kurumizaka

    NUCLEUS-AUSTIN   3 ( 1 ) 6 - 11  2012年01月  [査読有り]

     概要を見る

    Centromeres are epigenetically marked by the assembly of nucleosomes containing the centromere-specific histone H3 variant, CENP-A (CENP-A nucleosome) and their inheritance is probably dictated by the architecture of the centromeric nucleosome. We previously determined the crystal structure of the human CENP-A nucleosome. CENP-A forms a histone octamer containing two each of histones H2A, H2B, H4 and CENP-A and the DNA is left-handedly wrapped around the histone octamer, as in canonical nucleosomes containing histone H3. In the CENP-A nucleosome structure, 13 base pairs of the DNA ends are detached from the histone surface and two CENP-A regions, the alpha N helix and loop 1, adopt different structures from those in the H3 nucleosome. In this Extra View article, we provide a detailed structural comparison between CENP-A and H3 in nucleosomes and describe their distinctions and similarities.

    DOI J-GLOBAL

  • Structural basis for the DNA-binding activity of the bacterial Β-propeller protein YncE

    Kagawa W, Sagawa T, Niki H, Kurumizaka H

    Acta Crystallographica Section D: Biological Crystallography   67 ( 12 ) 1045 - 1053  2011年12月  [査読有り]

    DOI

  • Structure of the CENP-A nucleosome and its implications for centromeric chromatin architecture

    Hiroaki Tachiwana, Hitoshi Kurumizaka

    GENES & GENETIC SYSTEMS   86 ( 6 ) 357 - 364  2011年12月  [査読有り]

     概要を見る

    Centromeres are dictated by the epigenetic inheritance of the centromeric nucleosome containing the centromere-specific histone H3 variant, CENP-A. The structure of the CENP-A nucleosome has been considered to be the fundamental architecture of the centromeric chromatin. Controversy exists in the literature regarding the CENP-A nucleosome structures, with octasome, hemisome, compact octasome, hexasome, and tetrasome models being reported. Some of these CENP-A nucleosome models may correspond to transient intermediates for the assembly of the mature CENP-A nucleosome; however, their significances are still unclear. Therefore, the structure of the mature CENP-A nucleosome has been eagerly awaited. We reconstituted the human CENP-A nucleosome with its cognate centromeric DNA fragment, and determined its crystal structure. In this review, we describe the structure and the physical properties of the CENP-A nucleosome, and discuss their implications for centromeric chromatin architecture.

    DOI J-GLOBAL

  • Comprehensive Structural Analysis of Mutant Nucleosomes Containing Lysine to Glutamine (KQ) Substitutions in the H3 and H4 Histone-Fold Domains

    Wakana Iwasaki, Hiroaki Tachiwana, Koichiro Kawaguchi, Takehiko Shibata, Wataru Kagawa, Hitoshi Kurumizaka

    BIOCHEMISTRY   50 ( 36 ) 7822 - 7832  2011年09月  [査読有り]

     概要を見る

    Post-translational modifications (PTMs) of histones play important roles in regulating the structure and function of chromatin in eukaryotes. Although histone PTMs were considered to mainly occur at the N-terminal tails of histones, recent studies have revealed that PTMs also exist in the histone-fold domains, which are commonly shared among the core histones H2A, H2B, H3, and H4. The lysine residue is a major target for histone PTM, and the lysine to glutamine (KQ) substitution is known to mimic the acetylated states of specific histone lysine residues in vivo. Human histones H3 and H4 contain 11 lysine residues in their histone-fold domains (five for H3 and six for H4), and eight of these lysine residues are known to be targets for acetylation. In the present study, we prepared 11 mutant nucleosomes, in which each of the lysine residues of the H3 and H4 histone-fold domains was replaced by glutamine: H3 K56Q, H3 K64Q, H3 K79Q, H3 K115Q, H3 K122Q, H4 K31Q, H4 K44Q, H4 K59Q, H4 K77Q, H4 K79Q, and H4 K91Q. The crystal structures of these mutant nucleosomes were determined at 2.4-3.5 angstrom resolutions. Some of these amino acid substitutions altered the local protein-DNA interactions and the interactions between amino acid residues within the nucleosome. Interestingly, the C-terminal region of H2A was significantly disordered in the nucleosome containing H4 K44Q These results provide an important structural basis for understanding how histone modifications and mutations affect chromatin structure and function.

    DOI J-GLOBAL

  • Purification of the Human SMN-GEMIN2 Complex and Assessment of Its Stimulation of RAD51-Mediated DNA Recombination Reactions

    Motoki Takaku, Takashi Tsujita, Naoki Horikoshi, Yoshimasa Takizawa, Yong Qing, Kouji Hirota, Masae Ikura, Tsuyoshi Ikura, Shunichi Takeda, Hitoshi Kurumizaka

    BIOCHEMISTRY   50 ( 32 ) 6797 - 6805  2011年08月  [査読有り]

     概要を見る

    A deficiency in the SMN gene product causes the motor neuron degenerative disease spinal muscular atrophy. GEMIN2 was identified as an SMN-interacting protein, and the SMN-GEMIN2 complex constitutes part of the large SMN complex, which promotes the assembly of the spliceosomal small nuclear ribonucleoprotein (snRNP). In addition to its splicing function, we previously found that GEMIN2 alone stimulates RAD51-mediated recombination in vitro, and functions in DNA double-strand-break (DSB) repair through homologous recombination in vivo. However, the function of SMN in homologous recombination has not been reported. In the present study, we successfully purified the SMN-GEMIN2 complex as a fusion protein. The SMN-GEMIN2 fusion protein complemented the growth-defective phenotype of GEMIN2-knockout cells. The purified SMN-GEMIN2 fusion protein enhanced the RAD51-mediated homologous pairing much more efficiently than GEMIN2 alone. SMN-GEMIN2 possessed DNA-binding activity, which was not observed with the GEMIN2 protein, and significantly stimulated the secondary duplex DNA capture by the RAD51-single-stranded DNA complex during homologous pairing. These results provide the first evidence that the SMN-GEMIN2 complex plays a role in homologous recombination, in addition to spliceosomal snRNP assembly.

    DOI

  • Tracking epigenetic histone modifications in single cells using Fab-based live endogenous modification labeling

    Yoko Hayashi-Takanaka, Kazuo Yamagata, Teruhiko Wakayama, Timothy J. Stasevich, Takashi Kainuma, Toshiki Tsurimoto, Makoto Tachibana, Yoichi Shinkai, Hitoshi Kurumizaka, Naohito Nozaki, Hiroshi Kimura

    NUCLEIC ACIDS RESEARCH   39 ( 15 ) 6475 - 6488  2011年08月  [査読有り]

     概要を見る

    Histone modifications play an important role in epigenetic gene regulation and genome integrity. It remains largely unknown, however, how these modifications dynamically change in individual cells. By using fluorescently labeled specific antigen binding fragments (Fabs), we have developed a general method to monitor the distribution and global level of endogenous histone H3 lysine modifications in living cells without disturbing cell growth and embryo development. Fabs produce distinct nuclear patterns that are characteristic of their target modifications. H3K27 trimethylation-specific Fabs, for example, are concentrated on inactive X chromosomes. As Fabs bind their targets transiently, the ratio of bound and free molecules depends on the target concentration, allowing us to measure changes in global modification levels. High-affinity Fabs are suitable for mouse embryo imaging, so we have used them to monitor H3K9 and H3K27 acetylation levels in mouse preimplantation embryos produced by in vitro fertilization and somatic cell nuclear transfer. The data suggest that a high level of H3K27 acetylation is important for normal embryo development. As Fab-based live endogenous modification labeling (FabLEM) is broadly useful for visualizing any modification, it should be a powerful tool for studying cell signaling and diagnosis in the future.

    DOI J-GLOBAL

  • Crystal structure of the human centromeric nucleosome containing CENP-A

    Hiroaki Tachiwana, Wataru Kagawa, Tatsuya Shiga, Akihisa Osakabe, Yuta Miya, Kengo Saito, Yoko Hayashi-Takanaka, Takashi Oda, Mamoru Sato, Sam-Yong Park, Hiroshi Kimura, Hitoshi Kurumizaka

    NATURE   476 ( 7359 ) 232 - U135  2011年08月  [査読有り]

     概要を見る

    In eukaryotes, accurate chromosome segregation during mitosis and meiosis is coordinated by kinetochores, which are unique chromosomal sites for microtubule attachment(1,2). Centromeres specify the kinetochore formation sites on individual chromosomes, and are epigenetically marked by the assembly of nucleosomes containing the centromere-specific histone H3 variant, CENP-A(3-12). Although the underlying mechanism is unclear, centromere inheritance is probably dictated by the architecture of the centromeric nucleosome. Here we report the crystal structure of the human centromeric nucleosome containing CENP-A and its cognate alpha-satellite DNA derivative (147 base pairs). In the human CENP-A nucleosome, the DNA is wrapped around the histone octamer, consisting of two each of histones H2A, H2B, H4 and CENP-A, in a left-handed orientation. However, unlike the canonical H3 nucleosome, only the central 121 base pairs of the DNA are visible. The thirteen base pairs from both ends of the DNA are invisible in the crystal structure, and the alpha N helix of CENP-A is shorter than that of H3, which is known to be important for the orientation of the DNA ends in the canonical H3 nucleosome(13). A structural comparison of the CENP-A and H3 nucleosomes revealed that CENP-A contains two extra amino acid residues (Arg 80 and Gly 81) in the loop 1 region, which is completely exposed to the solvent. Mutations of the CENP-A loop 1 residues reduced CENP-A retention at the centromeres in human cells. Therefore, the CENP-A loop 1 may function in stabilizing the centromeric chromatin containing CENP-A, possibly by providing a binding site for trans-acting factors. The structure provides the first atomic-resolution picture of the centromere-specific nucleosome.

    DOI J-GLOBAL

  • Direct inhibition of TNF-α promoter activity by Fanconi anemia protein FANCD2.

    Matsushita N, Endo Y, Sato K, Kurumizaka H, Yamashita T, Takata M, Yanagi S

    PLoS One   6 ( 8 ) e23324  2011年08月  [査読有り]

    DOI J-GLOBAL

  • Biochemical analysis of the human ENA/VASP-family proteins, MENA, VASP and EVL, in homologous recombination

    Motoki Takaku, Hiroyuki Ueno, Hitoshi Kurumizaka

    JOURNAL OF BIOCHEMISTRY   149 ( 6 ) 721 - 729  2011年06月  [査読有り]

     概要を見る

    MENA, VASP and EVL are members of the ENA/VASP family of proteins and are involved in cytoplasmic actin remodeling. Previously, we found that EVL directly interacts with RAD51, an essential protein in the homologous recombinational repair of double-strand breaks (DSBs) and stimulates the RAD51-mediated recombination reactions in vitro. The EVL-knockdown MCF7 cells exhibited a clear reduction in RAD51-foci formation, suggesting that EVL may function in the DSB repair pathway through RAD51-mediated homologous recombination. However, the DSB repair defects were less significant in the EVL-knockdown cells, implying that two EVL paralogues, MENA and VASP, may complement the EVL function in human cells. Therefore, in the present study, we purified human MENA, VASP and EVL as recombinant proteins, and compared their biochemical activities in vitro. We found that all three proteins commonly exhibited the RAD51 binding, DNA binding and DNA-annealing activities. Stimulation of the RAD51-mediated homologous pairing was also observed with all three proteins. In addition, surface plasmon resonance analyses revealed that MENA, VASP and EVL mutually interacted. These results support the ideas that the ENA/VASP-family proteins are functionally redundant in homologous recombination, and that all three may be involved in the DSB repair pathway in humans.

    DOI J-GLOBAL

  • Structures of human nucleosomes containing major histone H3 variants

    Hiroaki Tachiwana, Akihisa Osakabe, Tatsuya Shiga, Yuta Miya, Hiroshi Kimura, Wataru Kagawa, Hitoshi Kurumizaka

    ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY   67 ( Pt6 ) 578 - 583  2011年06月  [査読有り]

     概要を見る

    The nucleosome is the fundamental repeating unit of chromatin, via which genomic DNA is packaged into the nucleus in eukaryotes. In the nucleosome, two copies of each core histone, H2A, H2B, H3 and H4, form a histone octamer which wraps 146 base pairs of DNA around itself. All of the core histones except for histone H4 have nonallelic isoforms called histone variants. In humans, eight histone H3 variants, H3.1, H3.2, H3.3, H3T, H3.5, H3.X, H3.Y and CENP-A, have been reported to date. Previous studies have suggested that histone H3 variants possess distinct functions in the formation of specific chromosome regions and/or in the regulation of transcription and replication. H3.1, H3.2 and H3.3 are the most abundant H3 variants. Here, crystal structures of human nucleosomes containing either H3.2 or H3.3 have been solved. The structures were essentially the same as that of the H3.1 nucleosome. Since the amino-acid residues specific for H3.2 and H3.3 are located on the accessible surface of the H3/H4 tetramer, they may be potential interaction sites for H3.2- and H3.3-specific chaperones.

    DOI J-GLOBAL

  • Vital Roles of the Second DNA-binding Site of Rad52 Protein in Yeast Homologous Recombination

    Naoto Arai, Wataru Kagawa, Kengo Saito, Yoshinori Shingu, Tsutomu Mikawa, Hitoshi Kurumizaka, Takehiko Shibata

    JOURNAL OF BIOLOGICAL CHEMISTRY   286 ( 20 ) 17607 - 17617  2011年05月  [査読有り]

     概要を見る

    RecA/Rad51 proteins are essential in homologous DNA recombination and catalyze the ATP-dependent formation of D-loops from a single-stranded DNA and an internal homologous sequence in a double-stranded DNA. RecA and Rad51 require a "recombination mediator" to overcome the interference imposed by the prior binding of single-stranded binding protein/replication protein A to the single-stranded DNA. Rad52 is the prototype of recombination mediators, and the human Rad52 protein has two distinct DNA-binding sites: the first site binds to single-stranded DNA, and the second site binds to either double-or single-stranded DNA. We previously showed that yeast Rad52 extensively stimulates Rad51-catalyzed D-loop formation even in the absence of replication protein A, by forming a 2: 1 stoichiometric complex with Rad51. However, the precise roles of Rad52 and Rad51 within the complex are unknown. In the present study, we constructed yeast Rad52 mutants in which the amino acid residues corresponding to the second DNA-binding site of the human Rad52 protein were replaced with either alanine or aspartic acid. We found that the second DNA-binding site is important for the yeast Rad52 function in vivo. Rad51-Rad52 complexes consisting of these Rad52 mutants were defective in promoting the formation of D-loops, and the ability of the complex to associate with double-stranded DNA was specifically impaired. Our studies suggest that Rad52 within the complex associates with double-stranded DNA to assist Rad51-mediated homologous pairing.

    DOI

  • Halenaquinone, a chemical compound that specifically inhibits the secondary DNA binding of RAD51

    Motoki Takaku, Takashi Kainuma, Takako Ishida-Takaku, Shintaro Ishigami, Hidekazu Suzuki, Satoshi Tashiro, Rob W. M. van Soest, Yoichi Nakao, Hitoshi Kurumizaka

    GENES TO CELLS   16 ( 4 ) 427 - 436  2011年04月  [査読有り]

     概要を見る

    Mutations and single-nucleotide polymorphisms affecting RAD51 gene function have been identified in several tumors, suggesting that the inappropriate expression of RAD51 activity may cause tumorigenesis. RAD51 is an essential enzyme for the homologous recombinational repair (HRR) of DNA double-strand breaks. In the HRR pathway, RAD51 catalyzes the homologous pairing between single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), which is the central step of the HRR pathway. To identify a chemical compound that regulates the homologous-pairing activity of RAD51, in the present study, we screened crude extract fractions from marine sponges by the RAD51-mediated homologous-pairing assay. Halenaquinone was identified as an inhibitor of the RAD51 homologous-pairing activity. A surface plasmon resonance analysis indicated that halenaquinone directly bound to RAD51. Intriguingly, halenaquinone specifically inhibited dsDNA binding by RAD51 alone or the RAD51-ssDNA complex, but only weakly affected the RAD51-ssDNA binding. In vivo, halenaquinone significantly inhibited the retention of RAD51 at double-strand break sites. Therefore, halenaquinone is a novel type of RAD51 inhibitor that specifically inhibits the RAD51-dsDNA binding.

    DOI J-GLOBAL

  • Involvement of SLX4 in interstrand cross-link repair is regulated by the Fanconi anemia pathway

    Kimiyo N. Yamamoto, Shunsuke Kobayashi, Masataka Tsuda, Hitoshi Kurumizaka, Minoru Takata, Koichi Kono, Josef Jiricny, Shunichi Takeda, Kouji Hirota

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   108 ( 16 ) 6492 - 6496  2011年04月  [査読有り]

     概要を見る

    Interstrand cross-links (ICLs) block replication and transcription and thus are highly cytotoxic. In higher eukaryotes, ICLs processing involves the Fanconi anemia (FA) pathway and homologous recombination. Stalled replication forks activate the eight-subunit FA core complex, which ubiquitylates FANCD2-FANCI. Once it is posttranslationally modified, this heterodimer recruits downstream members of the ICL repairosome, including the FAN1 nuclease. However, ICL processing has been shown to also involve MUS81-EME1 and XPF-ERCC1, nucleases known to interact with SLX4, a docking protein that also can bind another nuclease, SLX1. To investigate the role of SLX4 more closely, we disrupted the SLX4 gene in avian DT40 cells. SLX4 deficiency caused cell death associated with extensive chromosomal aberrations, including a significant fraction of isochromatid-type breaks, with sister chromatids broken at the same site. SLX4 thus appears to play an essential role in cell proliferation, probably by promoting the resolution of interchromatid homologous recombination intermediates. Because ubiquitylation plays a key role in the FA pathway, and because the N-terminal region of SLX4 contains a ubiquitin-binding zinc finger (UBZ) domain, we asked whether this domain is required for ICL processing. We found that SLX4(-/-) cells expressing UBZ-deficient SLX4 were selectively sensitive to ICL-inducing agents, and that the UBZ domain was required for interaction of SLX4 with ubiquitylated FANCD2 and for its recruitment to DNA-damage foci generated by ICL-inducing agents. Our findings thus suggest that ubiquitylated FANCD2 recruits SLX4 to DNA damage sites, where it mediates the resolution of recombination intermediates generated during the processing of ICLs.

    DOI J-GLOBAL

  • Structural and biochemical analyses of the human PAD4 variant encoded by a functional haplotype gene

    Naoki Horikoshi, Hiroaki Tachiwana, Kengo Saito, Akihisa Osakabe, Mamoru Sato, Michiyuki Yamada, Satoko Akashi, Yoshifumi Nishimura, Wataru Kagawa, Hitoshi Kurumizaka

    ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY   67 ( Pt 2 ) 112 - 118  2011年02月  [査読有り]

     概要を見る

    PAD4 is a peptidylarginine deiminase that catalyzes citrullination, a type of post-translational modification. In this reaction, arginine residues in proteins are converted to citrulline. PAD4 promotes the deimination of arginine residues in histones and may regulate transcription in the context of the chromatin. Single-nucleotide polymorphisms (SNP) in the gene encoding PAD4 identified it as one of the genes associated with susceptibility to rheumatoid arthritis. The PAD4 SNP involve three amino-acid substitutions: Ser55 to Gly, Ala82 to Val and Ala112 to Gly. Autoantibodies for improperly citrullinated proteins have been found in rheumatoid arthritis patients, suggesting that the PAD4SNP mRNA is more stable than the conventional PAD4 mRNA and/or the PAD4SNP protein possesses a higher citrullination activity than the PAD4 protein. In order to study the effects of the three amino-acid substitutions found in PAD4SNP, the crystal structure of PAD4SNP was determined and it was found that the amino-acid substitutions in PAD4SNP only induced conformational changes within the N-terminal domain, not in the active centre for citrullination located in the C-terminal domain. Biochemical analyses also suggested that the citrullination activity of PAD4SNP may not substantially differ from that of conventional PAD4. These structural and biochemical findings suggested that the improper protein citrullination found in rheumatoid arthritis patients is not caused by defects in the citrullination activity of PAD4SNP but by other reasons such as enhanced PAD4SNP mRNA stability.

    DOI J-GLOBAL

  • KIAA1018/FAN1 nuclease protects cells against genomic instability induced by interstrand cross-linking agents

    Kazunori Yoshikiyo, Katja Kratz, Kouji Hirota, Kana Nishihara, Minoru Takata, Hitoshi Kurumizaka, Satoshi Horimoto, Shunichi Takeda, Josef Jiricny

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   107 ( 50 ) 21553 - 21557  2010年12月  [査読有り]

     概要を見る

    Fanconi anemia (FA) is a rare genetic disease characterized by congenital defects, bone marrow failure, chromosomal instability, and cancer susceptibility. One hallmark of cells from FA patients is hypersensitivity to interstrand cross-linking agents, such as the chemotherapeutics cisplatin and mitomycin C (MMC). We have recently characterized a FANCD2/FANCI-associated nuclease, KIAA1018/FAN1, the depletion of which sensitizes human cells to these agents. However, as the down-regulation of FAN1 in human cells was mediated by siRNA and thus only transient, we were unable to study the long-term effects of FAN1 loss on chromosomal stability. We now describe the generation of chicken DT40 B cells, in which the FAN1 locus was disrupted by gene targeting. FAN1-null cells are highly sensitive to cisplatin and MMC, but not to ionizing or UV radiation, methyl methanesulfonate, or camptothecin. The cells do not display elevated sister chromatid exchange frequencies, either sporadic or MMC-induced. Interestingly, MMC treatment causes chromosomal instability that is quantitatively, but not qualitatively, comparable to that seen in FA cells. This finding, coupled with evidence showing that DT40 cells deficient in both FAN1 and FANCC, or FAN1 and FANCJ, exhibited increased sensitivity to cisplatin compared with cells lacking only FAN1, suggests that, despite its association with FANCD2/FANCI, FAN1 in DT40 cells participates in the processing of damage induced by interstrand cross-linking-generating agents also independently of the classical FA pathway.

    DOI J-GLOBAL

  • Single-stranded DNA catenation mediated by human EVL and a type I topoisomerase

    Motoki Takaku, Daisuke Takahashi, Shinichi Machida, Hiroyuki Ueno, Noriko Hosoya, Shukuko Ikawa, Kiyoshi Miyagawa, Takehiko Shibata, Hitoshi Kurumizaka

    NUCLEIC ACIDS RESEARCH   38 ( 21 ) 7579 - 7586  2010年11月  [査読有り]

     概要を見る

    The human Ena/Vasp-like (EVL) protein is considered to be a bifunctional protein, involved in both actin remodeling and homologous recombination. In the present study, we found that human EVL forms heat-stable multimers of circular single-stranded DNA (ssDNA) molecules in the presence of a type I topoisomerase in vitro. An electron microscopic analysis revealed that the heat-stable ssDNA multimers formed by EVL and topoisomerase were ssDNA catemers. The ssDNA catenation did not occur when either EVL or topoisomerase was omitted from the reaction mixture. A deletion analysis revealed that the ssDNA catenation completely depended on the annealing activity of EVL. Human EVL was captured from a human cell extract by TOPO III alpha-conjugated beads, and the interaction between EVL and TOPO III alpha was confirmed by a surface plasmon resonance analysis. Purified TOPO III alpha catalyzed the ssDNA catenation with EVL as efficiently as the Escherichia coli topoisomerase I. Since the ssDNA cutting and rejoining reactions, which are the sub-steps of ssDNA catenation, may be an essential process in homologous recombination, EVL and TOPO III alpha may function in the processing of DNA intermediates formed during homologous recombination.

    DOI J-GLOBAL

  • GEMIN2 promotes accumulation of RAD51 at double-strand breaks in homologous recombination

    Yoshimasa Takizawa, Yong Qing, Motoki Takaku, Takako Ishida, Yuichi Morozumi, Takashi Tsujita, Toshiaki Kogame, Kouji Hirota, Masayuki Takahashi, Takehiko Shibata, Hitoshi Kurumizaka, Shunichi Takeda

    NUCLEIC ACIDS RESEARCH   38 ( 15 ) 5059 - 5074  2010年08月  [査読有り]

     概要を見る

    RAD51 is a key factor in homologous recombination (HR) and plays an essential role in cellular proliferation by repairing DNA damage during replication. The assembly of RAD51 at DNA damage is strictly controlled by RAD51 mediators, including BRCA1 and BRCA2. We found that human RAD51 directly binds GEMIN2/SIP1, a protein involved in spliceosome biogenesis. Biochemical analyses indicated that GEMIN2 enhances the RAD51-DNA complex formation by inhibiting RAD51 dissociation from DNA, and thereby stimulates RAD51-mediated homologous pairing. GEMIN2 also enhanced the RAD51-mediated strand exchange, when RPA was pre-bound to ssDNA before the addition of RAD51. To analyze the function of GEMIN2, we depleted GEMIN2 in the chicken DT40 line and in human cells. The loss of GEMIN2 reduced HR efficiency and resulted in a significant decrease in the number of RAD51 subnuclear foci, as observed in cells deficient in BRCA1 and BRCA2. These observations and our biochemical analyses reveal that GEMIN2 regulates HR as a novel RAD51 mediator.

    DOI J-GLOBAL

  • Deimination stabilizes histone H2A/H2B dimers as revealed by electrospray ionization mass spectrometry

    Shingo Shimoyama, Aritaka Nagadoi, Hiroaki Tachiwana, Michiyuki Yamada, Mamoru Sato, Hitoshi Kurumizaka, Yoshifumi Nishimura, Satoko Akashi

    JOURNAL OF MASS SPECTROMETRY   45 ( 8 ) 900 - 908  2010年08月  [査読有り]

     概要を見る

    Post-translational modifications of histones for reversibly changing chromosomal structures in promoter regions of genes are a prerequisite for transcriptional activation and repression of genes. Peptidylarginine deiminase 4 (PAD4), which mediates histone deimination by converting arginine residues into citrulline residues, is involved in the repression of gene transcription. However, the mechanism is still unclear. We studied the effects of deimination on the reconstituted histone H2A/H2B dimer structure by electrospray ionization mass spectrometry. Deimination of the H2A/H2B dimer by PAD4 indicated that the mass of H2A increased 2.7 Da, suggesting that two or three Arg residues of H2A were deiminated. Deimination of H2A monomer alone showed a 6.6-Da increase in mass. This indicates that about four more Arg residues of H2A are modified in the monomer state than in the H2A/H2B dimer state. Taking account of the finding that the unstructured portions in proteins are susceptible to deimination by PAD4, it is likely that H2A in the monomer state has a more flexible structure than that in the dimer state. Furthermore, analysis of the association of the H2A/H2B dimer in 2 or 4 M ammonium acetate with nano-electrospray ionization mass spectrometry showed that a modified H2A/H2B dimer was less dissociated into H2A and H2B monomers than an unmodified dimer when high voltages were applied to the sample cone. This study provides convincing evidence that PAD4 deimination stabilizes the histone H2A/H2B dimer. Copyright (c) 2010 John Wiley & Sons, Ltd.

    DOI J-GLOBAL

  • 発癌防御に重要な相同組換えの分子機構

    齋藤健吾, 香川亘, 胡桃坂仁志

    最新医学   65   76 - 88  2010年06月  [査読有り]  [招待有り]

  • The putative nuclear localization signal of the human RAD52 protein is a potential sumoylation site

    Kengo Saito, Wataru Kagawa, Takehiro Suzuki, Hidekazu Suzuki, Shigeyuki Yokoyama, Hisato Saitoh, Satoshi Tashiro, Naoshi Dohmae, Hitoshi Kurumizaka

    JOURNAL OF BIOCHEMISTRY   147 ( 6 ) 833 - 842  2010年06月  [査読有り]

     概要を見る

    RAD52, a key factor in homologous recombination (HR), plays important roles in both RAD51-dependent and -independent HR pathways. Several studies have suggested a link between the functional regulation of RAD52 and the protein modification by a small ubiquitin-like modifier (SUMO). However, the molecular mechanism underlying the regulation of RAD52 by SUMO is unknown. To begin investigating this mechanism, we identified possible target sites for sumoylation in the human RAD52 protein by preparing a RAD52-SUMO complex using an established Escherichia coli sumoylation system. Mass spectrometry and amino acid sequencing of the enzymatically digested fragments of the purified complex revealed that the putative nuclear localization signal located near the C terminus of RAD52 was sumoylated. Biochemical studies of the RAD52-SUMO complex suggested that sumoylation at the identified site has no apparent effect on the DNA binding, D-loop formation, ssDNA annealing and RAD51-binding activities of RAD52. On the other hand, visualization of the GFP-fused RAD52 protein in the human cell that contained mutations at the identified sumoylation sites showed clear differences in the cytosolic and nuclear distributions of the protein. These results suggest the possibility of sumoylation playing an important role in the nuclear transport of RAD52.

    DOI J-GLOBAL

  • Structural basis of instability of the nucleosome containing a testis-specific histone variant, human H3T

    Hiroaki Tachiwana, Wataru Kagawa, Akihisa Osakabe, Koichiro Kawaguchi, Tatsuya Shiga, Yoko Hayashi-Takanaka, Hiroshi Kimura, Hitoshi Kurumizaka

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   107 ( 23 ) 10454 - 10459  2010年06月  [査読有り]

     概要を見る

    A histone H3 variant, H3T, is highly expressed in the testis, suggesting that it may play an important role in the chromatin reorganization required for meiosis and/or spermatogenesis. In the present study, we found that the nucleosome containing human H3T is significantly unstable both in vitro and in vivo, as compared to the conventional nucleosome containing H3.1. The crystal structure of the H3T nucleosome revealed structural differences in the H3T regions on both ends of the central alpha 2 helix, as compared to those of H3.1. The H3T-specific residues (Met71 and Val111) are the source of the structural differences observed between H3T and H3.1. A mutational analysis revealed that these residues are responsible for the reduced stability of the H3T-containing nucleosome. These physical and structural properties of the H3T-containing nucleosome may provide the basis of chromatin reorganization during spermatogenesis.

    DOI J-GLOBAL

  • Nucleosome Formation Activity of Human Somatic Nuclear Autoantigenic Sperm Protein (sNASP)

    Akihisa Osakabe, Hiroaki Tachiwana, Takaaki Matsunaga, Tatsuya Shiga, Ryu-Suke Nozawa, Chikashi Obuse, Hitoshi Kurumizaka

    JOURNAL OF BIOLOGICAL CHEMISTRY   285 ( 16 ) 11913 - 11921  2010年04月

     概要を見る

    NASP (nuclear autoantigenic sperm protein) is a member of the N1/N2 family, which is widely conserved among eukaryotes. Human NASP reportedly prefers to bind to histones H3.H4 and the linker histone H1, as compared with H2A.H2B, and is anticipated to function as an H3.H4 chaperone for nucleosome assembly. However, the direct nucleosome assembly activity of human NASP has not been reported so far. In humans, two spliced isoforms, somatic and testicular NASPs (sNASP and tNASP, respectively) were identified. In the present study we purified human sNASP and found that sNASP efficiently promoted the assembly of nucleosomes containing the conventional H3.1, H3.2, H3.3, or centromere-specific CENP-A. On the other hand, sNASP inefficiently promoted nucleosome assembly with H3T, a testis-specific H3 variant. Mutational analyses revealed that the Met-71 residue of H3T is responsible for this inefficient nucleosome formation by sNASP. Tetrasomes, composed of the H3.H4 tetramer and DNA without H2A.H2B, were efficiently formed by the sNASP-mediated nucleosome-assembly reaction. A deletion analysis of sNASP revealed that the central region, amino acid residues 26-325, of sNASP is responsible for nucleosome assembly in vitro. These experiments are the first demonstration that human NASP directly promotes nucleosome assembly and provide compelling evidence that sNASP is a bona fide histone chaperone for H3.H4.

    DOI

  • Nucleosome Formation Activity of Human Somatic Nuclear Autoantigenic Sperm Protein (sNASP)

    Akihisa Osakabe, Hiroaki Tachiwana, Takaaki Matsunaga, Tatsuya Shiga, Ryu-Suke Nozawa, Chikashi Obuse, Hitoshi Kurumizaka

    JOURNAL OF BIOLOGICAL CHEMISTRY   285 ( 16 ) 11913 - 11921  2010年04月  [査読有り]

     概要を見る

    NASP (nuclear autoantigenic sperm protein) is a member of the N1/N2 family, which is widely conserved among eukaryotes. Human NASP reportedly prefers to bind to histones H3.H4 and the linker histone H1, as compared with H2A.H2B, and is anticipated to function as an H3.H4 chaperone for nucleosome assembly. However, the direct nucleosome assembly activity of human NASP has not been reported so far. In humans, two spliced isoforms, somatic and testicular NASPs (sNASP and tNASP, respectively) were identified. In the present study we purified human sNASP and found that sNASP efficiently promoted the assembly of nucleosomes containing the conventional H3.1, H3.2, H3.3, or centromere-specific CENP-A. On the other hand, sNASP inefficiently promoted nucleosome assembly with H3T, a testis-specific H3 variant. Mutational analyses revealed that the Met-71 residue of H3T is responsible for this inefficient nucleosome formation by sNASP. Tetrasomes, composed of the H3.H4 tetramer and DNA without H2A.H2B, were efficiently formed by the sNASP-mediated nucleosome-assembly reaction. A deletion analysis of sNASP revealed that the central region, amino acid residues 26-325, of sNASP is responsible for nucleosome assembly in vitro. These experiments are the first demonstration that human NASP directly promotes nucleosome assembly and provide compelling evidence that sNASP is a bona fide histone chaperone for H3.H4.

    DOI

  • Holliday junction-binding activity of human SPF45

    Naoki Horikoshi, Yuichi Morozumi, Motoki Takaku, Yoshimasa Takizawa, Hitoshi Kurumizaka

    GENES TO CELLS   15 ( 4 ) 373 - 383  2010年04月  [査読有り]

     概要を見る

    SPF45 is considered to be a bifunctional protein that functions in splicing and DNA repair. A previous genetic study reported that Drosophila SPF45 participates in the DNA-repair pathway with a RAD51-family protein, RAD201, suggesting that SPF45 may function in DNA repair by the homologous-recombination pathway. To study the function of SPF45 in homologous recombination, we purified human SPF45 and found that it preferentially binds to the Holliday junction, which is a key DNA intermediate in the homologous-recombination pathway. Deletion analyses revealed that the RNA recognition motif, which is located in the C-terminal region of human SPF45, is not involved in DNA binding. On the other hand, alanine-scanning mutagenesis identified the N-terminal lysine residues, which may be involved in Holliday junction binding by human SPF45. We also found that human SPF45 significantly binds to a RAD51 paralog, RAD51B, although it also binds to RAD51 and DMC1 with lower affinity. These biochemical results support the idea that human SPF45 functions in DNA repair by homologous recombination.

    DOI J-GLOBAL

  • From meiosis to postmeiotic events: Uncovering the molecular roles of the meiosis-specific recombinase Dmc1

    Wataru Kagawa, Hitoshi Kurumizaka

    FEBS JOURNAL   277 ( 3 ) 590 - 598  2010年02月  [査読有り]

     概要を見る

    In meiosis, the accurate segregation of maternal and paternal chromosomes is accomplished by homologous recombination. A central player in meiotic recombination is the Dmc1 recombinase, a member of the RecA/Rad51 recombinase superfamily, which is widely conserved from viruses to humans. Dmc1 is a meiosis-specific protein that functions with the ubiquitously expressed homolog, the Rad51 recombinase, which is essential for both mitotic and meiotic recombination. Since its discovery, it has been speculated that Dmc1 is important for unique aspects of meiotic recombination. Understanding the distinctive properties of Dmc1, namely, the features that distinguish it from Rad51, will further clarify the mechanisms of meiotic recombination. Recent structural, biochemical, and genetic findings are now revealing the molecular mechanisms of Dmc1-mediated homologous recombination and its regulation by various recombination mediators.

    DOI J-GLOBAL

  • Dynamics in the transmission of genetic information: from meiosis to postmeiotic events

    Wataru Kagawa, Hitoshi Kurumizaka

    FEBS JOURNAL   277 ( 3 ) 564 - 564  2010年02月  [査読有り]

    DOI J-GLOBAL

  • Enhancing Survival of Mouse Oocytes Following Chemotherapy or Aging by Targeting Bax and Rad51

    Loro L. Kujjo, Tiina Laine, Ricardo J. G. Pereira, Wataru Kagawa, Hitoshi Kurumizaka, Shigeyuki Yokoyama, Gloria I. Perez

    PLOS ONE   5 ( 2 ) e9204  2010年02月  [査読有り]

     概要を見る

    Background: Therapeutic approaches to preserve fertility in females undergoing cancer treatments are currently ineffective. This is partly due to limited knowledge of the molecular mechanisms that injured germ cells elicit to repair damage and survive or to abort repair and activate biochemical pathways leading to death. So far, we know that following spontaneously occurring or drug-induced DNA damage, the efficiency of DNA repair is a critical determinant of the cell&apos;s fate. The protein encoded by the Rad51 gene is one of several components recruited for homologous recombination-dependent DNA double-strand break repair in both somatic cells and germ cells. Recently, we showed that microinjection of recombinant Rad51 into AKR/J mouse oocytes decreased the extent of spontaneous DNA double-strand breaks, suppressed apoptosis, and restored the developmental competence in AKR/J embryos. Herein we characterized the nature of chemotherapy-induced lesions in oocytes, and the associated individual components of the DNA damage sensor and repair apparatus. For comparison, we also assessed parallel spontaneous changes in aging oocytes.
    Methods: Data collected were derived from: analysis of apoptosis; immunodepletion; oocyte microinjections; immunocytochemistry; immunofluorescence; and CHIP-like assays.
    Results: Our data show that: (i) DNA damage in oocytes can be induced by both chemotherapy and spontaneously by the aging process; (ii) oocytes possess the machinery and capability for repairing such DNA damage; (iii) Rad51 is a critical player in the repair of both chemotherapy-induced and spontaneously-sustained DNA damage; and (iv) in response to damage, oocytes exhibit an inverse functional relationship between presence of Bax and activity of Rad51.
    Conclusion/Significance: Our results establish Rad51 and/or Bax as potential candidates that can be targeted for development of individualized chemotherapeutic interventions that are effective, but minimal in toxicity. The use of Rad51 and Bax modulating compounds could offer women the opportunity to maintain fully functional germ cells despite cancer treatments or aging.

    DOI J-GLOBAL

  • Erratum: Deimination stabilizes histone H2A/H2B dimers as revealed by electrospray ionizationmass spectrometry (Journal of Mass Spectrometry (2010) 45:8 (900-908) DOI: 10.1002/jms.1778)

    Shimoyama S, Nagadoi A, Tachiwan H, Yamad M, Sato M, Kurumizaka H, Nishimura Y, Akashi S

    Journal of Mass Spectrometry   45 ( 10 )  2010年  [査読有り]

    DOI

  • Biochemical analysis of the human EVL domains in homologous recombination

    Motoki Takaku, Shinichi Machida, Shugo Nakayama, Daisuke Takahashi, Hitoshi Kurumizaka

    FEBS JOURNAL   276 ( 20 ) 5841 - 5848  2009年10月  [査読有り]

     概要を見る

    EVL is a member of the ENA/VASP family, which is involved in actin-remodeling processes. Previously, we reported that human EVL directly interacts with RAD51, which is an essential protein in the homologous recombinational repair of DNA double-strand breaks, and stimulates RAD51-mediated recombination reactions in vitro. To identify the EVL domain required for the recombination function, we purified the EVL fragments EVL(1-221) and EVL(222-418), which contain the EVH1 and Pro-rich domains and the EVH2 domain, respectively. We found that EVL(222-418) possesses DNA-binding and RAD51-binding activities, and also stimulates RAD51-mediated homologous pairing. In contrast, EVL(1-221) did not exhibit any of these activities. Therefore, the EVH2 domain, which is highly conserved among the ENA/VASP family proteins, may be responsible for the recombination function of EVL.

    DOI J-GLOBAL

  • The ATR-Chk1 pathway plays a role in the generation of centrosome aberrations induced by Rad51C dysfunction

    Mari Katsura, Takanori Tsuruga, Osamu Date, Takashi Yoshihara, Mari Ishida, Yoshitaka Tomoda, Miyuki Okajima, Motoki Takaku, Hitoshi Kurumizaka, Aiko Kinomura, Hiromu K. Mishima, Kiyoshi Miyagawa

    NUCLEIC ACIDS RESEARCH   37 ( 12 ) 3959 - 3968  2009年07月  [査読有り]

     概要を見る

    Rad51C is a central component of two complexes formed by five Rad51 paralogs in vertebrates. These complexes are involved in repairing DNA double-strand breaks through homologous recombination. Despite accumulating evidence suggesting that the paralogs may prevent aneuploidy by controlling centrosome integrity, Rad51C&apos;s role in maintaining chromosome stability remains unclear. Here we demonstrate that Rad51C deficiency leads to both centrosome aberrations in an ATR-Chk1-dependent manner and increased aneuploidy in human cells. While it was reported that Rad51C deficiency did not cause centrosome aberrations in interphase in hamster cells, such aberrations were observed in interphase in HCT116 cells with Rad51C dysfunction. Caffeine treatment and down-regulation of ATR, but not that of ATM, reduced the frequency of centrosome aberrations in the mutant cells. Silencing of Rad51C by RNA interference in HT1080 cells resulted in similar aberrations. Treatment with a Chk1 inhibitor and silencing of Chk1 also reduced the frequency in HCT116 mutants. Accumulation of Chk1 at the centrosome and nuclear foci of gamma H2AX were increased in the mutants. Moreover, the mutant cells had a higher frequency of aneuploidy. These findings indicate that the ATR-Chk1 pathway plays a role in increased centrosome aberrations induced by Rad51C dysfunction.

    DOI J-GLOBAL

  • Human PSF binds to RAD51 and modulates its homologous-pairing and strand-exchange activities

    Yuichi Morozumi, Yoshimasa Takizawa, Motoki Takaku, Hitoshi Kurumizaka

    NUCLEIC ACIDS RESEARCH   37 ( 13 ) 4296 - 4307  2009年07月  [査読有り]

     概要を見る

    RAD51, a eukaryotic recombinase, catalyzes homologous-pairing and strand-exchange reactions, which are essential steps in homologous recombination and recombinational repair of double strand breaks. On the other hand, human PSF was originally identified as a component of spliceosomes, and its multiple functions in RNA processing, transcription and DNA recombination were subsequently revealed. In the present study, we found that PSF directly interacted with RAD51. PSF significantly enhanced RAD51-mediated homologous pairing and strand exchange at low RAD51 concentrations; however, in contrast, it inhibited these RAD51-mediated recombination reactions at the optimal RAD51 concentration. Deletion analyses revealed that the N-terminal region of PSF possessed the RAD51- and DNA-binding activities, but the central region containing the RNA-recognition motifs bound neither RAD51 nor DNA. These results suggest that PSF may have dual functions in homologous recombination and RNA processing through its N-terminal and central regions, respectively.

    DOI J-GLOBAL

  • DIDS, a chemical compound that inhibits RAD51-mediated homologous pairing and strand exchange

    Takako Ishida, Yoshimasa Takizawa, Takashi Kainuma, Jin Inoue, Tsutomu Mikawa, Takehiko Shibata, Hidekazu Suzuki, Satoshi Tashiro, Hitoshi Kurumizaka

    NUCLEIC ACIDS RESEARCH   37 ( 10 ) 3367 - 3376  2009年06月  [査読有り]

     概要を見る

    RAD51, an essential eukaryotic DNA recombinase, promotes homologous pairing and strand exchange during homologous recombination and the recombinational repair of double strand breaks. Mutations that up- or down-regulate RAD51 gene expression have been identified in several tumors, suggesting that inappropriate expression of the RAD51 activity may cause tumorigenesis. To identify chemical compounds that affect the RAD51 activity, in the present study, we performed the RAD51-mediated strand exchange assay in the presence of 185 chemical compounds. We found that 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS) efficiently inhibited the RAD51-mediated strand exchange. DIDS also inhibited the RAD51-mediated homologous pairing in the absence of RPA. A surface plasmon resonance analysis revealed that DIDS directly binds to RAD51. A gel mobility shift assay showed that DIDS significantly inhibited the DNA-binding activity of RAD51. Therefore, DIDS may bind near the DNA binding site(s) of RAD51 and compete with DNA for RAD51 binding.

    DOI J-GLOBAL

  • c-ABL tyrosine kinase stabilizes RAD51 chromatin association

    Hiroko Shimizu, Milena Popova, Fabrice Fleury, Masahiko Kobayashi, Naoyuki Hayashi, Isao Sakane, Hitoshi Kurumizaka, Ashok R. Venkitaraman, Masayuki Takahashi, Ken-ichi Yamamoto

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   382 ( 2 ) 286 - 291  2009年05月  [査読有り]

     概要を見る

    The assembly of RAD51 recombinase on DNA substrates at sites of breakage is essential for their repair by homologous recombination repair (HRR). The signaling pathway that triggers RAD51 assembly at damage sites to form subnuclear foci is unclear. Here, we provide evidence that c-ABL, a tyrosine kinase activated by DNA damage which phosphorylates RAD51 on Tyr-315, works at a previously unrecognized, proximal step to initiate RAD51 assembly. We first show that c-ABL associates with chromatin after DNA damage in a manner dependent on its kinase activity. Using RAD51 mutants that are unable to oligomerize to form a nucleoprotein filament, we separate RAD51 assembly on DNA to form foci into two steps: stable chromatin association followed by oligomerization. We show that phosphorylation on Tyr-315 by c-ABL is required for chromatin association of oligomerization-defective RAD51 mutants, but is insufficient to restore oligomerization. Our findings suggest a new model for the regulation of early steps of HRR. (C) 2009 Elsevier Inc. All rights reserved.

    DOI J-GLOBAL

  • Recombination Activator Function of the Novel RAD51-and RAD51B-binding Protein, Human EVL

    Motoki Takaku, Shinichi Machida, Noriko Hosoya, Shugo Nakayama, Yoshimasa Takizawa, Isao Sakane, Takehiko Shibata, Kiyoshi Miyagawa, Hitoshi Kurumizaka

    JOURNAL OF BIOLOGICAL CHEMISTRY   284 ( 21 ) 14326 - 14336  2009年05月  [査読有り]

     概要を見る

    The RAD51 protein is a central player in homologous recombinational repair. The RAD51B protein is one of five RAD51 paralogs that function in the homologous recombinational repair pathway in higher eukaryotes. In the present study, we found that the human EVL (Ena/Vasp-like) protein, which is suggested to be involved in actin-remodeling processes, unexpectedly binds to the RAD51 and RAD51B proteins and stimulates the RAD51-mediated homologous pairing and strand exchange. The EVL knockdown cells impaired RAD51 assembly onto damaged DNA after ionizing radiation or mitomycin C treatment. The EVL protein alone promotes single-stranded DNA annealing, and the recombination activities of the EVL protein are further enhanced by the RAD51B protein. The expression of the EVL protein is not ubiquitous, but it is significantly expressed in breast cancer-derived MCF7 cells. These results suggest that the EVL protein is a novel recombination factor that may be required for repairing specific DNA lesions, and that may cause tumor malignancy by its inappropriate expression.

    DOI J-GLOBAL

  • Biochemical analysis of the human DMC1-I37N polymorphism

    Juri Hikiba, Yoshimasa Takizawa, Shukuko Ikawa, Takehiko Shibata, Hitoshi Kurumizaka

    FEBS JOURNAL   276 ( 2 ) 457 - 465  2009年01月  [査読有り]

     概要を見る

    The DMC1 protein, a meiosis-specific DNA recombinase, promotes homologous pairing and strand exchange. The I37N single nucleotide polymorphism of the human DMC1 protein was reported as a result of human genome sequencing projects. In this study, we purified the human DMC1-I37N variant, as a recombinant protein. The DMC1 protein is known to require DNA for efficient ATP hydrolysis. By contrast, the DMC1-I37N variant efficiently hydrolyzed ATP in the absence of DNA. Like the conventional DMC1 protein, the DMC1-I37N variant promoted strand exchange, but it required a high Ca(2+) concentration (4-8 mm), a condition that inactivates the strand-exchange activity of the conventional DMC1 protein. These biochemical differences between the DMC1 and DMC1-I37N proteins suggest that the DMC1-I37N polymorphism may be a source of improper meiotic recombination, causing meiotic defects in humans.

    DOI J-GLOBAL

  • Structural Analysis of the Human Rad51 Protein-DNA Complex Filament by Tryptophan Fluorescence Scanning Analysis: Transmission of Allosteric Effects between ATP Binding and DNA Binding

    Axelle Renodon-Corniere, Yoshimasa Takizawa, Sebastien Conilleau, Vinh Tran, Shigenori Iwai, Hitoshi Kurumizaka, Masayuki Takahashi

    JOURNAL OF MOLECULAR BIOLOGY   383 ( 3 ) 575 - 587  2008年11月

     概要を見る

    Human Rad51 (HsRad51) catalyzes the strand exchange reaction, a crucial step in homologous recombination, by forming a filamentous complex with DNA. The structure of this filament is modified by ATP, which is required and hydrolyzed for the reaction. We analyzed the structure and the ATP-promoted conformational change of this filament. We systematically replaced aromatic residues in the protein, one at a time, with tryptophan, a fluorescent probe, and examined its effect on the activities (DNA binding, ATPase, ATP-promoted conformational change, and strand exchange reaction) and the fluorescence changes upon binding of ATP and DNA. Some residues were also replaced with alanine. We thus obtained structural information about various positions of the protein in solution. All the proteins conserved, at least partially, their activities. However, the replacement of histidine at position 294 (H294) and phenylalanine at 129 (F129) affected the ATP-induced conformational change of the DNA-HsRad51 filament, although it did not prevent DNA binding. F129 is considered to be close to the ATP-binding site and to H294 of a neighboring subunit. ATP probably modifies the structure around F129 and affects the subunit/subunit contact around H294 and the structure of the DNA-binding site. The replacement also reduced the DNA-dependent ATPase activity, suggesting that these residues are also involved in the transmission of the allosteric effect of DNA to the ATP-binding site, which is required for the stimulation of ATPase activity by DNA. The fluorescence analyses supported the structural change of the DNA-binding site by ATP and that of the ATP-binding site by DNA. This information will be useful to build a molecular model of the Rad51-DNA complex and to understand the mechanism of activation of Rad51 by ATP and that of the Rad51-promoted strand exchange reaction. (C) 2008 Elsevier Ltd. All rights reserved.

    DOI

  • Structural Analysis of the Human Rad51 Protein-DNA Complex Filament by Tryptophan Fluorescence Scanning Analysis: Transmission of Allosteric Effects between ATP Binding and DNA Binding

    Axelle Renodon-Corniere, Yoshimasa Takizawa, Sebastien Conilleau, Vinh Tran, Shigenori Iwai, Hitoshi Kurumizaka, Masayuki Takahashi

    JOURNAL OF MOLECULAR BIOLOGY   383 ( 3 ) 575 - 587  2008年11月  [査読有り]

     概要を見る

    Human Rad51 (HsRad51) catalyzes the strand exchange reaction, a crucial step in homologous recombination, by forming a filamentous complex with DNA. The structure of this filament is modified by ATP, which is required and hydrolyzed for the reaction. We analyzed the structure and the ATP-promoted conformational change of this filament. We systematically replaced aromatic residues in the protein, one at a time, with tryptophan, a fluorescent probe, and examined its effect on the activities (DNA binding, ATPase, ATP-promoted conformational change, and strand exchange reaction) and the fluorescence changes upon binding of ATP and DNA. Some residues were also replaced with alanine. We thus obtained structural information about various positions of the protein in solution. All the proteins conserved, at least partially, their activities. However, the replacement of histidine at position 294 (H294) and phenylalanine at 129 (F129) affected the ATP-induced conformational change of the DNA-HsRad51 filament, although it did not prevent DNA binding. F129 is considered to be close to the ATP-binding site and to H294 of a neighboring subunit. ATP probably modifies the structure around F129 and affects the subunit/subunit contact around H294 and the structure of the DNA-binding site. The replacement also reduced the DNA-dependent ATPase activity, suggesting that these residues are also involved in the transmission of the allosteric effect of DNA to the ATP-binding site, which is required for the stimulation of ATPase activity by DNA. The fluorescence analyses supported the structural change of the DNA-binding site by ATP and that of the ATP-binding site by DNA. This information will be useful to build a molecular model of the Rad51-DNA complex and to understand the mechanism of activation of Rad51 by ATP and that of the Rad51-promoted strand exchange reaction. (C) 2008 Elsevier Ltd. All rights reserved.

    DOI

  • Biochemical analysis of the N-terminal domain of human RAD54B

    Naoyuki Sarai, Wataru Kagawa, Norie Fujikawa, Kengo Saito, Juri Hikiba, Kozo Tanaka, Kiyoshi Miyagawa, Hitoshi Kurumizaka, Shigeyuki Yokoyama

    NUCLEIC ACIDS RESEARCH   36 ( 17 ) 5441 - 5450  2008年10月  [査読有り]

     概要を見る

    The human RAD54B protein is a paralog of the RAD54 protein, which plays important roles in homologous recombination. RAD54B contains an N-terminal region outside the SWI2/SNF2 domain that shares less conservation with the corresponding region in RAD54. The biochemical roles of this region of RAD54B are not known, although the corresponding region in RAD54 is known to physically interact with RAD51. In the present study, we have biochemically characterized an N-terminal fragment of RAD54B, consisting of amino acid residues 26-225 (RAD54B(26-225)). This fragment formed a stable dimer in solution and bound to branched DNA structures. RAD54B(26-225) also interacted with DMC1 in both the presence and absence of DNA. Ten DMC1 segments spanning the entire region of the DMC1 sequence were prepared, and two segments, containing amino acid residues 153-214 and 296-340, were found to directly bind to the N-terminal domain of RAD54B. A structural alignment of DMC1 with the Methanococcus voltae RadA protein, a homolog of DMC1 in the helical filament form, indicated that these RAD54B-binding sites are located near the ATP-binding site at the monomer-monomer interface in the DMC1 helical filament. Thus, RAD54B binding may affect the quaternary structure of DMC1. These observations suggest that the N-terminal domain of RAD54B plays multiple roles of in homologous recombination.

    DOI J-GLOBAL

  • Filament formation and robust strand exchange activities of the rice DMC1A and DMC1B proteins

    Isao Sakane, Chiaki Kamataki, Yoshimasa Takizawa, Marina Nakashima, Seiichi Toki, Hiroaki Ichikawa, Shukuko Ikawa, Takehiko Shibata, Hitoshi Kurumizaka

    NUCLEIC ACIDS RESEARCH   36 ( 13 ) 4266 - 4276  2008年08月

     概要を見る

    The DMC1 protein, a meiosis-specific DNA recombinase, catalyzes strand exchange between homologous chromosomes. In rice, two Dmc1 genes, Dmc1A and Dmc1B, have been reported. Although the Oryza sativa DMC1A protein has been partially characterized, however the biochemical properties of the DMC1B protein have not been defined. In the present study, we expressed the Oryza sativa DMC1A and DMC1B proteins in bacteria and purified them. The purified DMC1A and DMC1B proteins formed helical filaments along single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), and promoted robust strand exchange between ssDNA and dsDNA over five thousand base pairs in the presence of RPA, as a co-factor. The DMC1A and DMC1B proteins also promoted strand exchange in the absence of RPA with long DNA substrates containing several thousand base pairs. In contrast, the human DMC1 protein strictly required RPA to promote strand exchange with these long DNA substrates. The strand-exchange activity of the Oryza sativa DMC1A protein was much higher than that of the DMC1B protein. Consistently, the DNA-binding activity of the DMC1A protein was higher than that of the DMC1B protein. These biochemical differences between the DMC1A and DMC1B proteins may provide important insight into their functional differences during meiosis in rice.

    DOI

  • Filament formation and robust strand exchange activities of the rice DMC1A and DMC1B proteins

    Isao Sakane, Chiaki Kamataki, Yoshimasa Takizawa, Marina Nakashima, Seiichi Toki, Hiroaki Ichikawa, Shukuko Ikawa, Takehiko Shibata, Hitoshi Kurumizaka

    NUCLEIC ACIDS RESEARCH   36 ( 13 ) 4266 - 4276  2008年08月  [査読有り]

     概要を見る

    The DMC1 protein, a meiosis-specific DNA recombinase, catalyzes strand exchange between homologous chromosomes. In rice, two Dmc1 genes, Dmc1A and Dmc1B, have been reported. Although the Oryza sativa DMC1A protein has been partially characterized, however the biochemical properties of the DMC1B protein have not been defined. In the present study, we expressed the Oryza sativa DMC1A and DMC1B proteins in bacteria and purified them. The purified DMC1A and DMC1B proteins formed helical filaments along single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), and promoted robust strand exchange between ssDNA and dsDNA over five thousand base pairs in the presence of RPA, as a co-factor. The DMC1A and DMC1B proteins also promoted strand exchange in the absence of RPA with long DNA substrates containing several thousand base pairs. In contrast, the human DMC1 protein strictly required RPA to promote strand exchange with these long DNA substrates. The strand-exchange activity of the Oryza sativa DMC1A protein was much higher than that of the DMC1B protein. Consistently, the DNA-binding activity of the DMC1A protein was higher than that of the DMC1B protein. These biochemical differences between the DMC1A and DMC1B proteins may provide important insight into their functional differences during meiosis in rice.

    DOI

  • Identification of a second DNA binding site in the human Rad52 protein

    Wataru Kagawa, Ako Kagawa, Kengo Saito, Shukuko Ikawa, Takehiko Shibata, Hitoshi Kurumizaka, Shigeyuki Yokoyama

    JOURNAL OF BIOLOGICAL CHEMISTRY   283 ( 35 ) 24264 - 24273  2008年08月  [査読有り]

     概要を見る

    Rad52 plays essential roles in homology-dependent double-strand break repair. Various studies have established the functions of Rad52 in Rad51-dependent and Rad51-independent repair processes. However, the precise molecular mechanisms of Rad52 in these processes remain unknown. In the present study we have identified a novel DNA binding site within Rad52 by a structure-based alanine scan mutagenesis. This site is closely aligned with the putative single-stranded DNA binding site determined previously. Mutations in this site impaired the ability of the Rad52-single-stranded DNA complex to form a ternary complex with double-stranded DNA and subsequently catalyze the formation of D-loops. We found that Rad52 introduces positive supercoils into double-stranded DNA and that the second DNA binding site is essential for this activity. Our findings suggest that Rad52 aligns two recombining DNA molecules within the first and second DNA binding sites to stimulate the homology search and strand invasion processes.

    DOI J-GLOBAL

  • Structural and functional analyses of the DMC1-M200V polymorphism found in the human population

    Juri Hikiba, Kouji Hirota, Wataru Kagawa, Shukuko Ikawa, Takashi Kinebuchi, Isao Sakane, Yoshimasa Takizawa, Shigeyuki Yokoyama, Beatrice Mandon-Pepin, Alain Nicolas, Takehiko Shibata, Kunihiro Ohta, Hitoshi Kurumizaka

    NUCLEIC ACIDS RESEARCH   36 ( 12 ) 4181 - 4190  2008年07月  [査読有り]

     概要を見る

    The M200V polymorphism of the human DMC1 protein, which is an essential, meiosis-specific DNA recombinase, was found in an infertile patient, raising the question of whether this homozygous human DMC1-M200V polymorphism may cause infertility by affecting the function of the human DMC1 protein. In the present study, we determined the crystal structure of the human DMC1-M200V variant in the octameric-ring form. Biochemical analyses revealed that the human DMC1-M200V variant had reduced stability, and was moderately defective in catalyzing in vitro recombination reactions. The corresponding M194V mutation introduced in the Schizosaccharomyces pombe dmc1 gene caused a significant decrease in the meiotic homologous recombination frequency. Together, these structural, biochemical and genetic results provide extensive evidence that the human DMC1-M200V mutation impairs its function, supporting the previous interpretation that this single-nucleotide polymorphism is a source of human infertility.

    DOI J-GLOBAL

  • 構造から理解する相同DNA組換え機構

    胡桃坂仁志, 美川 努, 引場樹里, 柴田武彦

    生化学   80 ( 6 ) 511 - 520  2008年06月

  • Inhibition of filament formation of human Rad51 protein by a small peptide derived from the BRC-motif of the BRCA2 protein

    Julian Nomme, Yoshimasa Takizawa, Susan F. Martinez, Axelle Renodon-Corniere, Fabrice Fleury, Pierre Weigel, Ken-ichi Yamamoto, Hitoshi Kurumizaka, Masayuki Takahashi

    GENES TO CELLS   13 ( 5 ) 471 - 481  2008年05月  [査読有り]

     概要を見る

    Human Rad51 is a key element of recombinational DNA repair and is related to the resistance of cancer cells to chemo- and radiotherapies. The protein is thus a potential target of anti-cancer treatment. The crystallographic analysis shows that the BRC-motif of the BRCA2 tumor suppressor is in contact with the subunit-subunit interface of Rad51 and could thus prevent filament formation of Rad51. However, biochemical analysis indicates that a BRC-motif peptide of 69 amino acids preferentially binds to the N-terminal part of Rad51. We show experimentally that a short peptide of 28 amino acids derived from the BRC4 motif binds to the subunit-subunit interface and dissociates its filament, both in the presence and absence of DNA, certainly by binding to dissociated monomers. The inhibition is efficient and specific for Rad51: the peptide does not even interact with Rad51 homologs or prevent their interaction with DNA. Neither the N-terminal nor the C-terminal half of the peptide interacts with human Rad51, indicating that both parts are involved in the interaction, as expected from the crystal structure. These results suggest the possibility of developing inhibitors of human Rad51 based on this peptide.

    DOI J-GLOBAL

  • Nucleosome formation with the testis-specific histone H3 variant, H3t, by human nucleosome assembly proteins in vitro

    Hiroaki Tachiwana, Akihisa Osakabe, Hiroshi Kimura, Hitoshi Kurumizaka

    NUCLEIC ACIDS RESEARCH   36 ( 7 ) 2208 - 2218  2008年04月  [査読有り]

     概要を見る

    Five non-allelic histone H3 variants, H3.1, H3.2, H3.3, H3t and CENP-A, have been identified in mammals. H3t is robustly expressed in the testis, and thus was assigned as the testis-specific H3 variant. However, recent proteomics and tissue-specific RT-PCR experiments revealed a small amount of H3t expression in somatic cells. In the present study, we purified human H3t as a recombinant protein, and showed that H3t/H4 forms nucleosomes with H2A/H2B by the salt-dialysis method, like the conventional H3.1/H4. We found that H3t/H4 is not efficiently incorporated into the nucleosome by human Nap1 (hNap1), due to its defective H3t/H4 deposition on DNA. In contrast, human Nap2 (hNap2), a paralog of hNap1, promotes nucleosome assembly with H3t/H4. Mutational analyses revealed that the Ala111 residue, which is conserved among H3.1, H3.2 and H3.3, but not in H3t, is the essential residue for the hNap1-mediated nucleosome assembly. These results suggest that H3t may be incorporated into chromatin by a specific chaperone-mediated pathway.

    DOI J-GLOBAL

  • A common mechanism for the ATP-DnaA-dependent formation of open complexes at the replication origin

    Shogo Ozaki, Hironori Kawakami, Kenta Nakamura, Norie Fujikawa, Wataru Kagawa, Sam-Yong Park, Shigeyuki Yokoyama, Hitoshi Kurumizaka, Tsutomu Katayama

    JOURNAL OF BIOLOGICAL CHEMISTRY   283 ( 13 ) 8351 - 8362  2008年03月  [査読有り]

     概要を見る

    Initiation of chromosomal replication and its cell cycle-coordinated regulation bear crucial and fundamental mechanisms in most cellular organisms. Escherichia coli DnaA protein forms a homomultimeric complex with the replication origin (oriC). ATP-DnaA multimers unwind the duplex within the oriC unwinding element (DUE). In this study, structural analyses suggested that several residues exposed in the central pore of the putative structure of DnaA multimers could be important for unwinding. Using mutation analyses, we found that, of these candidate residues, DnaA Val-211 and Arg-245 are prerequisites for initiation in vivo and in vitro. Whereas DnaA V211A and R245A proteins retained normal affinities for ATP/ADP and DNA and activity for the ATP-specific conformational change of the initiation complex in vitro, oriC complexes of these mutant proteins were inactive in DUE unwinding and in binding to the single-stranded DUE. Unlike oriC complexes including ADP-DnaA or the mutant DnaA, ATP-DnaA-oriC complexes specifically bound the upper strand of single-stranded DUE. Specific T-rich sequences within the strand were required for binding. The corresponding conserved residues of the DnaA ortholog in Thermotoga maritima, an ancient eubacterium, were also required for DUE unwinding, consistent with the idea that the mechanism and regulation for DUE unwinding can be evolutionarily conserved. These findings provide novel insights into mechanisms for pore-mediated origin unwinding, ATP/ADP-dependent regulation, and helicase loading of the initiation complex.

    DOI J-GLOBAL

  • In vivo and in vitro footprinting of nucleosomes and transcriptional activators using an infrared-fluorescence DNA sequencer

    Nobuyuki Morohashi, Kumiko Nakajima, Shunsuke Kuwana, Hiroaki Tachiwana, Hitoshi Kurumizaka, Mitsuhiro Shimizu

    BIOLOGICAL & PHARMACEUTICAL BULLETIN   31 ( 2 ) 187 - 192  2008年02月  [査読有り]

     概要を見る

    The analysis of nucleosome positions and transcription factor binding in chromatin is a central issue for understanding the mechanisms of gene expression in eukaryotes. Here, we have developed a footprinting technique, using multi-cycle primer extension with an infrared-fluorescence DNA sequencer, to analyze chromatin structure in isolated yeast nuclei and transcriptional activator binding in living yeast cells. Using this technique, the binding of the yeast activators Hap1 and Hap2/3/4/5 to their cognate sites was detectable as hypersensitive sites by in vivo UV-photofootprinting, and the locations of nucleosomes in yeast minichromosomes were determined by micrococcal nuclease mapping. We also applied this method to determine the position of the nucleosome in the 5S DNA fragment reconstituted in vitro. This technique allowed us to eliminate the use of radioactive materials and to perform experiments on common benches. Thus, the footprinting procedure established in this study will be useful to researchers studying DNA-protein interactions and chromatin structure in vivo and in vitro.

    DOI J-GLOBAL

  • The actin-related protein hArp8 accumulates on the mitotic chromosomes and functions in chromosome alignment

    Naoki Aoyama, Asako Oka, Kumiko Kitayama, Hitoshi Kurumizaka, Masahiko Harata

    EXPERIMENTAL CELL RESEARCH   314 ( 4 ) 859 - 868  2008年02月  [査読有り]

     概要を見る

    The actin family consists of conventional actin and various actin-related proteins (Arps). Some of these Arps are localized in the nucleus, and a fraction of each of these nuclear Arps is functionally involved in chromatin remodeling and histone acetyltransferase complexes. On the other hand, in mitotic cells, the localization and function of the nuclear Arps are largely unknown. Human Arp8 (hArp8), an ortholog of yeast nuclear Arp8, was recently found to be associated with the hIN080-chromatin remodeling complex along with hArp5. Here we report that hArp8, but not hArp5, accumulates on mitotic chromosomes. This is the first example where a member of the actin family is found to be associated with mitotic chromosomes. Expression of truncated hArp8 proteins and depletion of endogenous hArp8 by RNA interference caused misalignment of mitotic chromosomes, suggesting that chromosome-associated hArp8 has a role in chromosome behavior. In contrast, depletion of hIno80 and hArp5 did not cause misalignment of chromosomes, suggesting that the role of hArp8 at mitotic chromosomes is independent of the activity of hINO80 complexes. These findings provide the first insight into a novel function of actin family members in mitosis. (C) 2007 Elsevier Inc. All rights reserved.

    DOI J-GLOBAL

  • Structural and biochemical aspects of homologous genetic (DNA) recombination

    Kurumizaka H, Mikawa T, Hikiba J, Shibata T

    Seikagaku   80 ( 6 ) 511 - 520  2008年  [査読有り]

  • The Lys313 residue of the human Rad51 protein negatively regulates the strand-exchange activity

    Takako Ishida, Yoshimasa Takizawa, Isao Sakane, Hitoshi Kurumizaka

    GENES TO CELLS   13 ( 1 ) 91 - 103  2008年01月  [査読有り]

     概要を見る

    The Rad51 protein, which catalyzes homologous-pairing and strand-exchange reactions, is an essential enzyme for homologous recombinational repair (HRR) and meiotic homologous recombination in eukaryotes. In humans, the conventional Rad51 (HsRad51) protein has a Lys residue at position 313; however, the HsRad51-Q313 protein, in which the Lys313 residue is replaced by Gln, was reported as an isoform, probably corresponding to a polymorphic variant. In this study, we purified the HsRad51-K313 and HsRad51-Q313 isoforms and analyzed their biochemical activities in vitro. Compared to the conventional HsRad51-K313 protein, the HsRad51-Q313 protein exhibited significantly enhanced strand-exchange activity under conditions with Ca2+, although the difference was not observed without Ca2+. A double-stranded DNA (dsDNA) unwinding assay revealed that the HsRad51-Q313 protein clearly showed enhanced DNA unwinding activity, probably due to its enhanced filament-formation ability. Mutational analyses of the HsRad51-Lys313 residue revealed that positively charged residues (Lys and Arg), but not negatively charged, polar and hydrophobic residues (Glu, Gln and Met, respectively), at position 313 reduced the strand-exchange and DNA unwinding abilities of the HsRad51 protein. These results suggest that the electrostatic environment around position 313 is important for the regulation of the HsRad51 recombinase activity.

    DOI J-GLOBAL

  • Altered DNA binding by the human Rad51-R150Q mutant found in breast cancer patients

    Takako Ishida, Yoshimasa Takizawa, Isao Sakane, Hitoshi Kurumizaka

    BIOLOGICAL & PHARMACEUTICAL BULLETIN   30 ( 8 ) 1374 - 1378  2007年08月  [査読有り]

     概要を見る

    The human Rad51 protein (HsRad51) catalyzes homologous pairing and strand exchange between single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) during recombinational repair of double-stranded DNA breaks. An HsRad51 mutation that results in the substitution of Gin for Arg150 (R150Q) was found in bilateral breast cancer patients; however, the consequences of this R150Q mutation have not been elucidated. To determine how this HsRad51(R150Q) mutation affects HsRad51 function, in the present study, we purified the HsRad51(R150Q) mutant. The purified HsRad51(R150Q) was completely proficient in the ATP-hydrolyzing activity. A gel filtration analysis revealed that HsRad51(R150Q) also retained the polymer formation ability. In contrast, the ssDNA- and dsDNA-binding abilities of HsRad51(R150Q) were clearly reduced, as compared to those of HsRad51. These differences in the DNA-binding properties between HsRad51(R150Q) and HsRad51 may be important to account for the tumorigenesis in breast cancer patients with the HsRad51(R150Q) mutation.

    DOI J-GLOBAL

  • The interaction of DiaA and DnaA regulates the replication cycle in E. coli by directly promoting ATP-DnaA-specific initiation complexes

    Kenji Keyamura, Norie Fujikawa, Takuma Ishida, Shogo Ozaki, Masayuki Su'etsugu, Kazuyuki Fujimitsu, Wataru Kagawa, Shigeyuki Yokoyama, Hitoshi Kurumizaka, Tsutomu Katayama

    GENES & DEVELOPMENT   21 ( 16 ) 2083 - 2099  2007年08月  [査読有り]

     概要を見る

    Escherichia coli DiaA is a DnaA-binding protein that is required for the timely initiation of chromosomal replication during the cell cycle. In this study, we determined the crystal structure of DiaA at 1.8 angstrom resolution. DiaA forms a homotetramer consisting of a symmetrical pair of homodimers. Mutational analysis revealed that the DnaA-binding activity and formation of homotetramers are required for the stimulation of initiation by DiaA. DiaA tetramers can bind multiple DnaA molecules simultaneously. DiaA stimulated the assembly of multiple DnaA molecules on oriC, conformational changes in ATP-DnaA-specific initiation complexes, and unwinding of oriC duplex DNA. The mutant DiaA proteins are defective in these stimulations. DiaA associated also with ADP-DnaA, and stimulated the assembly of inactive ADP-DnaA oriC complexes. Specific residues in the putative phosphosugar-binding motif of DiaA were required for the stimulation of initiation and formation of ATP-DnaA-specific-oriC complexes. Our data indicate that DiaA regulates initiation by a novel mechanism, in which DiaA tetramers most likely bind to multiple DnaA molecules and stimulate the assembly of specific ATP-DnaA-oriC complexes. These results suggest an essential role for DiaA in the promotion of replication initiation in a cell cycle coordinated manner.

    DOI J-GLOBAL

  • Genetic variance modifies apoptosis susceptibility in mature oocytes via alterations in DNA repair capacity and mitochondrial ultrastructure

    G. I. Perez, B. M. Acton, A. Jurisicova, G. A. Perkins, A. White, J. Brown, A. M. Trbovich, M. -R Kim, R. Fissore, J. Xu, A. Ahmady, S. D. D'Estaing, H. Li, W. Kagawa, H. Kurumizaka, S. Yokoyama, H. Okada, T. W. Mak, M. H. Ellisman, R. F. Casper, J. L. Tilly

    CELL DEATH AND DIFFERENTIATION   14 ( 3 ) 524 - 533  2007年03月  [査読有り]

     概要を見る

    Although the identification of specific genes that regulate apoptosis has been a topic of intense study, little is known of the role that background genetic variance plays in modulating cell death. Using germ cells from inbred mouse strains, we found that apoptosis in mature (metaphase II) oocytes is affected by genetic background through at least two different mechanisms. The first, manifested in AKR/J mice, results in genomic instability. This is reflected by numerous DNA double- strand breaks in freshly isolated oocytes, causing a high apoptosis susceptibility and impaired embryonic development following fertilization. Microinjection of Rad51 reduces DNA damage, suppresses apoptosis and improves embryonic development. The second, manifested in FVB mice, results in dramatic dimorphisms in mitochondrial ultrastructure. This is correlated with cytochrome c release and a high apoptosis susceptibility, the latter of which is suppressed by pyruvate treatment, Smac/DIABLO deficiency, or microinjection of 'normal' mitochondria. Therefore, background genetic variance can profoundly affect apoptosis in female germ cells by disrupting both genomic DNA and mitochondrial integrity.

    DOI J-GLOBAL

  • HIV-1 Vpr induces ATM-dependent cellular signal with enhanced homologous recombination

    C. Nakai-Murakami, M. Shimura, M. Kinomoto, Y. Takizawa, K. Tokunaga, T. Taguchi, S. Hoshino, K. Miyagawa, T. Sata, H. Kurumizaka, A. Yuo, Y. Ishizaka

    ONCOGENE   26 ( 4 ) 477 - 486  2007年01月  [査読有り]

     概要を見る

    An ATM-dependent cellular signal, a DNA-damage response, has been shown to be involved during infection of human immunodeficiency virus type-1 (HIV-1), and a high incidence of malignant tumor development has been observed in HIV-1-positive patients. Vpr, an accessory gene product of HIV-1, delays the progression of the cell cycle at the G2/M phase, and ATR-Chk1-Wee-1, another DNA-damage signal, is a proposed cellular pathway responsible for the Vpr-induced cell cycle arrest. In this study, we present evidence that Vpr also activates ATM, and induces expression of gamma-H2AX and phosphorylation of Chk2. Strikingly, Vpr was found to stimulate the focus formation of Rad51 and BRCA1, which are involved in repair of DNA double-strand breaks (DSBs) by homologous recombination (1411), and biochemical analysis revealed that Vpr dissociates the interaction of p53 and Rad51 in the chromatin fraction, as observed under irradiation-induced DSBs. Vpr was consistently found to increase the rate of HR in the locus of I-Scel, a rare cutting-enzyme site that had been introduced into the genome. An increase of the HR rate enhanced by Vpr was attenuated by an ATM inhibitor, KU55933, suggesting that Vpr-induced DSBs activate ATM-dependent cellular signal that enhances the intracellular recombination potential. In context with a recent report that KU55933 attenuated the integration of HIV-1 into host genomes, we discuss the possible role of Vpr-induced DSBs in viral integration and also in HIV-1 associated malignancy.

    DOI J-GLOBAL

  • Functional and structural analysis of DiaA protein, a novel stimulator for initiation of chromosomal replication in Esherichia coli

    Takuma Ishida, Hitoshi Kurumizaka, Tsutomu Katayama

    GENES & GENETIC SYSTEMS   81 ( 6 ) 407 - 407  2006年12月  [査読有り]

  • Stimulation of Dmc1-mediated DNA strand exchange by the human Rad54B protein

    Naoyuki Sarai, Wataru Kagawa, Takashi Kinebuchi, Ako Kagawa, Kozo Tanaka, Kiyoshi Miyagawa, Shukuko Ikawa, Takehiko Shibata, Hitoshi Kurumizaka, Shigeyuki Yokoyama

    NUCLEIC ACIDS RESEARCH   34 ( 16 ) 4429 - 4437  2006年09月  [査読有り]

     概要を見る

    The process of homologous recombination is indispensable for both meiotic and mitotic cell division, and is one of the major pathways for double-strand break (DSB) repair. The human Rad54B protein, which belongs to the SWI2/SNF2 protein family, plays a role in homologous recombination, and may function with the Dmc1 recombinase, a meiosis-specific Rad51 homolog. In the present study, we found that Rad54B enhanced the DNA strand-exchange activity of Dmc1 by stabilizing the Dmc1-single-stranded DNA (ssDNA) complex. Therefore, Rad54B may stimulate the Dmc1-mediated DNA strand exchange by stabilizing the nucleoprotein filament, which is formed on the ssDNA tails produced at DSB sites during homologous recombination.

    DOI J-GLOBAL

  • Roles of the human Rad51 L1 and L2 loops in DNA binding

    Yusuke Matsuo, Isao Sakane, Yoshimasa Takizawa, Masayuki Takahashi, Hitoshi Kurumizaka

    FEBS JOURNAL   273 ( 14 ) 3148 - 3159  2006年07月  [査読有り]

     概要を見る

    The human Rad51 protein, a eukaryotic ortholog of the bacterial RecA protein, is a key enzyme that functions in homologous recombination and recombinational repair of double strand breaks. The Rad51 protein contains two flexible loops, L1 and L2, which are proposed to be sites for DNA binding, based on a structural comparison with RecA. In the present study, we performed mutational and fluorescent spectroscopic analyses on the L1 and L2 loops to examine their role in DNA binding. Gel retardation and DNA-dependent ATP hydrolysis measurements revealed that the substitution of the tyrosine residue at position 232 (Tyr232) within the L1 loop with alanine, a short side chain amino acid, significantly decreased the DNA-binding ability of human Rad51, without affecting the protein folding or the salt-induced, DNA-independent ATP hydrolysis. Even the conservative replacement with tryptophan affected the DNA binding, indicating that Tyr232 is involved in DNA binding. The importance of the L1 loop was confirmed by the fluorescence change of a tryptophan residue, replacing the Asp231, Ser233, or Gly236 residue, upon DNA binding. The alanine replacement of phenylalanine at position 279 (Phe279) within the L2 loop did not affect the DNA-binding ability of human Rad51, unlike the Phe203 mutation of the RecA L2 loop. The Phe279 side chain may not be directly involved in the interaction with DNA. However, the fluorescence intensity of the tryptophan replacing the Rad51-Phe279 residue was strongly reduced upon DNA binding, indicating that the L2 loop is also close to the DNA-binding site.

    DOI J-GLOBAL

  • The DnaA homolog of the hyperthermophilic eubacterium Thermotoga maritima forms an open complex with a minimal 149-bp origin region in an ATP-dependent manner

    S Ozaki, K Fujimitsu, H Kurumizaka, T Katayama

    GENES TO CELLS   11 ( 4 ) 425 - 438  2006年04月

     概要を見る

    In Escherichia coli, ATP-DnaA, but not ADP-DnaA, forms an initiation complex that undergoes site-specific duplex DNA unwinding, open complex formation. However, it remains unclear how highly the ATP-dependent activation of the initiation factor is conserved in evolution. The hyperthermophile Thermotoga maritima is one of the most ancient eubacteria in evolution. Here, we show that the DnaA homolog (tmaDnaA) of this bacterium forms open complexes with the predicted origin region (tma-oriC) in vitro. TmaDnaA has a strong and specific affinity for ATP/ADP as well as for 12-mer repeating sequences within the tma-oriC. Unlike ADP-tmaDnaA, ATP-tmaDnaA is highly cooperative in DNA binding and forms open complexes in a manner that depends on temperature and the superhelical tension of the tma-oriC-bearing plasmid. The minimal tma-oriC required for unwinding is a 149-bp region containing five repeats of the 12-mer sequence and two AT-rich 9-mer repeats. TmaDnaA-binding to the 12-mer motif provokes DNA bending. The 9-mer region is the duplex-unwinding site. The tmaDnaA-binding and unwinding motifs of tma-oriC share sequence homology with corresponding archaeal and eukaryotic sequences. These findings suggest that the ATP-dependent molecular switch of the initiator and the mechanisms in the replication initiation complex are highly conserved in eubacterial evolution.

    DOI

  • The DnaA homolog of the hyperthermophilic eubacterium Thermotoga maritima forms an open complex with a minimal 149-bp origin region in an ATP-dependent manner

    S Ozaki, K Fujimitsu, H Kurumizaka, T Katayama

    GENES TO CELLS   11 ( 4 ) 425 - 438  2006年04月  [査読有り]

     概要を見る

    In Escherichia coli, ATP-DnaA, but not ADP-DnaA, forms an initiation complex that undergoes site-specific duplex DNA unwinding, open complex formation. However, it remains unclear how highly the ATP-dependent activation of the initiation factor is conserved in evolution. The hyperthermophile Thermotoga maritima is one of the most ancient eubacteria in evolution. Here, we show that the DnaA homolog (tmaDnaA) of this bacterium forms open complexes with the predicted origin region (tma-oriC) in vitro. TmaDnaA has a strong and specific affinity for ATP/ADP as well as for 12-mer repeating sequences within the tma-oriC. Unlike ADP-tmaDnaA, ATP-tmaDnaA is highly cooperative in DNA binding and forms open complexes in a manner that depends on temperature and the superhelical tension of the tma-oriC-bearing plasmid. The minimal tma-oriC required for unwinding is a 149-bp region containing five repeats of the 12-mer sequence and two AT-rich 9-mer repeats. TmaDnaA-binding to the 12-mer motif provokes DNA bending. The 9-mer region is the duplex-unwinding site. The tmaDnaA-binding and unwinding motifs of tma-oriC share sequence homology with corresponding archaeal and eukaryotic sequences. These findings suggest that the ATP-dependent molecular switch of the initiator and the mechanisms in the replication initiation complex are highly conserved in eubacterial evolution.

    DOI

  • Stimulation of DNA strand exchange by the human TBPIP/Hop2-Mnd1 complex

    R Enomoto, T Kinebuchi, M Sato, H Yagi, H Kurumizaka, S Yokoyama

    JOURNAL OF BIOLOGICAL CHEMISTRY   281 ( 9 ) 5575 - 5581  2006年03月  [査読有り]

     概要を見る

    In Saccharomyces cerevisiae, the Hop2 protein forms a complex with the Mnd1 protein and is required for the alignment of homologous chromosomes during meiosis, probably through extensive homology matching between them. The Rad51 and Dmc1 proteins, the eukaryotic RecA orthologs, promote strand exchange and may function in the extensive matching of homology within paired DNA molecules. In the present study, we purified the human TBPIP/ Hop2-Mnd1 complex and found that it significantly stimulates the Dmc1- and Rad51-mediated strand exchange. The human Hop2-Mnd1 complex preferentially binds to a three-stranded DNA branch, which mimics the strand-exchange intermediate. These findings are consistent with genetic data, which showed that the Hop2 and Mnd1 proteins are required for homology matching between homologous chromosomes. Therefore, the human TBPIP/ Hop2-Mnd1 complex may ensure proper pairing between homologous chromosomes through its stimulation of strand exchange during meiosis.

    DOI J-GLOBAL

  • Erratum: Role of the N-terminal domain of the human DMC1 protein in octamer formation and DNA binding (Journal of Biological Chemistry (2005) 280, (28382-28387))

    Kinebuchi T, Kagawa W, Kurumizaka H, Yokoyama S

    Journal of Biological Chemistry   281 ( 50 )  2006年  [査読有り]

  • HIV-1 Vpr induces DNA double-strand breaks

    H Tachiwana, M Shimura, C Nakai-Murakami, K Tokunaga, Y Takizawa, T Sata, H Kurumizaka, Y Ishizaka

    CANCER RESEARCH   66 ( 2 ) 627 - 631  2006年01月  [査読有り]

     概要を見る

    Recent observations imply that HIV-1 infection induces chromosomal DNA damage responses. However, the precise molecular mechanism and biological relevance are not fully understood. Here, we report that HIV-1 infection causes double-strand breaks in chromosomal DNA. We further found that Vpr, an accessory gene product of HIV-1, is a major factor responsible for HIV-1-induced double-strand breaks. The purified Vpr protein promotes double-strand breaks when incubated with isolated nuclei, although it does not exhibit endonuclease activity in vitro. A carboxyl-terminally truncated Vpr mutant that is defective in DNA-binding activity is less car-able of Vpr-dependent double-strand break formation in isolated nuclei. The data suggest that double-strand breaks induced by Vpr depend on its DNA-binding activity and that Vpr may recruit unknown nuclear factor(s) with positive endonuclease activity to chromosomal DNA. This is the first direct evidence that Vpr induces double-strand breaks in HIV-1-infected cells. We discuss the possible roles of Vpr-induced DNA damage in HIV-1 infection and the involvement of Vpr in further acquired immunodeficiency syndrome-related tumor development.

    DOI J-GLOBAL

  • Human centromere protein B induces translational positioning of nucleosomes on alpha-satellite sequences

    Y Tanaka, H Tachiwana, K Yoda, H Masumoto, T Okazaki, H Kurumizaka, S Yokoyama

    JOURNAL OF BIOLOGICAL CHEMISTRY   280 ( 50 ) 41609 - 41618  2005年12月

     概要を見る

    The human centromere proteins A (CENP-A) and B (CENP-B) are the fundamental centromere components of chromosomes. CENP-A is the centromere-specific histone H3 variant, and CENP-B specifically binds a 17-base pair sequence (the CENP-B box), which appears within every other alpha-satellite DNA repeat. In the present study, we demonstrated centromere-specific nucleosome formation in vitro with recombinant proteins, including histones H2A, H2B, H4, CENP-A, and the DNA-binding domain of CENP-B. The CENP-A nucleosome wraps 147 base pairs of the alpha-satellite sequence within its nucleosome core particle, like the canonical H3 nucleosome. Surprisingly, CENP-B binds to nucleosomal DNA when the CENP-B box is wrapped within the nucleosome core particle and induces translational positioning of the nucleosome without affecting its rotational setting. This CENP-B-induced translational positioning only occurs when the CENP-B box sequence is settled in the proper rotational setting with respect to the histone octamer surface. Therefore, CENP-B may be a determinant for translational positioning of the centromere-specific nucleosomes through its binding to the nucleosomal CENP-B box.

    DOI

  • Human centromere protein B induces translational positioning of nucleosomes on α-satellite sequences

    Tanaka Y, Tachiwana H, Yoda K, Masumoto H, Okazaki T, Kurumizaka H, Yokoyama S

    Journal of Biological Chemistry   280 ( 50 ) 41609 - 41618  2005年12月  [査読有り]

    DOI

  • A FancD2-monoubiquitin fusion reveals hidden functions of Fanconi anemia core complex in DNA repair

    N Matsushita, H Kitao, M Ishiai, N Nagashima, S Hirano, K Okawa, T Ohta, DS Yu, PJ McHugh, ID Hickson, AR Venkitaraman, H Kurumizaka, M Takata

    MOLECULAR CELL   19 ( 6 ) 841 - 847  2005年09月  [査読有り]

     概要を見る

    In DNA damage responses, the Fanconi anemia (FA) protein, FancD2, is targeted to chromatin and forms nuclear foci following its monoubiquitination, a process likely catalyzed by the FA core complex. Here, we show that a chicken FancD2-ubiquitin fusion protein, carrying a Lys-Arg substitution removing the natural monoubiquitination site (D2KR-Ub), could reverse cisplatin hypersensitivity and localize to chromatin in FANCD2-deficient DT40 cells. Importantly, the chromatin targeting was dependent on three core complex components as well as the hydrophobic surface of ubiquitin that may direct protein-protein interactions. Furthermore, a constitutively chromatin bound fusion of D2KR-histone H2B could complement cisplatin sensitivity in FANCD2- but not FANCC-, FANCG-, or FANCL-deficient cells. Thus these core complex components have an additional function in the DNA repair, which is independent of the monoubiquitination and chromatin targeting of FancD2. These results define functional consequences of FancD2 monoubiquitination and reveal previously hidden functions for the FA protein core complex.

    DOI J-GLOBAL

  • Role of the N-terminal domain of the human DMC1 protein in octamer formation and DNA binding

    T Kinebuchi, W Kagawa, H Kurumizaka, S Yokoyama

    JOURNAL OF BIOLOGICAL CHEMISTRY   280 ( 31 ) 28382 - 28387  2005年08月  [査読有り]

     概要を見る

    The DMC1 protein, a eukaryotic homologue of RecA that shares significant amino acid identity with RAD51, exhibits two oligomeric DNA binding forms, an octameric ring and a helical filament. In the crystal structure of the octameric ring form, the DMC1 N-terminal domain (1-81 amino acid residues) was highly flexible, with multiple conformations. On the other hand, the N-terminal domain of Rad51 makes specific interactions with the neighboring ATPase domain in the helical filament structure. To gain insights into the functional role of the N-terminal domain of DMC1, we prepared a deletion mutant, DMC1-(82-340), that lacks the N-terminal 81 amino acid residues from the human DMC1 protein. Analytical ultracentrifugation experiments revealed that, whereas full-length DMC1 forms a octamer, DMC1(82-340) is a heptamer. Furthermore, DNA binding experiments showed that DMC1-(82-340) was completely defective in both single-stranded and double-stranded DNA binding activities. Therefore, the N-terminal domain of DMC1 is required for the formation of the octamer, which may support the proper DNA binding activity of the DMC1 protein.

    DOI J-GLOBAL

  • Independent and sequential recruitment of NHEJ and HR factors to DNA damage sites in mammalian cells

    JS Kim, TB Krasieva, H Kurumizaka, DJ Chen, AMR Taylor, K Yokomori

    JOURNAL OF CELL BIOLOGY   170 ( 3 ) 341 - 347  2005年08月  [査読有り]

     概要を見る

    Damage recognition by repair/checkpoint factors is the critical first step of the DNA damage response. DNA double strand breaks ( DSBs) activate checkpoint signaling and are repaired by nonhomologous end-joining (NHEJ) and homologous recombination (HR) pathways. However, in vivo kinetics of the individual factor responses and the mechanism of pathway choice are not well understood. We report cell cycle and time course analyses of checkpoint activation by ataxia-telangiectasia mutated and damage site recruitment of the repair factors in response to laser-induced DSBs. We found that MRN acts as a DNA damage marker, continuously localizing at unrepaired damage sites. Damage recognition by NHEJ factors precedes that of HR factors. HR factor recruitment is not influenced by NHEJ factor assembly and occurs throughout interphase. Damage site retention of NHEJ factors is transient, whereas HR factors persist at unrepaired lesions, revealing unique roles of the two pathways in mammalian cells.

    DOI J-GLOBAL

  • CpG methylation of the CENP-B box reduces human CENP-B binding

    Y Tanaka, H Kurumizaka, S Yokoyama

    FEBS JOURNAL   272 ( 1 ) 282 - 289  2005年01月  [査読有り]

     概要を見る

    In eukaryotes, CpG methylation is an epigenetic DNA modification that is important for heterochromatin formation. Centromere protein B (CENP-B) specifically binds to the centromeric 17 base-pair CENP-B box DNA, which contains two CpG dinucleotides. In this study, we tested complex formation by the DNA-binding domain of CENP-B with methylated and unmethylated CENP-B box DNAs, and found that CENP-B preferentially binds to the unmethylated CENP-B box DNA. Competition analyses revealed that the affinity of CENP-B for the CENP-B box DNA is reduced nearly to the level of nonspecific DNA binding by CpG methylation.

    DOI J-GLOBAL

  • Mutational analyses of the human Rad51-Tyr315 residue, a site for phosphorylation in leukaemia cells

    Y Takizawa, T Kinebuchi, W Kagawa, S Yokoyama, T Shibata, H Kurumizaka

    GENES TO CELLS   9 ( 9 ) 781 - 790  2004年09月  [査読有り]

     概要を見る

    The human Rad51 protein, which plays a central role in homologous recombination, catalyses homologous pairing. The Rad51-Tyr315 residue is known to be constitutively phosphorylated in leukaemia cells and is thought to reside within the subunit-subunit interface of the Rad51 filament. To study the function of the Tyr315 residue, we purified five Rad51 mutants, Y315D, Y315E, Y315R, Y315A and Y315F, in which the Tyr315 residue was replaced by Asp, Glu, Arg, Ala and Phe, respectively. Biochemical studies of these Rad51 mutants revealed that the Y315D and Y315E mutants are defective in homologous pairing due to their impaired ssDNA binding, but their dsDNA binding remained unaffected. The Y315D, Y315E and Y315R mutants are defective in dsDNA unwinding, which depends on Rad51-filament formation, suggesting that these mutants are defective in filament formation on dsDNA. Therefore, the Rad51-Tyr315 residue plays important roles in ssDNA binding and filament formation.

    DOI J-GLOBAL

  • Positive role of the mammalian TBPIP/HOP2 protein in DMC1-mediated homologous pairing

    R Enomoto, T Kinebuchi, M Sato, H Yagi, T Shibata, H Kurumizaka, S Yokoyama

    JOURNAL OF BIOLOGICAL CHEMISTRY   279 ( 34 ) 35263 - 35272  2004年08月  [査読有り]

     概要を見る

    In meiosis, homologous recombination preferentially occurs between homologous chromosomes rather than between sister chromatids, which is opposite to the bias of mitotic recombinational repair. The TBPIP/HOP2 protein is a factor that ensures the proper pairing of homologous chromosomes during meiosis. In the present study, we found that the purified mouse TBPIP/HOP2 protein stimulated homologous pairing catalyzed by the meiotic DMC1 recombinase in vitro. In contrast, TBPIP/HOP2 did not stimulate homologous pairing by RAD51, which is another homologous pairing protein acting in both meiotic and mitotic recombination. The positive effect of TBPIP/HOP2 in the DMC1-mediated homologous pairing was only observed when TBPIP/HOP2 first binds to double-stranded DNA, not to single-stranded DNA, before the initiation of the homologous pairing reaction. Deletion analyses revealed that the C-terminal basic region of TBPIP/HOP2 is required for efficient DNA binding and is also essential for its homologous pairing stimulation activity. Therefore, these results suggest that TBPIP/HOP2 directly binds to DNA and functions as an activator for DMC1 during the homologous pairing step in meiosis.

    DOI J-GLOBAL

  • Location of tyrosine 315, a target for phosphorylation by cAbl tyrosine kinase, at the edge of the subunit-subunit interface of the human Rad51 filament

    S Conilleau, Y Takizawa, H Tachiwana, F Fleury, H Kurumizaka, M Takahashi

    JOURNAL OF MOLECULAR BIOLOGY   339 ( 4 ) 797 - 804  2004年06月

     概要を見る

    Rad51 is a key element of recombinational DNA repair and its activity is regulated by phosphorylation of the tyrosine residue at position 315 by cAb1 kinase. This phosphorylation could be involved in the resistance of cancer cells to chemotherapy. We have investigated the role of this residue by comparing the three-dimensional structures of human Rad51 and its prokaryotic homologue, Escherichia coli RecA. The residue appeared to be on the edge of the subunit-subunit interacting site. The fluorescence intensity of the tryptophan residue inserted at position 315 of human Rad51 in the place of tyrosine was decreased by adding 3 M urea, although the protein was not unfolded as there was no large change in the fluorescence peak position or circular dichroism signal. This change in fluorescence occurred at a lower urea concentration when the protein was diluted, which favours dissociation. These results indicate that the change is related to the dissociation of Rad51 polymer and that residue 315 is close to the subunit-subunit interacting site. ATP and ADP, which affect the filament structure, caused a blue shift in the fluorescence peak. These nucleotides probably altered the subunit-subunit contacts and may thus affect the filament structure. Phosphorylation of this residue could therefore affect the formation and structure of the Rad51 filament. Correct prediction of subunit-subunit interface of Rad51 by simple comparison of structures of Rad51 and RecA supports the idea that Rad51 forms the filament in a similar way as does RecA. (C) 2004 Elsevier Ltd. All rights reserved.

    DOI

  • Location of tyrosine 315, a target for phosphorylation by cAbl tyrosine kinase, at the edge of the subunit-subunit interface of the human Rad51 filament

    S Conilleau, Y Takizawa, H Tachiwana, F Fleury, H Kurumizaka, M Takahashi

    JOURNAL OF MOLECULAR BIOLOGY   339 ( 4 ) 797 - 804  2004年06月  [査読有り]

     概要を見る

    Rad51 is a key element of recombinational DNA repair and its activity is regulated by phosphorylation of the tyrosine residue at position 315 by cAb1 kinase. This phosphorylation could be involved in the resistance of cancer cells to chemotherapy. We have investigated the role of this residue by comparing the three-dimensional structures of human Rad51 and its prokaryotic homologue, Escherichia coli RecA. The residue appeared to be on the edge of the subunit-subunit interacting site. The fluorescence intensity of the tryptophan residue inserted at position 315 of human Rad51 in the place of tyrosine was decreased by adding 3 M urea, although the protein was not unfolded as there was no large change in the fluorescence peak position or circular dichroism signal. This change in fluorescence occurred at a lower urea concentration when the protein was diluted, which favours dissociation. These results indicate that the change is related to the dissociation of Rad51 polymer and that residue 315 is close to the subunit-subunit interacting site. ATP and ADP, which affect the filament structure, caused a blue shift in the fluorescence peak. These nucleotides probably altered the subunit-subunit contacts and may thus affect the filament structure. Phosphorylation of this residue could therefore affect the formation and structure of the Rad51 filament. Correct prediction of subunit-subunit interface of Rad51 by simple comparison of structures of Rad51 and RecA supports the idea that Rad51 forms the filament in a similar way as does RecA. (C) 2004 Elsevier Ltd. All rights reserved.

    DOI

  • Structural basis for octameric ring formation and DNA interaction of the human homologous-pairing protein Dmc1

    T Kinebuchi, W Kagawa, R Enomoto, K Tanaka, K Miyagawa, T Shibata, H Kurumizaka, S Yokoyama

    MOLECULAR CELL   14 ( 3 ) 363 - 374  2004年05月  [査読有り]

     概要を見る

    The human Dmc1 protein, a RecA/Rad51 homolog, is a meiosis-specific DNA recombinase that catalyzes homologous pairing. RecA and Rad51 form helical filaments, while Dmc1 forms an octameric ring. In the present study, we crystallized the full-length human Dmc1 protein and solved the structure of the Dmc1 octameric ring. The monomeric structure of the Dmc1 protein closely resembled those of the human and archaeal Rad51 proteins. In addition to the polymerization motif that was previously identified in the Rad51 proteins, we found another hydrogen bonding interaction at the polymer interface, which could explain why Dmc1 forms stable octameric rings instead of helical filaments. Mutagenesis studies identified the inner and outer basic patches that are important for homologous pairing. The inner patch binds both single-stranded and double-stranded DNAs, while the outer one binds single-stranded DNA. Based on these results, we propose a model for the interaction of the Dmc1 rings with DNA.

    DOI

  • Expression and purification of recombinant human histones

    Y Tanaka, M Tawaramoto-Sasanuma, S Kawaguchi, T Ohta, K Yoda, H Kurumizaka, S Yokoyama

    METHODS   33 ( 1 ) 3 - 11  2004年05月  [査読有り]

     概要を見る

    Nucleosomes reconstituted from bacterially expressed histories are useful for functional and structural analyses of historic variants, histone mutants, and histone post-translational modifications. In the present study, we developed a new method for the expression and purification of recombinant human histones. The human historic H2A, H2B, and H3 genes were expressed well in Escherichia coli cells. but the human histone H4 gene was poorly expressed. Therefore, we designed a new historic H4 gene with codons optimized for the E coli expression system and constructed the H4 gene by chemically synthesized oligodeoxyribonucleotides. The recombinant human histories were expressed as hexahistidine-tagged proteins and were purified by one-step chromatography with nickel-nitrilotriacetic acid agarose in the presence of 6 M urea. The H2A/H2B dimer and the H3/H4 tetramer were refolded by dialysis against buffer without urea, and the hexahistidine-tags of the histories in the H2A/H2B dimer and the H3/H4 tetramer were removed by thrombin protease digestion. The H2A/H2B dimer and the H3/H4 tetramer obtained by this method were confirmed to be proficient in nucleosome formation by the salt dialysis method. The human CENP-A gene, the centromere-specific histone H3 variant, contains 28 minor codons for E. coli. A new CENP-A gene optimized for the E. coli expression system was also constructed. and we found that the purified recombinant CENP-A protein formed a nucleosome-like structure with histones H2A, H2B. and H4. (C) 2003 Elsevier Inc. All rights reserved.

    DOI J-GLOBAL

  • Structural basis for octameric ring formation and DNA interaction of the human homologous-pairing protein Dmc1

    T Kinebuchi, W Kagawa, R Enomoto, K Tanaka, K Miyagawa, T Shibata, H Kurumizaka, S Yokoyama

    MOLECULAR CELL   14 ( 3 ) 363 - 374  2004年05月  [査読有り]

     概要を見る

    The human Dmc1 protein, a RecA/Rad51 homolog, is a meiosis-specific DNA recombinase that catalyzes homologous pairing. RecA and Rad51 form helical filaments, while Dmc1 forms an octameric ring. In the present study, we crystallized the full-length human Dmc1 protein and solved the structure of the Dmc1 octameric ring. The monomeric structure of the Dmc1 protein closely resembled those of the human and archaeal Rad51 proteins. In addition to the polymerization motif that was previously identified in the Rad51 proteins, we found another hydrogen bonding interaction at the polymer interface, which could explain why Dmc1 forms stable octameric rings instead of helical filaments. Mutagenesis studies identified the inner and outer basic patches that are important for homologous pairing. The inner patch binds both single-stranded and double-stranded DNAs, while the outer one binds single-stranded DNA. Based on these results, we propose a model for the interaction of the Dmc1 rings with DNA.

    DOI J-GLOBAL

  • Preferential binding to branched DNA strands and strand-annealing activity of the human Rad51B, Rad51C, Rad51D and Xrcc2 protein complex

    H Yokoyama, N Sarai, W Kagawa, R Enomoto, T Shibata, H Kurumizaka, S Yokoyama

    NUCLEIC ACIDS RESEARCH   32 ( 8 ) 2556 - 2565  2004年04月  [査読有り]

     概要を見る

    The Rad51B, Rad51C, Rad51D and Xrcc2 proteins are Rad51 paralogs, and form a complex (BCDX2 complex) in mammalian cells. Mutant cells defective in any one of the Rad51-paralog genes exhibit spontaneous genomic instability and extreme sensitivity to DNA-damaging agents, due to inefficient recombinational repair. Therefore, the Rad51 paralogs play important roles in the maintenance of genomic integrity through recombinational repair. In the present study, we examined the DNA-binding preference of the human BCDX2 complex. Competitive DNA-binding assays using seven types of DNA substrates, single-stranded DNA (ssDNA), double-stranded DNA, 5'- and 3'-tailed duplexes, nicked duplex DNA, Y-shaped DNA and a synthetic Holliday junction, revealed that the BCDX2 complex preferentially bound to the two DNA substrates with branched structures (the Y-shaped DNA and the synthetic Holliday junction). Furthermore, the BCDX2 complex catalyzed the strand-annealing reaction between a long linear ssDNA (1.2 kb in length) and its complementary circular ssDNA. These properties of the BCDX2 complex may be important for its roles in the maintenance of chromosomal integrity.

    DOI J-GLOBAL

  • Erratum: Structural and biochemical analyses of hemimethylated DNA binding by the SeqA protein (Nucleic Acids Research (2003) vol. 32 (82-92))

    Fujikawa N, Kurumizaka H, Nureki O, Tanaka Y, Yamazoe M, Hiraga S, Yokoyama S

    Nucleic Acids Research   32 ( 2 )  2004年  [査読有り]

    DOI

  • Structural and biochemical analyses of hemimethylated DNA binding by the SeqA protein

    N Fujikawa, H Kurumizaka, O Nureki, Y Tanaka, M Yamazoe, S Hiraga, S Yokoyama

    NUCLEIC ACIDS RESEARCH   32 ( 1 ) 82 - 92  2004年01月  [査読有り]

     概要を見る

    The Escherichia coli SeqA protein recognizes the 11 hemimethylated G-(m)A-T-C sites in the oriC region of the chromosome, and prevents replication over-initiation within one cell cycle. The crystal structure of the SeqA C-terminal domain with hemimethylated DNA revealed the N-6-methyladenine recognition mechanism; however, the mechanism of discrimination between the hemimethylated and fully methylated states has remained elusive. In the present study, we performed mutational analyses of hemimethylated G-(m)A-T-C sequences with the minimal DNA-binding domain of SeqA (SeqA(71-181)), and found that SeqA(71-181) specifically binds to hemimethylated DNA containing a sequence with a mismatched (m)A:G base pair [G-(m)A(:G)-T-C] as efficiently as the normal hemimethylated G-(m)A(:T)-T-C sequence. We determined the crystal structures of SeqA(71-181) complexed with the mismatched and normal hemimethylated DNAs at 2.5 and 3.0 Angstrom resolutions, respectively, and found that the mismatched (m)A:G base pair and the normal (m)A:T base pair are recognized by SeqA in a similar manner. Furthermore, in both crystal structures, an electron density is present near the unmethylated adenine, which is only methylated in the fully methylated state. This electron density, which may be due to a water molecule or a metal ion, can exist in the hemimethylated state, but not in the fully methylated state, because of steric clash with the additional methyl group.

    DOI J-GLOBAL

  • Crystal structure of the human centromere protein B (CENP-B) dimerization domain at 1.65-angstrom resolution

    MS Tawaramoto, SY Park, Y Tanaka, O Nureki, H Kurumizaka, S Yokoyama

    JOURNAL OF BIOLOGICAL CHEMISTRY   278 ( 51 ) 51454 - 51461  2003年12月

     概要を見る

    The human centromere protein B (CENP-B), a centromeric heterochromatin component, forms a homodimer that specifically binds to a distinct DNA sequence (the CENP-B box), which appears within every other alpha-satellite repeat. Previously, we determined the structure of the human CENP-B DNA-binding domain, CENP-B-(1-129), complexed with the CENP-B box DNA. In the present study, we determined the crystal structure of its dimerization domain (CENP-B-(540-599)), another functional domain of CENP-B, at 1.65-Angstrom resolution. CENP- B-(540-599) contains two alpha-helices, which are folded into an antiparallel configuration. The CENP-B-(540-599) dimer formed a symmetrical, antiparallel, four-helix bundle structure with a large hydrophobic patch in which 23 residues of one monomer form van der Waals contacts with the other monomer. In the CENP- B-(540-599) dimer, the N-terminal ends of CENP-B-(540-599) are oriented on opposite sides of the dimer. This CENP- B dimer configuration may be suitable for capturing two distant CENP-B boxes during centromeric heterochromatin formation.

    DOI

  • Crystal Structure of the Human Centromere Protein B (CENP-B) Dimerization Domain at 1.65-Å Resolution

    Tawaramoto M.S, Park S.-Y, Tanaka Y, Nureki O, Kurumizaka H, Yokoyama S

    Journal of Biological Chemistry   278 ( 51 ) 51454 - 51461  2003年12月  [査読有り]

    DOI

  • Region and amino acid residues required for Rad51C binding in the human Xrcc3 protein

    H Kurumizaka, R Enomoto, M Nakada, K Eda, S Yokoyama, T Shibata

    NUCLEIC ACIDS RESEARCH   31 ( 14 ) 4041 - 4050  2003年07月  [査読有り]

     概要を見る

    The Xrcc3 protein, which is required for the homologous recombinational repair of damaged DNA, forms a complex with the Rad51C protein in human cells. Mutations in either the Xrcc3 or Rad51C gene cause extreme sensitivity to DNA-damaging agents and generate the genomic instability frequently found in tumors. In the present study, we found that the Xrcc3 segment containing amino acid residues 63-346, Xrcc3(63-346), is the Rad51C-binding region. Biochemical analyses revealed that Xrcc3(63-346) forms a complex with Rad51C, and the Xrcc3(63-346)- Rad51C complex possesses ssDNA and dsDNA binding abilities comparable to those of the full-length Xrcc3-Rad51C complex. Based on the structure of RecA, which is thought to be the ancestor of Xrcc3, six Xrcc3 point mutants were designed. Two-hybrid and biochemical analyses of the Xrcc3 point mutants revealed that Tyr139 and Phe249 are essential amino acid residues for Rad51C binding. Superposition of the Xrcc3 Tyr139 and Phe249 residues on the RecA structure suggested that Tyr139 may function to ensure proper folding and Phe249 may be important to constitute the Rad51C-binding interface in Xrcc3.

    DOI J-GLOBAL

  • ヒトDNA組換え酵素Rad52の11量体リング構造

    香川亘, 胡桃坂仁志, 横山茂之

    日本結晶学会誌   45 ( 2 ) 119 - 123  2003年04月  [査読有り]  [招待有り]

    DOI J-GLOBAL

  • Structural basis of replication origin recognition by the DnaA protein

    N Fujikawa, H Kurumizaka, O Nureki, T Terada, M Shirouzu, T Katayama, S Yokoyama

    NUCLEIC ACIDS RESEARCH   31 ( 8 ) 2077 - 2086  2003年04月  [査読有り]

     概要を見る

    Escherichia coli DnaA binds to 9 bp sequences (DnaA boxes) in the replication origin, oriC, to form a complex initiating chromosomal DNA replication. In the present study, we determined the crystal structure of its DNA-binding domain (domain IV) complexed with a DnaA box at 2.1 Angstrom resolution. DnaA domain IV contains a helix-turn-helix motif for DNA binding. One helix and a loop of the helix-turn-helix motif are inserted into the major groove and 5 bp (3' two-thirds of the DnaA box sequence) are recognized through base-specific hydrogen bonds and van der Waals contacts with the C5-methyl groups of thymines. In the minor groove, Arg399, located in the loop adjacent to the motif, recognizes three more base pairs (5' one-third of the DnaA box sequence) by base-specific hydrogen bonds. DNA bending by similar to28degrees was also observed in the complex. These base-specific interactions explain how DnaA exhibits higher affinity for the strong DnaA boxes (R1, R2 and R4) than the weak DnaA boxes (R3 and M) in the replication origin.

    DOI J-GLOBAL

  • Incorporation of DUF/FACT into chromatin enhances the accessibility of nucleosomal DNA

    H Seo, K Okuhara, H Kurumizaka, T Yamada, T Shibata, K Ohta, T Akiyama, H Murofushi

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   303 ( 1 ) 8 - 13  2003年03月  [査読有り]

     概要を見る

    DNA unwinding. factor (DUF) was discovered as an essential DNA replication factor in Xenopus egg extracts. DUF consists of an HMG protein and a homolog of Cdc68p/Spt16p, and has the capability of unwinding dsDNA. Here we have examined the interaction of DUF with chromatin. DUF was incorporated into chromatin assembled from sperm heads and from plasmid DNA in egg extracts. It was revealed that the chromatin assembled in egg extracts immunodepleted of DUF is less sensitive to micrococcal nuclease (NNase) digestion than that assembled in control extracts, indicating that chromatin containing DUF has more decompact structure than that without DUF. Also we found that DUF has a high affinity for core histones in vitro. We suggest that the function of DUF may be to make the chromatin structure accessible to replication factors. (C) 2003 Elsevier Science (USA). All rights reserved.

    DOI J-GLOBAL

  • Identification of functional domains of the Escherichia coli SeqA protein

    N Fujikawa, H Kurumizaka, M Yamazoe, S Hiraga, S Yokoyama

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   300 ( 3 ) 699 - 705  2003年01月  [査読有り]

     概要を見る

    The Escherichia coli SeqA protein, a negative regulator of chromosomal DNA replication, prevents the overinitiation of replication within one cell cycle by binding to hemimethylated G-(m)A-T-C sequences in the replication origin, oriC. In addition to the hemimethylated DNA-binding activity, the SeqA protein has a self-association activity, which is also considered to be essential for its regulatory function in replication initiation. To study the functional domains responsible for the DNA-binding and self-association activities, we performed a deletion analysis of the SeqA protein and found that the N-terminal (amino acid residues 1-59) and the C-terminal (amino acid residues 71-181) regions form structurally distinct domains. The N-terminal domain, which is not involved in DNA binding, has the self-association activity. In contrast, the C-terminal domain, which lacks the self-association activity, specifically binds to the hemimethylated G-(m)A-T-C sequence. Therefore, two essential SeqA activities, self-association and DNA-binding, are independently performed by the structurally distinct N-terminal and C-terminal domains, respectively. (C) 2002 Elsevier Science (USA). All rights reserved.

    DOI J-GLOBAL

  • Holliday junctilon binding activity of the human Rad51B protein

    H Yokoyama, H Kurumizaka, S Ikawa, S Yokoyama, T Shibata

    JOURNAL OF BIOLOGICAL CHEMISTRY   278 ( 4 ) 2767 - 2772  2003年01月  [査読有り]

     概要を見る

    The human Rad51B protein is involved in the recombinational repair of damaged DNA. Chromosomal rearrangements of the Rad51B gene have been found in uterine leiomyoma patients, suggesting that the Rad51B gene suppresses tumorigenesis. In the present study, we found that the purified Rad51B protein bound to single-stranded DNA and double-stranded DNA in the presence of ATP and either Mg2+ or Mn2+ and hydrolyzed ATP in a DNA-dependent manner. When the synthetic Holliday junction was present along with the half-cruciform and double-stranded oligonucleotides, the Rad51B protein only bound to the synthetic Holliday junction, which mimics a key intermediate in homologous recombination. In contrast, the human Rad51 protein bound to all three DNA substrates with no obvious preference. Therefore, the Rad51B protein may have a specific function in Holliday junction processing in the homologous recombinational repair pathway in humans.

    DOI J-GLOBAL

  • Crystal structure of the homologous-pairing domain from the human Rad52 recombinase in the undecameric form

    W Kagawa, H Kurumizaka, R Ishitani, S Fukai, O Nureki, T Shibata, S Yokoyama

    MOLECULAR CELL   10 ( 2 ) 359 - 371  2002年08月  [査読有り]

     概要を見る

    The human Rad52 protein forms a heptameric ring that catalyzes homologous pairing. The N-terminal half of Rad52 is the catalytic domain for homologous pairing and the ring formed by the domain fragment was reported to be approximately decameric. Splicing variants of Rad52 and a yeast homolog (Rad59) are composed mostly of this domain. In this study, we determined the crystal structure of the homologous-pairing domain of human Rad52 and revealed that the domain forms an undecameric ring. Each monomer has a beta-beta-beta-alpha fold, which consists of highly conserved amino acid residues among Rad52 homologs. A mutational analysis revealed that the amino acid residues located between the beta-beta-beta-alpha fold and the characteristic hairpin loop are essential for ssDNA and dsDNA binding.

    DOI J-GLOBAL

  • Homologous pairing and ring and filament structure formation activities of the human Xrcc2-Rad51D complex

    H Kurumizaka, S Ikawa, M Nakada, R Enomoto, W Kagawa, T Kinebuchi, M Yamazoe, S Yokoyama, T Shibata

    JOURNAL OF BIOLOGICAL CHEMISTRY   277 ( 16 ) 14315 - 14320  2002年04月  [査読有り]

     概要を見る

    The Xrcc2 and Rad51D/Rad51L3 proteins, which belong to the Rad51 paralogs, are required for homologous recombinational repair (HRR) in vertebrates. The Xrcc2 and Rad51D/Rad51L3 genes, whose products interact with each other, have essential roles in ensuring normal embryonic development. In the present study, we coexpressed the human Xrcc2 and Rad51D/Rad51L3 proteins (Xrcc2 and Rad51D, respectively) in Escherichia coli, and purified the Xrcc2-Rad51D complex to homogeneity. The Xrcc2-Rad51D complex catalyzed homologous pairing between single-stranded and double-stranded DNA, similar to the function of the Xrcc3-Rad51C complex, which is another complex of the Rad51 paralogs. An electron microscopic analysis showed that Xrcc2-Rad51D formed a multimeric ring structure in the absence of DNA. In the presence of ssDNA, Xrcc2-Rad51D formed a filamentous structure, which is commonly observed among the human homologous pairing proteins, Rad51, Rad52, and Xrcc3-Rad51C.

    DOI J-GLOBAL

  • Human Rad5413 is a double-stranded DNA-dependent ATPase and has biochemical properties different from its structural homolog in yeast, Tid1/Rdh54

    K Tanaka, W Kagawa, T Kinebuchi, H Kurumizaka, K Miyagawa

    NUCLEIC ACIDS RESEARCH   30 ( 6 ) 1346 - 1353  2002年03月  [査読有り]

     概要を見る

    The RAD52 epistasis group genes are involved in homologous recombination, and they are conserved from yeast to humans. We have cloned a novel human gene, RAD54B, which is homologous to yeast and human RAD54. Human Rad54B (hRad54B) shares high homology with human Rad54 (hRad54) in the central region containing the helicase motifs characteristic of the SNF2/SWI2 family of proteins, but the N-terminal domain is less conserved. In yeast, another RAD54 homolog, TID1/RDH54, plays a role in recombination. Tid1/Rdh54 interacts with yeast Rad51 and a meiosis-specific Rad51 homolog, Dmc1. The N-terminal domain of hRad54B shares homology with that of Tid1/Rdh54, suggesting that Rad54B may be the human counterpart of Tid1/Rdh54. We purified the hRad54 and hRad54B proteins from baculovirus-infected insect cells and examined their biochemical properties. hRad54B, like hRad54, is a DNA-binding protein and hydrolyzes ATP in the presence of double-stranded DNA, though its rate of ATP hydrolysis is lower than that of hRad54. Human Rad51 interacts with hRad54 and enhances its ATPase activity. In contrast, neither human Rad51 nor Dmc1 directly interacts with hRad54B. Although hRad54B is the putative counterpart of Tid1/Rdh54, our findings suggest that hRad54B behaves differently from Tid1/Rdh54.

    DOI

  • Human Rad54B is a double-stranded DNA-dependent ATPase and has biochemical properties different from its structural homolog in yeast, Tid1/Rdh54

    Tanaka K, Kagawa W, Kinebuchi T, Kurumizaka H, Miyagawa K

    Nucleic Acids Research   30 ( 6 ) 1346 - 1353  2002年03月  [査読有り]

    DOI J-GLOBAL

  • Structural and functional analyses of proteins involved in translation, transcription, replication, and recombination

    Kurumizaka, H, Yokoyama, S

    Progress in Biotechnology   22  2002年

    DOI

  • Structural and functional analyses of proteins involved in translation, DNA recombination, chromosome architecture, and signal transduction

    Kurumizaka H, Yokoyama S

    Progress in Biotechnology   22 ( C ) 169 - 180  2002年  [査読有り]

    DOI

  • Crystal structure of the CENP-B protein-DNA complex: the DNA-binding domains of CENP-B induce kinks in the CENP-B box DNA

    Y Tanaka, O Nureki, H Kurumizaka, S Fukai, S Kawaguchi, M Ikuta, J Iwahara, T Okazaki, S Yokoyama

    EMBO JOURNAL   20 ( 23 ) 6612 - 6618  2001年12月  [査読有り]

     概要を見る

    The human centromere protein B (CENP-B), one of the centromere components, specifically binds a 17 bp sequence (the CENP-B box), which appears in every other alpha -satellite repeat. In the present study, the crystal structure of the complex of the DNA-binding region (129 residues) of CENP-B and the CENP-B box DNA has been determined at 2.5 Angstrom resolution. The DNA-binding region forms two helix-turn-helix domains, which are bound to adjacent major grooves of the DNA. The DNA is kinked at the two recognition helix contact sites, and the DNA region between the kinks is straight. Among the major groove protein-bound DNAs, this 'kink-straight-kink' bend contrasts with ordinary 'round bends' (gradual bending between two protein contact sites). The larger kink (43 degrees) is induced by a novel mechanism, 'phosphate bridging by an arginine-rich helix': the recognition helix with an arginine cluster is inserted perpendicularly into the major groove and bridges the groove through direct interactions with the phosphate groups. The overall bending angle is 59 degrees, which may be important for the centromere-specific chromatin structure.

    DOI J-GLOBAL

  • Homologous pairing promoted by the human Rad52 protein

    W Kagawa, H Kurumizaka, S Ikawa, S Yokoyama, T Shibata

    JOURNAL OF BIOLOGICAL CHEMISTRY   276 ( 37 ) 35201 - 35208  2001年09月  [査読有り]

     概要を見る

    The Rad52 protein, which is unique to eukaryotes, plays important roles in the Rad51-dependent and the Rad51-independent pathways of DNA recombination. In the present study, we have biochemically characterized the homologous pairing activity of the HsRad52 protein (Homo sapiens Rad52 and found that the presynaptic complex formation with ssDNA is essential in its catalysis of homologous pairing. We have identified an N-terminal fragment (amino acid residues 1-237, HsRad52(1-237)) that is defective in binding to the human Rad51 protein, which catalyzed homologous pairing as efficiently as the wild type HsRad52. Electron microscopic visualization revealed that HsRad52 and HsRad52(1-237) both formed nucleoprotein filaments with single-stranded DNA. These lines of evidence suggest the role of HsRad52 in the homologous pairing step of the Rad51-independent recombination pathway. Our results reveal the striking similarity between HsRad52 and the Escherichia coli RecT protein, which functions in a RecA-independent recombination pathway.

    DOI J-GLOBAL

  • Homologous genetic recombination as an intrinsic dynamic property of a DNA structure induced by RecA/Rad51-family proteins: A possible advantage of DNA over RNA as genomic material

    T Shibata, T Nishinaka, T Mikawa, H Aihara, H Kurumizaka, S Yokoyama, Y Ito

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   98 ( 15 ) 8425 - 8432  2001年07月  [査読有り]

     概要を見る

    Heteroduplex joints are general intermediates of homologous genetic recombination in DNA genomes, A heteroduplex joint is formed between a single-stranded region (or tail), derived from a cleaved parental double-stranded DNA, and homologous regions in another parental double-stranded DNA, in a reaction mediated by the RecA/Rad51-family of proteins. In this reaction, a RecA/Rad51-family protein first forms a filamentous complex with the single-stranded DNA, and then interacts with the double-stranded DNA in a search for homology, Studies of the three-dimensional structures of single-stranded DNA bound either to Escherichia coli RecA or Saccharomyces cerevisiae Rad51 have revealed a novel extended DNA structure. This structure contains a hydrophobic interaction between the 2 ' methylene moiety of each deoxyribose and the aromatic ring of the following base, which allows bases to rotate horizontally through the interconversion of sugar puckers. This base rotation explains the mechanism of the homology search and base-pair switch between double-stranded and singlestranded DNA during the formation of heteroduplex joints, The pivotal role of the 2 ' methylene-base interaction in the heteroduplex joint formation is supported by comparing the recombination of RNA genomes with that of DNA genomes, Some simple organisms with DNA genomes induce homologous recombination when they encounter conditions that are unfavorable for their survival. The extended DNA structure confers a dynamic property on the otherwise chemically and genetically stable double-stranded DNA, enabling gene segment rearrangements without disturbing the coding frame (i.e., protein-segment shuffling). These properties may give an extensive evolutionary advantage to DNA.

    DOI J-GLOBAL

  • Homologous-pairing activity of the human DNA-repair proteins Xrcc3.Rad51C.

    Kurumizaka, H, Ikawa, S, Nakada, M, Eda, K, Kagawa, W, Takata, M, Takeda, S, Yokoyama, S, Shibata, T

    Proceedings of National Academy of Science of the United States of America   98 ( 10 ) 5538 - 5543  2001年05月  [査読有り]

    DOI J-GLOBAL

  • Specific defects in double-stranded DNA unwinding and homologous pairing of a mutant RecA protein

    H Kurumizaka, H Aihara, S Ikawa, T Shibata

    FEBS LETTERS   477 ( 1-2 ) 129 - 134  2000年07月  [査読有り]

     概要を見る

    The DNA molecules bound to RecA filaments are extended 1.5-fold relative to B-form DNA, This extended DNA structure may be important in the recognition of homology between single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). In this study, we show that the K286N mutation specifically impaired the dsDNA unwinding and homologous pairing activities of RecA, without an apparent effect on dsDNA binding itself. In contrast, the R243Q mutation caused defective dsDNA unwinding, due to the defective dsDNA binding of the C-terminal domain of RecA, These results provide new evidence that dsDNA unwinding is essential to homology recognition between ssDNA and dsDNA during homologous pairing, (C) 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

    DOI J-GLOBAL

  • Identification of Rad51 alteration in patients with bilateral breast cancer

    M Kato, K Yano, F Matsuo, H Saito, T Katagiri, H Kurumizaka, M Yoshimoto, F Kasumi, F Akiyama, G Sakamoto, H Nagawa, Y Nakamura, Y Miki

    JOURNAL OF HUMAN GENETICS   45 ( 3 ) 133 - 137  2000年  [査読有り]

     概要を見る

    The human Rad51 gene, HsRAD51, is a homolog of RecA of Escherichia coli and functions in recombination and DNA repair. BRCA1 and BRCA2 proteins form a complex with Rad51, and these genes are thought to participate in a common DNA damage response pathway associated with the activation of homologous recombination and double-strand break repair. Additionally, we have shown that the pattern of northern blot analysis of the Rad51 gene is closely similar to those of the BRCA1 and BRCA2 genes. It is therefore possible that alterations of the Rad51 gene may be involved in the development of hereditary breast cancer. To investigate this possibility, we screened Japanese patients with hereditary breast cancer for Rad51 mutations and found a single alteration in exon 6, This was determined to be present in the germline in two patients with bilateral breast cancer, one with synchronous bilateral breast cancer and the other with synchronous bilateral multiple breast cancer. In both patients, blood DNAs showed a G-to-A transition in the second nucleotide of codon 150. which results in the substitution of glutamine for arginine. As this alteration was not present in any patients with breast or colon cancer examined, we assume that this missense alteration is likely to be a disease-causing mutation.

    DOI J-GLOBAL

  • Structural genomics projects in Japan

    S Yokoyama, Y Matsuo, H Hirota, T Kigawa, M Shirouzu, Y Kuroda, H Kurumizaka, S Kawaguchi, Y Ito, T Shibata, M Kainosho, Y Nishimura, Y Inoue, S Kuramitsu

    PROGRESS IN BIOPHYSICS & MOLECULAR BIOLOGY   73 ( 5 ) 363 - 376  2000年  [査読有り]

    DOI J-GLOBAL

  • Human Rad51 amino acid residues required for Rad52 binding.

    Kurumizaka H, Aihara H, Kagawa W, Shibata T, Yokoyama S

    Journal of Molecular Biology/Academic Press   291 ( 3 ) 537 - 548  1999年08月  [査読有り]

    DOI J-GLOBAL

  • The N-terminal domain of the human Rad51 protein binds DNA: Structure and a DNA binding surface as revealed by NMR

    H Aihara, Y Ito, H Kurumizaka, S Yokoyama, T Shibata

    JOURNAL OF MOLECULAR BIOLOGY   290 ( 2 ) 495 - 504  1999年07月  [査読有り]

     概要を見る

    Human Rad51 protein (HsRad51) is a homolog of Escherichia coli RecA protein, and functions in DNA repair and recombination. Ln higher eukaryotes, Rad51 protein is essential for cell viability. The N-terminal region of HsRad51 is highly conserved among eukaryotic Rad51 proteins but is absent from RecA, suggesting a Rad51-specific function for this region. Here, we have determined the structure of the N-terminal part of HsRad51 by NMR spectroscopy. The N-terminal region forms a compact domain consisting of five short helices, which shares structural similarity with a domain of endonuclease III, a DNA repair enzyme of E. coli. NMR experiments did not support the involvement of the N-terminal domain in HsRad51-HsBrca2 interaction or the self-association of HsRad51 as proposed by previous studies. However, NMR tiration experiments demonstrated a physical interaction of the domain with DNA, and allowed mapping of the DNA binding surface. Mutation analysis showed that the DNA binding surface is essential for double-stranded and single-stranded DNA binding of HsRad51. Our results suggest the presence of a DNA binding site on the outside surface of the HsRad51 filament and provide a possible explanation for the regulation of DNA binding by phosphorylation within the N-terminal domain. (C) 1999 Academic Press.

    DOI J-GLOBAL

  • The mutant RecA proteins, RecAR243Q and RecAK245N, exhibit defective DNA binding in homologous pairing

    H Kurumizaka, S Ikawa, A Sarai, T Shibata

    ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS   365 ( 1 ) 83 - 91  1999年05月  [査読有り]

     概要を見る

    In homologous pairing, the RecA protein sequentially binds to single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), aligning the two DNA molecules within the helical nucleoprotein filament. To identify the DNA binding region, which stretches from the outside to the inside of the filament, we constructed two mutant RecA proteins, RecAR243Q and RecAK245N, with the amino acid substitutions of Arg243 to Gin and Lys245 to Asn, respectively. These amino acids are exposed to the solvent in the crystal structure of the RecA protein and are located in the central domain, which is believed to be the catalytic center of the homologous pairing activity. The mutations of Arg243 to Gin (RecAR243Q) and Lys245 to Asn (RecAK245N) impair the repair of UV-damaged DNA in vivo and cause defective homologous pairing of ssDNA and dsDNA in vitro. Although RecAR243Q is only slightly defective and RecAK245N is completely proficient in ssDNA binding to form the presynaptic filament, both mutant RecA proteins are defective in the formation of the three-component complex including ssDNA, dsDNA, and RecA protein, The ability to form dsDNA from complementary single strands is also defective in both RecAR243Q and RecAK245N. These results suggest that the region including Arg243 and Lys245 may be involved in the path of secondary DNA binding to the presynaptic filament. (C) 1999 Academic Press.

    DOI J-GLOBAL

  • The nucleosome: A powerful regulator of transcription

    AP Wolffe, H Kurumizaka

    PROGRESS IN NUCLEIC ACID RESEARCH AND MOLECULAR BIOLOGY, VOL 61   61   379 - 422  1998年  [査読有り]

  • Sin mutations of histone H3: Influence on nucleosome core structure and function

    H Kurumizaka, AP Wolffe

    MOLECULAR AND CELLULAR BIOLOGY   17 ( 12 ) 6953 - 6969  1997年12月  [査読有り]

     概要を見る

    Sin mutations in Saccharomyces cerevisiae alleviate transcriptional defects that result from the inactivation of the yeast SWI/SNF complex. We have investigated the structural and functional consequences for the nucleosome of Sin mutations in histone H3. We directly test the hypothesis that mutations in histone H3 leading to a SWI/SNF-independent (Sin) phenotype in yeast lead to nucleosomal destabilization. In certain instances this is shown to be true; however, nucleosomal destabilization does not always occur. Topoisomerase I-mediated relaxation of minichromosomes assembled with either mutant histone H3 or wild-type H3 together with histones H2A, H2B, and H4 indicates that DNA is constrained into nucleosomal structures containing either mutant or wild-type proteins. However, nucleosomes containing particular mutant H3 molecules (R116-H and T118-I) are more accessible to digestion by micrococcal nuclease and do not constrain DNA in a precise rotational position, as revealed by digestion with DNase I. This result establishes that Sin mutations in histone H3 located close to the dyad axis can destabilize histone-DNA contacts at the periphery of the nucleosome core. Other nucleosomes containing a distinct mutant H3 molecule (E105-K) associated with a Sin phenotype show very little change in nucleosome structure and stability compared to wild-type nucleosomes. Both mutant and wild-type nucleosomes continue to restrict the binding of either TATA-binding protein/transcription factor IIA (TFIIA) or the RNA polymerase III transcription machinery. Thus, different Sin mutations in histone H3 alter the stability of histone-DNA interactions to various extents in the nucleosome while maintaining the fundamental architecture of the nucleosome and contributing to a common Sin phenotype.

  • Sin mutations of histone H3: Influence on nucleosome core structure and function

    H Kurumizaka, AP Wolffe

    MOLECULAR AND CELLULAR BIOLOGY   17 ( 12 ) 6953 - 6969  1997年12月  [査読有り]

     概要を見る

    Sin mutations in Saccharomyces cerevisiae alleviate transcriptional defects that result from the inactivation of the yeast SWI/SNF complex. We have investigated the structural and functional consequences for the nucleosome of Sin mutations in histone H3. We directly test the hypothesis that mutations in histone H3 leading to a SWI/SNF-independent (Sin) phenotype in yeast lead to nucleosomal destabilization. In certain instances this is shown to be true; however, nucleosomal destabilization does not always occur. Topoisomerase I-mediated relaxation of minichromosomes assembled with either mutant histone H3 or wild-type H3 together with histones H2A, H2B, and H4 indicates that DNA is constrained into nucleosomal structures containing either mutant or wild-type proteins. However, nucleosomes containing particular mutant H3 molecules (R116-H and T118-I) are more accessible to digestion by micrococcal nuclease and do not constrain DNA in a precise rotational position, as revealed by digestion with DNase I. This result establishes that Sin mutations in histone H3 located close to the dyad axis can destabilize histone-DNA contacts at the periphery of the nucleosome core. Other nucleosomes containing a distinct mutant H3 molecule (E105-K) associated with a Sin phenotype show very little change in nucleosome structure and stability compared to wild-type nucleosomes. Both mutant and wild-type nucleosomes continue to restrict the binding of either TATA-binding protein/transcription factor IIA (TFIIA) or the RNA polymerase III transcription machinery. Thus, different Sin mutations in histone H3 alter the stability of histone-DNA interactions to various extents in the nucleosome while maintaining the fundamental architecture of the nucleosome and contributing to a common Sin phenotype.

  • An interaction between a specified surface of the C-terminal domain of RecA protein and double-stranded DNA for homologous pairing.

    Aihara H, Ito Y, Kurumizaka H, Terada T, Yokoyama S, Shibata T

    Journal of Molecular Biology/Academic Press   274 ( 2 ) 213 - 221  1997年11月  [査読有り]

    DOI J-GLOBAL

  • Histone acetylation: influence on transcription, nucleosome mobility and positioning, and linker histone-dependent transcriptional repression.

    Ura K, Kurumizaka H, Dimitrov S, Almouzni G, Wolffe AP

    The EMBO Journal/Oxford University Press   16 ( 8 ) 2096 - 2107  1997年04月  [査読有り]

    DOI J-GLOBAL

  • A possible role of the C-terminal domain of the RecA protein. A gateway model for double-stranded DNA binding.

    Kurumizaka H, Aihara H, Ikawa S, Kashima T, Bazemore LR, Kawasaki K, Sarai A, Radding CM, Shibata T

    The Journal of Biological Chemistry/the American Society for Biochemistry and Molecular Biology   271 ( 52 ) 33515 - 33524  1996年12月  [査読有り]

    DOI J-GLOBAL

  • Functional domains for assembly of histones H3 and H4 into the chromatin of Xenopus embryos

    L Freeman, H Kurumizaka, AP Wolffe

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   93 ( 23 ) 12780 - 12785  1996年11月  [査読有り]

     概要を見る

    Histones H3 and H4 have a well defined structural role in the nucleosome and an established role in the regulation of transcription. We have made use of a microinjection strategy using Xenopus embryos to define the minimal structural components of H3 and H4 necessary for nucleosome assembly into metazoan chromosomes in vivo. we find that both the N-terminal tail of H4, including all sites of acetylation, and the C-terminal alpha-helix of the H4 histone fold domain are dispensable for chromatin assembly. The N-terminal tail and an N-terminal alpha-helix of H3 are also dispensable for chromatin assembly. However, the remainder of the H3 and H4 histone folds are essential for incorporation of these proteins into chromatin. We suggest that elements of the histone fold domain maintain both nucleosomal integrity and have distinct functions essential for cell viability.

    DOI J-GLOBAL

  • Homologous recognition by RecA protein using non-equivalent three DNA-strand-binding sites

    H Kurumizaka, T Shibata

    JOURNAL OF BIOCHEMISTRY   119 ( 2 ) 216 - 223  1996年02月  [査読有り]

     概要を見る

    A key step in homologous recombination is the formation of a heteroduplex joint between double-stranded DNA and single-stranded DNA by the homologous pairing and strand-exchange, and this step is also important in recombinational repair of damaged DNA in various organisms. The homologous pairing and the strand-exchange are promoted in vivo and in vitro by RecA protein of Escherichia coli or its homologues of bacteria, virus, and lower and higher eukaryotes. A central question on the mechanism of homologous recombination is how RecA protein (and its homologues) recognizes homologous sequences between single-stranded DNA and double-stranded DNA. Recent studies suggest that RecA protein promotes homologous recognition between these DNA molecules by the formation of a transient and additional pairing of identical sequences via non-Watson-Crick interactions to the Watson-Crick-type duplex DNA, and that RecA protein uses three non-equivalent DNA-strand-binding sites in this reaction.

  • Homologous recognition by RecA protein using non-equivalent three DNA-strand-binding sites

    Kurumizaka H, Shibata T

    Journal of Biochemistry   119 ( 2 ) 216 - 223  1996年  [査読有り]

  • Solution structure of the DNA binding domain of a nucleoid-associated protein, H-NS, from Escherichia coli.

    Shindo H, Iwaki T, Ieda R, Kurumizaka H, Ueguchi C, Mizuno T, Morikawa S, Nakamura H, Kuboniwa H

    FEBS Letters/Elsevier   360 ( 2 ) 125 - 131  1995年02月  [査読有り]

    DOI J-GLOBAL

  • Mechanisms of the recognition of homologous DNA molecules by RecA protein

    Kurumizaka H, Shibata T

    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme   40 ( 10 ) 1647 - 1655  1995年  [査読有り]

  • A chimeric Rec-A protein that implicates non-Watson-Crick interactions in homologous pairing.

    Kurumizaka H, Rao BJ, Ogawa T, Radding CM, Shibata T

    Nucleic Acids Research/Oxford University Press   22 ( 16 ) 3387 - 3391  1994年08月  [査読有り]

    DOI J-GLOBAL

  • A CHIMERIC RECA PROTEIN EXHIBITS ALTERED DOUBLE-STRANDED DNA-BINDING

    H KURUMIZAKA, S IKAWA, T IKEYA, T OGAWA, T SHIBATA

    JOURNAL OF BIOLOGICAL CHEMISTRY   269 ( 4 ) 3068 - 3075  1994年01月  [査読有り]

     概要を見る

    RecAc38 protein, a chimeric RecA protein of Escherichia coli and Pseudomonas aeruginosa, is proficient in the renaturation from complementary single strands. However, RecAc38 protein showed a significant deficiency in promoting homologous pairing of single-stranded DNA and double-stranded DNA. RecAc38 protein was able to remove the secondary structure of single-stranded DNA, the first step of homologous pairing, at a slightly reduced rate. RecAc38 protein-single-stranded DNA-complex (presynaptic complex) was found to be deficient in the sequence-independent binding to double-stranded DNA which is a step in the search for the homology. On the other hand, once unwinding was initiated, RecAc38 protein was able to propagate the unwinding of the double helix at the same extent as wild-type Rec-A protein. These defects and the proficiency of RecAc38 protein are explained by a model showing that RecA protein has three distinct DNA strand-binding sites (a site for the primary binding to single-stranded DNA, a site for the binding to a strand of second single- or double-stranded DNA, and a site required for the binding to the other strand of the double-stranded DNA) and that RecAc38 protein has a defect in the third site.

  • A CHIMERIC RECA PROTEIN EXHIBITS ALTERED DOUBLE-STRANDED DNA-BINDING

    H KURUMIZAKA, S IKAWA, T IKEYA, T OGAWA, T SHIBATA

    JOURNAL OF BIOLOGICAL CHEMISTRY   269 ( 4 ) 3068 - 3075  1994年01月  [査読有り]

     概要を見る

    RecAc38 protein, a chimeric RecA protein of Escherichia coli and Pseudomonas aeruginosa, is proficient in the renaturation from complementary single strands. However, RecAc38 protein showed a significant deficiency in promoting homologous pairing of single-stranded DNA and double-stranded DNA. RecAc38 protein was able to remove the secondary structure of single-stranded DNA, the first step of homologous pairing, at a slightly reduced rate. RecAc38 protein-single-stranded DNA-complex (presynaptic complex) was found to be deficient in the sequence-independent binding to double-stranded DNA which is a step in the search for the homology. On the other hand, once unwinding was initiated, RecAc38 protein was able to propagate the unwinding of the double helix at the same extent as wild-type Rec-A protein. These defects and the proficiency of RecAc38 protein are explained by a model showing that RecA protein has three distinct DNA strand-binding sites (a site for the primary binding to single-stranded DNA, a site for the binding to a strand of second single- or double-stranded DNA, and a site required for the binding to the other strand of the double-stranded DNA) and that RecAc38 protein has a defect in the third site.

  • Proton NMR study on a histone-like protein, HU alpha, from Escherichia coli and its complex with oligo DNAs.

    Shindo H, Kurumizaka H, Furubayashi A, Sakuma C, Matsumoto U, Yanagida A, Goshima N, Kano Y, Imamoto F

    Biological Pharmaceutical Bulletin/Japanese Sciety for Pharmacy   16;5  1993年05月

    DOI

  • Proton NMR study on a histone-like protein, HU<inf>α</inf>, From escherichia coli and its complex with oligo DNAs.

    Shindo H, Kurumizaka H, Furubayashi A, Sakuma C, Matsumoto U, Yanagida A, Goshima N, Kano Y, Imamoto F

    Biological and Pharmaceutical Bulletin   16 ( 5 ) 437 - 443  1993年05月  [査読有り]

    DOI

  • SPECIFIC AND NONSPECIFIC INTERACTIONS OF INTEGRATION HOST FACTOR WITH OLIGO DNAS AS REVEALED BY CIRCULAR-DICHROISM SPECTROSCOPY AND FILTER BINDING ASSAY

    H KURUMIZAKA, F KANKE, U MATSUMOTO, H SHINDO

    ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS   295 ( 2 ) 297 - 301  1992年06月  [査読有り]

    DOI J-GLOBAL

  • Proton NMR study on a type II DNA binding protein huα from escherichia coli and its complex with oligo DNA

    Shindo H, Kurumizaka H, Furubayashi A, Sakuma C, Matumoto U, Yanagida A, Goshima N, Kano Y, Imamoto F

    Analytical Sciences   7 ( Supplement ) 849 - 852  1991年  [査読有り]

    DOI

▼全件表示

書籍等出版物

  • 臨床免疫・アレルギー科 第75巻第6号【話題】 「クロマチンを形成したゲノムDNAが自然免疫DNAセンサーcGASの監視から逃れる仕組み」

    鯨井智也, 胡桃坂仁志

    科学評論社  2021年06月

  • 遺伝子医学MOOK36号 エピゲノムで新たな解明が進む「先天性疾患」第1章 エピゲノム総論 3.クロマチンモデリング因子

    大角 健, 鯨井智也, 胡桃坂仁志

    株式会社メディカル ドゥ  2021年04月

  • 生体の化学 vol.71 No.4 「ヌクレオソームによるクロマチンの構造多様性」

    藤田理紗, 胡桃坂仁志

    医学書院  2020年08月

  • 月刊細胞5月号【遺伝子制御の基盤となる細胞核・クロマチン構造】木村宏・編 「転写におけるクロマチン構造研究」

    鯨井智也, 胡桃坂仁志

    ニューサイエンス社  2020年05月

  • 【イメージング時代の構造生命科学 細胞の動態、膜のないオルガネラ、分子の構造変化をトランススケールに観る】(第1章)近年の技術革新と解かれた構造 クライオ電子顕微鏡によるクロマチンダイナミクス研究

    鯨井 智也, 滝沢 由政, 胡桃坂 仁志

    (株)羊土社  2020年03月

  • Preparation of various nucleosomes for biochemical and structural analyses

    Dacher M, Iikura Y, Kujirai T, Kurumizaka H

    2020年01月

  • 医学のあゆみ Vol.267 No.13 企画 田中啓二 「クロマチンによるエピジェネティックな転写制御」

    胡桃坂仁志, 鯨井智也

    医歯薬出版  2018年12月

  • 日本の科学者 Vol. 53 No. 8「遺伝子の働きを制御するヒストン」

    小山昌子, 胡桃坂仁志

    本の泉社  2018年08月 ISBN: 9784780713381

  • 教科書を書き換えろ!染色体の新常識 : ポリマー・相分離から疾患・老化まで

    平野 達也, 胡桃坂 仁志

    羊土社  2018年 ISBN: 9784758103749

  • あなたのタンパク質精製、大丈夫ですか? : 貴重なサンプルをロスしないための達人の技

    胡桃坂 仁志, 有村 泰宏

    羊土社  2018年 ISBN: 9784758122382

  • 生体の科学「パイオニア転写因子によるクロマチン構造変換」

    小山昌子, 胡桃坂仁志

    医学書院  2017年06月

  • ナノバイオ・メディシン: 細胞核内反応とゲノム編集

    宇理須恒雄, 佐久間哲史, 高田望, 竹中繁織, 小澤岳昌, 吉村英哲, 胡桃坂仁志, 越阪部晃永, 原田昌彦, 束田裕一, 宮成悠介, 塩見美喜子, 大西遼

    近代科学社  2017年

  • Annual Review 呼吸器 2016『ヒストンH2Aバリアントによる細胞機能制御』

    堀越直樹, 有村泰宏, 胡桃坂仁志

    中外医学社  2016年

  • Surgery frontier『ヒストンバリアントとは』

    有村泰宏, 胡桃坂仁志

    メディカルレビュー社  2015年

  • 実験医学Vol32 No.13.2014[通巻541号]

    胡桃坂仁志

    羊土社  2014年08月

  • 構造生命科学で何がわかるのか,何ができるのか

    越阪部晃永, 堀越直樹, 胡桃坂仁志, 田中啓二, 若槻壮市

    羊土社  2014年06月

  • エピジェネティクスキーワード事典

    胡桃坂仁志, 越阪部晃永, 有村泰宏

    羊土社  2013年11月

  • 染色体と細胞核のダイナミクス

    胡桃坂仁志, 木村宏

    化学同人  2013年11月

  • がん基盤生物学

    高久誉大, 町田晋一, 胡桃坂仁志

    南山堂  2013年10月

  • 基礎コース 細胞生物学

    胡桃坂仁志, 市川雄一

    東京化学同人  2013年09月

  • 実験医学 Vol.31 No.4 絵で見る先端分子生物学

    胡桃坂仁志, 松本亮平

    羊土社  2013年03月

  • 実験医学 Vol.31 No.3 絵で見る先端分子生物学

    胡桃坂仁志, 松本亮平

    羊土社  2013年02月

  • 実験医学 Vol.30 No.11 絵で見る先端分子生物学

    胡桃坂仁志, 松本亮平

    羊土社  2012年07月

  • 実験医学 Vol.30 No.9 絵で見る先端分子生物学

    胡桃坂仁志, 松本亮平

    羊土社  2012年06月

  • 実験医学 Vol.30 No.8 絵で見る先端分子生物学

    胡桃坂仁志, 松本亮平

    羊土社  2012年05月

  • 実験医学 Vol.30 No.6 絵で見る先端分子生物学

    胡桃坂仁志, 松本亮平

    羊土社  2012年04月

  • 実験医学 Vol.30 No.4 絵で見る先端分子生物学

    胡桃坂仁志, 松本亮平

    羊土社  2012年03月

  • 実験医学 Vol.30 No.3 絵で見る先端分子生物学

    胡桃坂仁志, 松本亮平

    羊土社  2012年02月

  • 実験医学 Vol.30 No.1 絵で見る先端分子生物学

    胡桃坂仁志, 松本亮平

    羊土社  2012年01月

  • SPring-8 Research Frontiers

    H.Tachiwana, W.Kagawa, H.Kurumizaka

    ROKKO Publishing & Sale Co.  2011年08月

  • 実験医学別冊 目的別で選べる核酸実験の原理とプロトコール

    胡桃坂仁志

    羊土社  2011年06月

  • 最新医学 第65巻/6月増刊号

    齊藤健吾, 香川亘, 胡桃坂仁志

    最新医学社  2010年06月

  • タンパク質実験マニュアル

    胡桃坂仁志

    朝倉書店  2006年09月

  • 基本がわかれば面白い! バイオの授業

    胡桃坂仁志

    羊土社  2006年02月

  • Structural and biochemical analyses of human proteins A and B

    Tanaka, Y, Kurumizaka, H

    DNA Structure, Chromatin and Gene Expression, TRANSWORLD RESEARCH NETWORK  2006年

  • イラストでみる やさしい先端バイオ

    胡桃坂仁志

    羊土社  2002年

▼全件表示

Misc

  • DNA修復タンパク質RAD52のクライオ電子顕微鏡解析

    荻野 駿, 五月女 美香, 鴨井 一輝, 滝沢 由政, 胡桃坂 仁志, 香川 亘

    日本生化学会大会プログラム・講演要旨集   92回   [1T13m - 03]  2019年09月

  • ヒストンメチルトランスフェラーゼPR-Set7の生化学的および構造的解析(Biochemical and structural analyses of histone methyltransferase PR-Set7)

    何 承翰, 滝沢 由政, 小林 航, 石井 初芽, 平野 里奈, 有村 泰宏, 胡桃坂 仁志

    日本生化学会大会プログラム・講演要旨集   92回   [2T13a - 02]  2019年09月

  • CENP-Aを含むトリヌクレオソームのクライオ電子顕微鏡解析

    何 承翰, 滝沢 由政, 小林 航, 立和名 博昭, マティアス・ウォルフ, 胡桃坂 仁志

    日本生化学会大会プログラム・講演要旨集   91回   [1P - 243]  2018年09月

  • 細胞周期中のCCANの構成の動的な変化(Dynamic Change of CCAN organization during the cell cycle)

    Nagpal Harsh, 堀 哲也, Furukawa Ayako, 菅瀬 謙治, 越阪部 晃永, 胡桃坂 仁志, 深川 竜郎

    日本生化学会大会・日本分子生物学会年会合同大会講演要旨集   88回・38回   [2T23 - 13(2P0113)]  2015年12月

  • GATA3 modulates chromatin structure to establish active enhancers in breast cancer cells

    Motoki Takaku, Sara A. Grimm, Takashi Shimbo, Lalith Perera, Shinichi Machida, Hitoshi Kurumizaka, Paul A. Wade

    CANCER RESEARCH   75  2015年08月

    研究発表ペーパー・要旨(国際会議)  

    DOI

  • Structural Characterization of the Histone Multimers in the Gas Phase using Ion Mobility Mass Spectrometry and Molecular Dynamics Simulation

    Kazumi Saikusa, Sotaro Fuchigami, Kyohei Takahashi, Yuuki Asano, Aritaka Nagadoi, Hiroaki Tachiwana, Hitoshi Kurumizaka, Mitsunori Ikeguchi, Yoshifumi Nishimura, Satoko Akashi

    BIOPHYSICAL JOURNAL   106 ( 2 ) 464A - 464A  2014年01月

    研究発表ペーパー・要旨(国際会議)  

  • Nucleosome-disfavoring properties of telomeric repeat sequences in vivo

    Y. Ichikawa, N. Morohashi, H. Mikai, Y. Nishimura, H. Kurumizaka, M. Shimizu

    MOLECULAR BIOLOGY OF THE CELL   24  2013年

    研究発表ペーパー・要旨(国際会議)  

  • クロマチン高次構造上における相同組換え反応の解析

    町田晋一, 高久誉大, 小林航, 越阪部晃永, 立和名博昭, 鈴木秀和, 浦聖恵, 井倉正枝, 井倉毅, 田代聡, 胡桃坂仁志

    第35 回分子生物学会年会 2012 年12 月12 日    2012年12月

  • 固体NNRを用いたヌクレオソームにおけるヒストンH2Aの構造解析

    戸所泰人, 森脇義仁, 長土居有隆, 立和名博昭, 胡桃坂仁志, 西村善文

    Abstr Annu Meet NMR Soc Jpn   51st   318 - 319  2012年10月

    J-GLOBAL

  • DNA損傷応答に関わる新規アセチル化標的タンパク質の同定

    安田武嗣, 齋藤健吾, 香川亘, 荻朋男, 鈴木健祐, 堂前直, 日野拓也, 中沢由華, 中邑愛, 羽澤勝治, 花岡文雄, 菅澤薫, 岡安隆一, 胡桃坂仁志, 田嶋克史

    日本遺伝学会大会プログラム・予稿集   84th   100  2012年08月

    J-GLOBAL

  • The Histone-fold CENP-T-W-S-X complex induces positive supercoils into DNA

    K. Takeuchi, T. Nishino, K. Mayanagi, N. Horikoshi, A. Osakabe, H. Tachiwana, T. Hori, H. Kurumizaka, T. Fukagawa

    MOLECULAR BIOLOGY OF THE CELL   23  2012年

    研究発表ペーパー・要旨(国際会議)  

  • DNA損傷によるヒトRad52タンパク質のアセチル化は,DNA二重鎖切断部位への集積に必要である

    安田武嗣, 齋藤健吾, 香川亘, 荻朋男, 鈴木健祐, 堂前直, 日野拓也, 中沢由華, 早乙女(中邑)愛, 加藤宝光, GENET Matthew, 羽澤勝治, 冨田雅典, 花岡文雄, 菅澤薫, JEGGO Penny, 岡安隆一, 田嶋克史, 胡桃坂仁志

    日本分子生物学会年会プログラム・要旨集(Web)   35th   2W5II-1 (WEB ONLY)  2012年

    J-GLOBAL

  • CENP‐T‐W‐S‐X複合体は,動原体タンパク質群の集合機構に重要な役割を担う

    竹内康造, 西野達哉, 堀哲也, 竹内康造, 西野達哉, 堀哲也, GASCOIGNE Karen E, GASCOIGNE Karen E, 立和名博昭, 越阪部晃永, 胡桃坂仁志, CHEESEMAN Iain M, CHEESEMAN Iain M, 深川竜郎, 深川竜郎

    日本分子生物学会年会プログラム・要旨集(Web)   34th   WEB ONLY 3T12PII-9  2011年

    J-GLOBAL

  • Deimination stabilizes histone H2A/H2B dimers as revealed by electrospray ionization mass spectrometry (vol 45, pg 900, 2010)

    Shingo Shimoyama, Aritaka Nagadoi, Hiroaki Tachiwana, Michiyuki Yamada, Mamoru Sato, Hitoshi Kurumizaka, Yoshifumi Nishimura, Satoko Akashi

    JOURNAL OF MASS SPECTROMETRY   45 ( 10 ) 1232 - 1232  2010年10月

    その他  

    DOI

  • DNA結合活性を有するCCANタンパク質群の機能解析

    竹内康造, 竹内康造, 堀哲也, 堀哲也, 西野達哉, 西野達哉, 立和名博昭, 越阪部晃永, 胡桃坂仁志, 深川竜郎, 深川竜郎

    生化学     ROMBUNNO.3P-0508  2010年

    J-GLOBAL

  • 新規RAD51B相互作用タンパク質EVLの相同組換えにおける機能

    高久 誉大, 細谷 紀子, 滝沢 由政, 町田 晋一, 井川 粛子, 柴田 武彦, 宮川 清, 胡桃坂 仁志

    日本生化学会大会・日本分子生物学会年会合同大会講演要旨集   81回・31回   2T4 - 2  2008年11月

  • Biochemical analyses of human histone H3 variant, H3t

    H. Tachiwana, A. Osakabe, H. Kimura, H. Kurumizaka

    FEBS JOURNAL   275   432 - 432  2008年06月

    研究発表ペーパー・要旨(国際会議)  

  • Identification of a novel Rad51B-interacting protein

    M. Takaku, Y. Takizawa, S. Nakayama, S. Machida, H. Kurumizaka

    FEBS JOURNAL   275   116 - 116  2008年06月

    研究発表ペーパー・要旨(国際会議)  

  • Identification and biochemical analysis of the novel Rad51-binding protein

    Y. Takizawa, M. Takaku, S. Tada, Y. Morozumi, T. Ishida, H. Kurumizaka

    FEBS JOURNAL   275   116 - 116  2008年06月

    研究発表ペーパー・要旨(国際会議)  

  • The functional differences between HsRad51 and its isoform

    T. Ishida, Y. Takizawa, I. Sakane, H. Kurumizaka

    FEBS JOURNAL   275   113 - 113  2008年06月

    研究発表ペーパー・要旨(国際会議)  

  • In vitro recombinase activities of the rice Dmc1A and Dmc1B proteins

    H. Kurumizaka, I. Sakane, C. Kamataki, Y. Takizawa

    FEBS JOURNAL   275   114 - 114  2008年06月

    研究発表ペーパー・要旨(国際会議)  

  • The human PSF protein is a novel Rad51-interacting protein

    Y. Morozumi, Y. Takizawa, H. Kurumizaka

    FEBS JOURNAL   275   114 - 114  2008年06月

    研究発表ペーパー・要旨(国際会議)  

  • Analyses of the polymorphic Dmc-1-M200V variant

    J. Hikiba, K. Hirota, W. Kagawa, S. Ikawa, T. Kinebuchi, I. Sakane, Y. Takizaw, S. Yokoyama, B. Mandon-Pipin, A. Nicolas, T. Shibata, K. Ohta, H. Kurumizaka

    FEBS JOURNAL   275   113 - 113  2008年06月

    研究発表ペーパー・要旨(国際会議)  

  • Analysis on a novel functional structure in DnaA for regulating the duplex unwinding of chromosomal replication origin

    Shogo Ozaki, Hironori Kawakami, Kenta Nakamura, Norie Fujikawa, Wataru Kagawa, Shigeyuki Yokoyama, Hitoshi Kurumizaka, Tsutomu Katayama

    GENES & GENETIC SYSTEMS   82 ( 6 ) 545 - 545  2007年12月

    研究発表ペーパー・要旨(国際会議)  

  • Regulatory mechanism for replicational initiation by DiaA, a DnaA-binding factor

    Tsutomu Katayama, Kenji Keyamura, Norie Fujikawa, Takuma Ishida, Shogo Ozaki, Masayuki Suetsugu, Kazuyuki Fujimitsu, Wataru Kagawa, Shigeyuki Yokoyama, Hitoshi Kurumizaka

    GENES & GENETIC SYSTEMS   82 ( 6 ) 508 - 508  2007年12月

    研究発表ペーパー・要旨(国際会議)  

  • Characterization of human Rad51 nucleoprotein filaments using a single molecule technique

    Judith Mine, Ludovic Disseau, Masayuki Takahashi, Hitoshi Kurumizaka, Marie Dutreix, Jean-Louis Viovy

    BIOPHYSICAL JOURNAL     168A - 168A  2007年01月

    研究発表ペーパー・要旨(国際会議)  

  • Role of the N-terminal domain of the human DMC1 protein in octamer formation and DNA binding. (vol 280, pg 28382, 2005)

    Takashi Kinebuchi, Wataru Kagawa, Hitoshi Kurumizaka, Shigeyuki Yokoyama

    JOURNAL OF BIOLOGICAL CHEMISTRY   281 ( 50 ) 38966 - 38966  2006年12月

    その他  

  • RAD51 rescues compromised developmental competence of female germ cells

    GI Perez, A Jurisicova, M Gertsenstein, W Kagawa, H Kurumizaka, S Yokoyama

    JOURNAL OF THE SOCIETY FOR GYNECOLOGIC INVESTIGATION   13 ( 2 ) 294A - 294A  2006年02月

    研究発表ペーパー・要旨(国際会議)  

▼全件表示

産業財産権

  • 選択的な染色体タンパク質のアシル化を行うための人工触媒システム

    特許第6675119号

    金井 求、川島 茂裕、山次 健三、天本 義史、青井 勇樹、須藤 宏城、永島 臨、胡桃坂 仁志、越阪部 晃永、有村 泰宏

    権利者: 国立大学法人 東京大学

    特許権

  • タンパク質又は遺伝子導入用試薬

    特許第5403324号

    武岡 真司、武田 直也、胡桃坂 仁志、坂根 勲、池ヶ谷 菜海子、小幡 洋輔、齋藤 俊介

    権利者: 学校法人 早稲田大学

    特許権

受賞

  • 科学技術分野の文部科学大臣表彰 科学技術賞 研究部門

    2021年04月   文部科学省  

    受賞者: 胡桃坂 仁志

  • 持田記念学術賞

    2020年11月   公益財団法人 持田記念医学薬学振興財団   エピゲノム創薬のクロマチン構造基盤の構築  

    受賞者: 胡桃坂 仁志

  • 柿内三郎記念賞

    2018年06月   公益社団法人 日本生化学会  

    受賞者: 胡桃坂 仁志

  • 名誉教授

    2018年04月   早稲田大学  

    受賞者: 胡桃坂 仁志

共同研究・競争的資金等の研究課題

  • 胡桃坂クロマチンアトラスプロジェクト

    JST  ERATO

    研究期間:

    2019年10月
    -
    2025年03月
     

  • ヌクレオソーム高次構造とダイナミクスによるクロマチン潜在能の解明

    科学研究費助成事業(東京大学)  科学研究費助成事業(新学術領域研究(研究領域提案型))

    研究期間:

    2018年06月
    -
    2023年03月
     

  • エピジェネティクス研究と創薬のための再構成クロマチンの生産と性状解析

    AMED  創薬等ライフサイエンス研究支援基盤事業

    研究期間:

    2017年
    -
    2022年03月
     

  • 細胞ポテンシャル測定システムの開発

    JST  CREST

    研究期間:

    2016年
    -
    2019年09月
     

    大川恭行

  • 動的クロマチン構造と機能

    科学研究費助成事業(東京大学)  科学研究費助成事業(新学術領域研究(研究領域提案型))

    研究期間:

    2018年04月
    -
    2019年03月
     

  • 性スペクトラム構築の基盤となるエピゲノム情報の機能発現機構

    科学研究費助成事業(東京大学)  科学研究費助成事業(新学術領域研究(研究領域提案型))

    研究期間:

    2018年04月
    -
    2018年06月
     

  • 機能的なクロマチンの再構成と高分解能3D構造・機能解析

    科学研究費助成事業(東京大学)  科学研究費助成事業(新学術領域研究(研究領域提案型))

    研究期間:

    2018年04月
    -
    2018年06月
     

  • 構造解析用核内タンパク質等の生産と評価

    AMED  創薬等ライフサイエンス研究支援基盤事業

  • クロマチン動構造の国際共同研究ネットワーク形成

    科学研究費助成事業(早稲田大学)  科学研究費助成事業(新学術領域研究(研究領域提案型))

  • 再構成ヌクレオソームを用いた動的クロマチン構造の解明

    科学研究費助成事業(早稲田大学)  科学研究費助成事業(新学術領域研究(研究領域提案型))

  • 動的クロマチン構造と機能

    科学研究費助成事業(早稲田大学)  科学研究費助成事業(新学術領域研究(研究領域提案型))

  • クロマチンDNAにおける相同的組換え機構に関する研究

    科学研究費助成事業(理化学研究所)  科学研究費助成事業(奨励研究(A))

  • ヒトにおける相同DNA組換えの分子機構に関する研究

    科学研究費助成事業(理化学研究所)  科学研究費助成事業(奨励研究(A))

  • 染色体の安定維持に重要なヒトRad51ファミリータンパク質の構造、機能解析

    科学研究費助成事業(理化学研究所)  科学研究費助成事業(特定領域研究(C))

  • DnaAドメインIVを用いた新規抗菌剤の探索開発プロセスの研究

    科学研究費助成事業(九州大学)  科学研究費助成事業(基盤研究(B))

  • がん化防御に重要なDNA組換え修復装置の構造および機能解析

    科学研究費助成事業(理化学研究所)  科学研究費助成事業(特定領域研究(C))

  • ヒトDNA組換え修復装置による発がん防御の分子機構の解析

    科学研究費助成事業(理化学研究所)  科学研究費助成事業(特定領域研究)

  • DNA組換え修複による発がん防御の分子メカニズム

    科学研究費助成事業(特殊法人理化学研究所)  科学研究費助成事業(特定領域研究)

  • 特殊なDNA構造に結合する新規タンパク質の同定とその立体構造予測に関する研究

    科学研究費助成事業(特殊法人理化学研究所)  科学研究費助成事業(特定領域研究)

  • ヒトDNA組換え装置サブユニットの高次構造と機能に関する研究

    科学研究費助成事業(特殊法人理化学研究所)  科学研究費助成事業(若手研究(B))

  • バクテリア・セントロメア様配列migSの染色体分配機構における分子機能の解明

    科学研究費助成事業(国立遺伝学研究所)  科学研究費助成事業(基盤研究(B))

  • 染色体複製開始とその制御を行う、動的かつ複合的蛋白質高次装置の機能構造特性

    科学研究費助成事業(九州大学)  科学研究費助成事業(基盤研究(B))

  • DNA組換え修復タンパク質群による発がん防御の分子機構

    科学研究費助成事業(早稲田大学)  科学研究費助成事業(特定領域研究)

  • 単鎖DNA結合タンパク質の新規立体構造モチーフ解析とデータベースからの検索

    科学研究費助成事業(早稲田大学)  科学研究費助成事業(特定領域研究)

  • 染色体ダイナミクスに関わるタンパク質の構造・機能解析

    科学研究費助成事業(早稲田大学)  科学研究費助成事業(特定領域研究)

  • 発がんの原因となる二重鎖切断を正確に修復する相同組換え装置の機能・構造解析

    科学研究費助成事業(早稲田大学)  科学研究費助成事業(特定領域研究)

  • 相同組換えタンパク質の高次構造ダイナミクスとその機能的役割の解明

    科学研究費助成事業(早稲田大学)  科学研究費助成事業(基盤研究(C))

  • 記号とパターンの統合によるin silicoバイオインフォマティックス

    科学研究費助成事業(早稲田大学)  科学研究費助成事業(基盤研究(A))

  • 発がん防御に重要な新規二重鎖切断修復タンパク質の探索とその機能・構造解析

    科学研究費助成事業(早稲田大学)  科学研究費助成事業(特定領域研究)

  • 染色体ダイナミクスの生化学・構造生物学的解析

    科学研究費助成事業(早稲田大学)  科学研究費助成事業(特定領域研究)

  • 遺伝情報の発現を制御するクロマチンドメインの分子構築と動態

    科学研究費助成事業(明星大学)  科学研究費助成事業(基盤研究(C))

  • 遺伝情報収納・発現・継承の時空間場

    科学研究費助成事業(大阪大学)  科学研究費助成事業(新学術領域研究(研究領域提案型))

  • 二重鎖切断修復に働く新規発がん防御因子の探索とその機能・構造解析

    科学研究費助成事業(早稲田大学)  科学研究費助成事業(特定領域研究)

  • 細胞核の機能的複合体の構造と動作メカニズム

    科学研究費助成事業(早稲田大学)  科学研究費助成事業(新学術領域研究(研究領域提案型))

  • セントロメア領域に特異的なヌクレオソームの結晶構造解析

    科学研究費助成事業(早稲田大学)  科学研究費助成事業(若手研究(スタートアップ))

  • 転写制御におけるクロマチンダイナミズムを規定するシス及びトランス因子の機能解析

    科学研究費助成事業(明星大学)  科学研究費助成事業(基盤研究(C))

  • 新規相同組換え因子の探索と組換え反応機構の解析

    科学研究費助成事業(早稲田大学)  科学研究費助成事業(基盤研究(B))

  • セントロメア領域のクロマチン構造基盤の解析

    科学研究費助成事業(早稲田大学)  科学研究費助成事業(若手研究(B))

  • クロマチンにおける相同組換えの分子機構に関する研究

    科学研究費助成事業(早稲田大学)  科学研究費助成事業(基盤研究(A))

▼全件表示

講演・口頭発表等

  • クロマチン構造における RNA ポリメラーゼ II による転写伸長機構

    胡桃坂仁志

    日本生化学会関東支部例会  

    発表年月: 2021年06月

  • クロマチンアトラスプロジェクト・キックオフシンポジウム

    胡桃坂仁志, 岡田由紀, 山口潤一郎, 吉川雅英, 藤芳暁

    発表年月: 2021年05月

  • クライオ電子顕微鏡によるクロマチン構造解析

    胡桃坂 仁志  [招待有り]

    令和2年度 よこはまNMR研究会総会  

    発表年月: 2021年03月

  • Structural Studies for Nucleosome Core Particle Complexed with Its Binding Factors

    胡桃坂 仁志  [招待有り]

    RIKEN BDR Symposium  

    発表年月: 2021年03月

    開催年月:
    2021年03月
     
     
  • Nucleosome contribution to epigenetic genome regulation

    胡桃坂 仁志  [招待有り]

    Biophysical Society 65th Annual Meeting  

    発表年月: 2021年02月

    開催年月:
    2021年02月
     
     
  • クロマチンダイナミクスの構造生物学的研究

    胡桃坂 仁志  [招待有り]

    第16回日本臨床プロテオゲノミクス研究会  

    発表年月: 2020年12月

  • クロマチン構造による遺伝子発現のエピジェネティック制御

    胡桃坂 仁志  [招待有り]

    第43回日本分子生物学会年会  

    発表年月: 2020年12月

    開催年月:
    2020年12月
     
     
  • クライオEM 単粒子解析によるクロマチンダイナミクスの理解

    胡桃坂 仁志  [招待有り]

    日本中性子科学会2020年会  

    発表年月: 2020年11月

    開催年月:
    2020年11月
     
     
  • エピジェネティクスの根幹を担うクロマチン機能構造の可視化

    胡桃坂 仁志  [招待有り]

    第93回日本生化学会大会  

    発表年月: 2020年09月

    開催年月:
    2020年09月
     
     
  • Cryo-EM studies for nucleosome and chromatin

    胡桃坂 仁志  [招待有り]

    第92回日本生化学会大会  

    発表年月: 2019年09月

    開催年月:
    2019年09月
     
     
  • 21世紀の新たな遺伝学 エピジェネティクス

    胡桃坂仁志  [招待有り]

    日本遺伝学会第91回大会公開市民講座「エピジェネティクスの視点から生命の謎に迫る」  

    発表年月: 2019年09月

  • Structural studies of chromatin towards the understanding of epigenetics mechanisms

    胡桃坂仁志  [招待有り]

    Telluride workshop  

    発表年月: 2019年07月

  • DNA配列に依存しない遺伝子制御機構 「エピジェネティクス」の構造基盤

    胡桃坂仁志  [招待有り]

    広島大学統合生命科学研究科開設記念シンポジウム〜ゲノムから地球環境まで  

    発表年月: 2019年06月

  • Cryo-EM imaging of the chromatin architecture

    胡桃坂仁志  [招待有り]

    第19回日本蛋白質科学会年会 第71回日本細胞生物学会大会 合同年次大会  

    発表年月: 2019年06月

  • ヌクレオソーム高次構造とダイナミクスの解析によるクロマチン潜在能の解明

    胡桃坂仁志

    新学術領域クロマチン潜在能第2回領域会議  

    発表年月: 2019年06月

  • 創薬の基盤となるエピジェネティクスの構造生物学

    胡桃坂仁志  [招待有り]

    第56回薬剤学懇談会研究討論会  

    発表年月: 2019年06月

  • Structural studies of the chromatin: Towards the understanding of epigenetics

    胡桃坂仁志  [招待有り]

    第13回日本エピジェネティクス研究会年会  

    発表年月: 2019年05月

  • Structural studies of chromatin: Toward understanding the regulation of genomic DNA

    胡桃坂 仁志  [招待有り]

    Multiscale Modeling of Chromatin: Bridging Experiment with Theory  

    発表年月: 2019年04月

  • クロマチン機能発現の構造基盤

    胡桃坂 仁志

    染色体研究の最前線2019  

    発表年月: 2019年03月

  • クロマチンに潜在する遺伝子制御能の構造基盤

    第41回日本分子生物学会年会  

    発表年月: 2018年11月

  • クロマチン基盤構造の多様性とDNA損傷修復

    日本放射線影響学会第61回大会  

    発表年月: 2018年11月

  • Structural studies of genomic DNA regulation by chromatin

    3R+3C:Replication, Recombination and Repair (3R) with special focus on Chromosome, Chromatin and Cell Cycle (3C)  

    発表年月: 2018年11月

  • 動的構造生物学によるクロマチン機能解析

     [招待有り]

    大阪大学蛋白質研究所セミナー 構造生物学と計算科学の融合による動的構造生物学の新しい展開  

    発表年月: 2018年09月

  • エピジェネティック創薬の基盤となるクロマチン構造とダイナミクスに関する研究

     [招待有り]

    第16回次世代を担う若手のためのフィジカル・ファーマフォーラム  

    発表年月: 2018年09月

  • クロマチンによる遺伝子発現制御の構造基盤

     [招待有り]

    第91回日本生化学会大会  

    発表年月: 2018年09月

  • Contribution of CENP-A into centromeric chromatin architecture

     [招待有り]

    Gordon Conference The Structure, Function and Evolution of Centromeres  

    発表年月: 2018年07月

  • Structural insights into the chromatin dynamics that underlie genome function

     [招待有り]

    FASEB Machines on Genes?  

    発表年月: 2018年06月

  • クロマチンコーディングの構造基盤

     [招待有り]

    染色体研究の最前線2018  

    発表年月: 2018年03月

  • Nucleosome Remodeling and Structure

     [招待有り]

    INDO-JAPAN Conference  

    発表年月: 2018年02月

  • クロマチンの高次構造とダイナミクスの相関構造解析

     [招待有り]

    第31回日本放射光学会年会・放射光科学合同シンポジウム  

    発表年月: 2018年01月

  • 再構成クロマチンによるエピジェネティクス機構の解析

     [招待有り]

    2017年度生命科学系学会合同年次大会  

    発表年月: 2017年12月

  • Structural biology of chromatin:Towards the understanding of epigenetics

     [招待有り]

    発表年月: 2017年11月

  • Structural Biology of Epigenetic Chromatin Regulation

     [招待有り]

    15th Chinese Biophysics Congress  

    発表年月: 2017年11月

  • Structural studies of reconstituted chromatin units

     [招待有り]

    HMGU-Japan Epigenetics and Chromatin Meeting  

    発表年月: 2017年09月

  • ヌクレオソームのリモデリング機構

     [招待有り]

    第76回日本癌学会学術総会  

    発表年月: 2017年09月

  • Structural studies of the nucleosome-nucleosome interaction

     [招待有り]

    EMBO Conference: The Nucleosome: From Atoms to Genomes  

    発表年月: 2017年08月

  • エピジェネティクスの制御基盤としてのクロマチン構造多様性

     [招待有り]

    構造生命科学のフロンティア -次世代の統合的研究に向けて-  

    発表年月: 2017年06月

  • Structural studies for dynamic chromatin architecture

     [招待有り]

    Pioneering Project "Dynamic Structural Biology" 第1回研究報告会  

    発表年月: 2017年06月

  • ヌクレオソームの構造と動的多様性によるゲノムDNA機構制御

     [招待有り]

    第17回日本蛋白質科学会年会  

    発表年月: 2017年06月

  • 細胞核でのクロマチン機能制御の構造基盤

     [招待有り]

    日本大学文理学部生命科学セミナー 細胞核機能の発現と制御  

    発表年月: 2017年06月

  • Chromatin contribution in DNA repair

     [招待有り]

    The 6th US-Japan DNA Repair Meeting  

    発表年月: 2017年05月

  • クライオ電験とX線結晶構造解析によるクロマチン高次構造とダイナミクス

     [招待有り]

    よこはまNMR研究会  

    発表年月: 2017年03月

  • Structural basis of epigenetic chromatin regulation

     [招待有り]

    The 5th International Symposium of the Mathematics on Chromatin Dynamics  

    発表年月: 2017年03月

  • Structural studies for functional chromatin

     [招待有り]

    Japan-Swiss Symposium Chromatin Structure and Dynamics  

    発表年月: 2017年01月

  • 染色体の構造基盤

     [招待有り]

    ワークショップ染色体研究の最前線  

    発表年月: 2017年01月

  • 岡崎フラグメント50周年シンポジウム

     [招待有り]

    発表年月: 2016年12月

  • Structural studies for epigenetic regulation of genomic DNA

     [招待有り]

    10th International 3R (Replication, Recombination and Repair) Symposium  

    発表年月: 2016年11月

  • Three dimensional structures and dynamics of chromatin

     [招待有り]

    第54回生物物理学会  

    発表年月: 2016年11月

  • クロマチン構造によるゲノムDNA制御

     [招待有り]

    平成28年度遺伝研研究会「クロマチン・細胞核の動的構造変換とエピジェネティック制御」  

    発表年月: 2016年10月

  • がん細胞で見られるヒストン変異のクロマチン構造・動態変動への影響

     [招待有り]

    第89回日本生化学会大会  

    発表年月: 2016年09月

  • Structural basis of chromatin dynamics

     [招待有り]

    Telluride workshop on chromatin structure and dynamics  

    発表年月: 2016年08月

  • Structural Versatility of Nucleosomes and Chromatin Dynamics

     [招待有り]

    Colorado Chromatin Meeting 2016  

    発表年月: 2016年08月

  • 遺伝子のすがたを原子レベルで見る

     [招待有り]

    一般公開シンポジウム「遺伝子のすがたーカラダの中で起こる不思議―」  

    発表年月: 2016年08月

  • Altered structures and physical characteristics of nucleosomes containing cancer-associated histone mutations

     [招待有り]

    DNA metabolism, genome stability and diseases  

    発表年月: 2016年06月

  • HISTONE CONTRIBUTIONS TO CHROMATIN DYNAMICS

     [招待有り]

    Chromatin, Epigenetics and Transcription  

    発表年月: 2016年05月

  • 天然変性ハブとしてのクロマチン構造

     [招待有り]

    横浜NMR研究会  

    発表年月: 2016年01月

  • 精巣特異的リンカーヒストンバリアントH1Tによる高次クロマチン構造形成と相同組換え反応への影響

    町田晋一, 林田亮太, 高久誉大, 福戸敦彦, 孫継英, 木野村愛子, 田代聡, 胡桃坂仁志

    発表年月: 2016年01月

  • 新生ヒストンの貯蔵に関与するヒストンシャペロンーヒストン複合体の機能解析

    加藤大貴, 越阪部晃永, 明石知子, 胡桃坂仁志

    発表年月: 2016年01月

  • ヒストンバリアントH3.6を含むヌクレオソームはDNAからヒストンが解離しやすい

    田口裕之, 堀越直樹, 佐藤浩一, 有村泰宏, 前原一満, 原田哲仁, 木村宏, 大川恭行, 胡桃坂仁志

    発表年月: 2016年01月

  • ヒストンバリアントH2A.Zによる転写活性化機構

    堀越直樹, 佐藤浩一, 日下部将之, 奥裕之, 原田昌彦, 木村宏, 胡桃坂仁志

    発表年月: 2016年01月

  • セントロメア特異的ヒストンバリアントCENP-Aの形成するクロマチン構造の解析

    立和名博昭, 鈴木翠, 胡桃坂仁志

    発表年月: 2016年01月

  • クロマチンにおける紫外線損傷DNA認識機構の解析

    越阪部晃永, 立和名博昭, 堀越直樹, 香川亘, 松本翔太, 山元淳平, 花岡文雄, 菅澤薫, 岩井成憲, 胡桃坂仁志

    発表年月: 2016年01月

  • がんゲノムデータベースより見出されたヒストン点変異がヌクレオソーム構造および細胞に及ぼす影響の解析

    有村泰宏, 野田真美子, 藤田理紗, 井倉(野村)正枝, 杉山正明, 木村宏, 井倉毅, 胡桃坂仁志

    発表年月: 2016年01月

  • DNA二重鎖切断損傷修復に重要なヒストン翻訳後修飾H2AK15ub,H4K20me2がクロマチン構造に与える影響

    関根 慧, 小林 航, 町田 晋一, 胡桃坂 仁志

    発表年月: 2016年01月

  • クロマチン構造とダイナミクスの分子機構

     [招待有り]

    国際高等研究所 クロマチン・デコーディング  

    発表年月: 2015年12月

  • クロマチン構造によるDNA機能のエピジェネティック制御

     [招待有り]

    第38回日本分子生物学会年会、第88回日本生化学会大会合同大会  

    発表年月: 2015年12月

  • Structural basis of chromatin dynamics

     [招待有り]

    4th International Symposium of the Mathematics on Chromatin Live Dynamics  

    発表年月: 2015年12月

  • 精巣特異的ヒストンバリアントH2AL2とTH2Bの機能解析

    浦浜嵩, Sophie Barral, 田中大貴, 両角佑一, 越阪部晃永, Saadi Khochbin, 胡桃坂仁志

    発表年月: 2015年12月

  • 相同組換えにおけるRPA2のユビキチン化を介した分解

    稲野 将二郎, 佐藤 浩一, 石合 正道, 勝木 陽子, 中田 慎一郎, 胡桃坂 仁志, 高田 穣

    発表年月: 2015年12月

  • 新規ヒストンH3バリアントH3mm7は骨格筋分化能を制御する

    原田哲仁, 前原一満, 田口裕之, 佐藤優子, 謝炎, 立花太郎, 木村宏, 胡桃坂仁志, 大川恭行

    発表年月: 2015年12月

  • ヒストンバリアントH2A.Bは、ヌクレオソーム中で自らを他のH2Aバリアントと置き換える

    有村泰宏, 矢島成人, 白山一義, 野田真美子, 藤田理紗, 胡桃坂仁志

    発表年月: 2015年12月

  • ヌクレオソームの新たな立体構造解析法の開発

    浦野一輝, 堀越直樹, 鯨井智也, 田口裕之, 胡桃坂仁志, 香川亘

    発表年月: 2015年12月

  • セントロメアタンパク質CENP-BとCENP-Aヌクレオソームの相互作用解析

    藤田理紗, 大竹興一郎, 有村泰宏, 宮優太, 越阪部晃永, 立和名博昭, 大関淳一郎, 舛本寛, 胡桃坂仁志

    発表年月: 2015年12月

  • シナプトネマ複合体による減数分裂期相同組換えの制御機構

    小林航, 高久誉大, 寺本睦美, 町田晋一, 立和名博昭, 細谷紀子, 宮川清, 前原一満, 大川恭行, 胡桃坂仁志

    発表年月: 2015年12月

  • Overlapping dinucleosomeの構造生物学的および物理化学的解析

    足立 風水也, 越阪部 晃永, 堀越 直樹, 有村 泰宏, 七種 和美, 明石 知子, 西村 善文, 杉山 正明, 松本 淳, 河野 秀俊, 胡桃坂 仁志

    発表年月: 2015年12月

  • ヒストンバリアントによるクロマチン動構造制御

     [招待有り]

    遺伝研研究会 クロマチン・細胞核構造の形成とダイナミクスによるゲノム機能制御  

    発表年月: 2015年10月

  • 相同組換え修復に重要なFANCI-FANCD2複合体の生化学的機能解析

    佐藤浩一, 下向真代, 胡桃坂仁志

    発表年月: 2015年10月

  • シナプトネマ複合体構成因子SYCP3の減数分裂期組換えにおける機能解析

    小林航, 町田晋一, 寺本睦美, 細谷紀子, 宮川清, 胡桃坂仁志

    発表年月: 2015年10月

  • Structural versatility and dynamics of chromatin

     [招待有り]

    第53回日本生物物理学会年会  

    発表年月: 2015年09月

  • Chromatin structure and dynamics regulated by histonesChromatin and Epigenetics

     [招待有り]

    Chromatin and Epigenetics -Dr. Robert T. Simpson memorial meeting-  

    発表年月: 2015年08月

  • Histone Contributions in Chromatin Dynamics

     [招待有り]

    International Symposium on Chromatin Structure, Dynamics and Function  

    発表年月: 2015年08月

  • The distincit property of histone H3.Y nucleosome

    ○Tomoya Kujirai, Naoki Horikoshi, Koichi Sato, Kazumitsu Maehara, Akihisa Osakabe, Shinichi Machida, Yasuyuki Ohkawa, Hiroshi Kimura, Hitoshi Kurumizaka

    発表年月: 2015年08月

  • The biochemical analyses of the chromatin containing mono-ubiquitinated nucleosomes

    ◯Shinichi Machida, Yuki Nishiyama, Hitoshi Kurumizaka

    発表年月: 2015年08月

  • The Importance of Chromatin Dynamics for Genetic Activities

    ○Frederic Berger, Takashi Urahama, Zdravko Lorkovic, Tomokazu Kawashima, Ramesh Yelagandula, Hiroyuki Taguchi, Naoki Horikoshi, Hitoshi Kurumizaka

    発表年月: 2015年08月

  • Structural, Biochemical, and Genome-Wide Analyses of Human Histone Variant H3.5

    Takashi Urahama, Akihito Harada, Kazumitsu Maehara, Naoki Horikoshi, Koichi Sato, Yuko Sato, Koji Shiraishi, Norihiro Sugino, Akihisa Osakabe, Hiroaki Tachiwana, Wataru Kagawa, Hiroshi Kimura, Yasuyuki Ohkawa, Hitoshi Kurumizaka

    発表年月: 2015年08月

  • Structural and biochemical analyses of the nucleosome containing a novel H3 variant, H3F3AP6

    ○Hiroyuki Taguchi, Naoki Horikoshi, Koichi Sato, Yasuhiro Arimura, Akihisa Osakabe, Kazumitsu Maehara, Hiroshi Kimura, Yasuyuki Ohkawa, Hitoshi Kurumizaka

    発表年月: 2015年08月

  • Structural and biochemical analyses for accommodation and recognition of UV-damaged bases within nucleosome

    ○Akihisa Osakabe, Hiroaki Tachiwana, Wataru Kagawa, Naoki Horikoshi, Syota Matsumoto, Junpei Yamamoto, Fumio Hanaoka, Kaoru Sugasawa, Shigenori Iwai, Hitoshi Kurumizaka

    発表年月: 2015年08月

  • Structural Insights into Rapid Assembly and Disassembly of the CRM1-Nuclear Export Complex

    ○Masako Koyama, Natsuki Shirai, Yoshiyuki Matsuura, Hitoshi Kurumizaka

    発表年月: 2015年08月

  • Reconstitution and structural analyses of the heterotypic nucleosome containing H2A.Z and H2A

    ○Naoki Horikoshi, Koichi Sato, Yasuhiro Arimura, Hiroyuki Oku, Masayuki Kusakabe, Masahiko Harata, Hiroshi Kimura, Hitoshi Kurumizaka

    発表年月: 2015年08月

  • Mouse Testis Specific Histone H3 Variant, H3mmT is Essential for Spermatogenesis and Ensures the Entry into Meiosis

    ○Jun Ueda, Takashi Urahama, Akihito Harada, Shinichi Machida, Kazumitsu Maehara, Naoki Horikoshi, Akihisa Osakabe, Hiroaki Tachiwana, Tatsuma Yao, Minami Yamada, Takashi Iwamoto, Masahito Ikawa, Taro Tachibana, Hiroshi Kimura, Yasuyuki Ohkawa, Hitoshi Kurumizaka, Kazuo Yamagata

    発表年月: 2015年08月

  • Intrinsic Histone Exchange Property of the Human Histone Variant H2A.B

    ○Yasuhiro Arimura, Kazuyoshi Shirayama, Naruto Yajima, Hitoshi Kurumizaka

    発表年月: 2015年08月

  • Histone Chaperone tNASP Promotes Nucleosome Assembly with H3.3

    ○Daiki Kato, Akihisa Osakabe, Hiroaki Tachiwana, Hiroki Tanaka, Hitoshi Kurumizaka

    発表年月: 2015年08月

  • Biochemical analysis of RAD51 and DMC1 in the synaptonemal complex

    ○Wataru Kobayashi, Motoki Takaku, Mutsumi Teramoto, Shinichi Machida, Hiroaki Tachiwana, Noriko Hosoya, Kiyoshi Miyagawa, Kazumitsu Maehara, Yasuyuki Ohkawa, Hitoshi Kurumizaka

    発表年月: 2015年08月

  • Biochemical analysis of DNA crosslink repair proteins, FANCI-FANCD2 complex in homologous recombination

    ◯Koichi Sato, Mayo Shimomuki, Hitoshi Kurumizaka

    発表年月: 2015年08月

  • Analyses of Structure and Dynamics with Model Chromatin

    Hiroaki Tachiwana, Yasuhiro Arimura, Ryohei Matsumoto, Yoshimasa Takizawa, Tetsuya Hori, Atsushi Matsumoto, Takashi Oda, Mamoru Sato, Hidetoshi Kono, Tatsuo Fukagawa, Melanie D. Ohi, Hitoshi Kurumiza

    発表年月: 2015年08月

  • ヒストン修飾特異的一本鎖抗体の結合解析

    鯨井智也, 堀越直樹, 佐藤優子, 木村宏, 胡桃坂仁志

    発表年月: 2015年07月

  • 第9回エピジェネティクス研究会年会 ランチョンセミナー

     [招待有り]

    発表年月: 2015年05月

  • Structural properties of the nucleosomes containing cancer-associated mutations

     [招待有り]

    大阪大学蛋白研セミナーNuclear organization and actin-dependent mechanisms in genome stability  

    発表年月: 2015年05月

  • ヒストンH3.Yが形成するクロマチン及びヌクレオソームの機能解析

    鯨井智也, 堀越直樹, 佐藤浩一, 前原一満, 越阪部晃永, 町田晋一, 有村泰宏, 立和名博昭, 大川恭行, 木村宏, 胡桃坂仁志

    発表年月: 2015年05月

  • がんで高頻度に検出されるヒストン点変異H2B E76Kを含むヌクレオソームの解析

    野田真美子, 有村泰宏, 藤田理紗, 白山一義, 木村宏, 胡桃坂仁志

    発表年月: 2015年05月

  • Overlapping dinucleosomeの再構成および生化学的解析

    足立風水也, 越阪部晃永, 堀越直樹, 有村泰宏, 七種和美, 明石知子, 西村善文, 杉山正明, 胡桃坂仁志

    発表年月: 2015年05月

  • 原子レベルで見る生命の設計図

    発表年月: 2015年01月

  • Strucuural and functional studies of various nucleosomes as fundamental building blocks for Chromatin

    発表年月: 2014年12月

  • Biochemical studies for homologous recombination reaction in chromatin

    発表年月: 2014年11月

  • クロマチンのエピジェネティック制御と創薬

    発表年月: 2014年11月

  • 第87回日本生化学会大会

    発表年月: 2014年10月

  • クロマチン動構造とヒストンバリアント

    発表年月: 2014年09月

  • クロマチン構造とダイナミクスの多様性による遺伝子のエピジェネティクス制御機構

    発表年月: 2014年08月

  • 再構成ヌクレオソームを用いた動的クロマチン構造の解明

    発表年月: 2014年07月

  • BIOCHEMICAL ANALYSES OF RICE DNA RECOMBINASES RAD51 AND DMC1

    発表年月: 2014年07月

  • Structures and physical properties of nucleosome isoforms with histone variants

    発表年月: 2014年06月

  • ワークショップ「クロマチンの動態構造とDNA機能発現機構」

    発表年月: 2014年06月

  • Structure and dynamics of the nucleosome isoforms

    発表年月: 2014年05月

  • エピジェネティクスにおけるヒストンバリアントの役割

    発表年月: 2014年05月

  • Structural basis for chromatin dynamics

    発表年月: 2014年03月

  • Structure and dynamics of the nucleosome isoforms

    発表年月: 2014年03月

  • Nucleosome-disfavoring properties of telomeric repeat sequences in vivo

    発表年月: 2013年12月

  • ICL修復に重要なFAN1ヌクレアーゼの生化学的機能解析

    発表年月: 2013年12月

  • Histone H3-ubiquitination by UHRF1 in maintenance of DNA methylation

    発表年月: 2013年12月

  • p53 isoform、∆ 1stTAD-p53の機能解析及び標的遺伝子の同定

    発表年月: 2013年12月

  • クロマチン機能発現の基盤としてのヌクレオソーム構造多様性

    発表年月: 2013年12月

  • CENP-Aヌクレオソームにおける動的なDNA構造とその制御機構

    発表年月: 2013年12月

  • メチル化DNAを含むヌクレオソームの再構成および生化学的物理学的解析

    発表年月: 2013年12月

  • リンカーヒストンを含む高次クロマチン構造における相同組換え機構

    発表年月: 2013年12月

  • ファンコニ貧血原因遺伝子産物FANCD2の示すヒストンシャペロン活性の生理的意義

    発表年月: 2013年12月

  • 生細胞蛍光イメージングに適した低分子蛍光色素の探索

    発表年月: 2013年12月

  • 出芽酵母Ume6における転写活性化および抑制に関与する天然変性領域の同定

    発表年月: 2013年12月

  • Cdt1結合蛋白質GRWD1は新規ヒストンシャペロンでありクロマチン構造とMCM結合を制御している

    発表年月: 2013年12月

  • 酵母Rad52 C末端側領域の相同組換えにおける役割

    発表年月: 2013年12月

  • ヘテロクロマチンタンパク質HP1のリン酸化修飾と高次クロマチン構造形成のダイナミクス

    発表年月: 2013年12月

  • CTGとATTCTリピート配列によるヌクレオソームポジショニング

    発表年月: 2013年12月

  • 分裂酵母ヒストンバリアント Pht1 およびCnp1 を含むヌクレオソームの 生化学的解析

    発表年月: 2013年11月

  • 新規ヒトヒストンバリアント H3F3AP6 を含むヌクレオソームの機能解析

    発表年月: 2013年11月

  • クロマチンでの相同組換え反応におけるDMC1の機能

    発表年月: 2013年11月

  • DNA 鎖間架橋修復に重要な FANCI-FANCD2 複合体の生化学的解析

    発表年月: 2013年11月

  • FAN1 ヌクレアーゼが担うDNA 鎖間架橋除去機構の解明

    発表年月: 2013年11月

  • 紫外線損傷DNAを含むヌクレオソームの構造生物学・生化学的解析

    発表年月: 2013年11月

  • Biochemical analysis of the ICL repair proteins, FANCI-FANCD2 complex

    発表年月: 2013年11月

  • Biochemical analysis of the ICL repair proteins, FANCI-FANCD2 complex

    発表年月: 2013年11月

  • Roles of Fanconi anemia protein FANCD2 in DNA cross link repair

    発表年月: 2013年11月

  • Compensatory functions and interdependency of the DNA-binding domain of BRCA2 with the BRCA1-PALB2 complex

    発表年月: 2013年11月

  • 分裂酵母ヒストンバリアント Pht1 および Cnp1 を含むヌクレオソームの 生化学的解析

    発表年月: 2013年11月

  • 新規ヒトヒストンバリアント H3F3AP6 を含むヌクレオソームの機能解析

    発表年月: 2013年11月

  • ヒトヒストンバリアントH2A.B を含むヌクレオソームの機能解析

    発表年月: 2013年11月

  • メチル化DNAを含むヌクレオソームの再構成および生化学的解析

    発表年月: 2013年11月

  • リンカーヒストンH1バリアントの機能解析

    発表年月: 2013年11月

  • セントロメアタンパク質CENP-BのCENP-B boxへの結合におけるCENP-Aヌクレオソームの影響

    発表年月: 2013年11月

  • In vivoにおけるヌクレオソーム形成に及ぼすテロメア反復配列とその結合因子TRF1の影響

    発表年月: 2013年11月

  • ヘテロクロマチンタンパク質HP1 のリン酸化修飾と高次クロマチン構造形成のダイナミクス

    発表年月: 2013年11月

  • Homologous recombination reaction in higher ordered chromatin

    発表年月: 2013年10月

  • エピジェネティクス機構の理解を目指したクロマチン構造生物学

    発表年月: 2013年10月

  • クロマチン高次構造形成におけるヌクレオソーム構造多様性

    発表年月: 2013年10月

  • ヒストンバリアントによるエピジェネティクスの構造基盤

    発表年月: 2013年09月

  • 減数分裂期特異的な相同組換え酵素DMC1のクロマチンにおける反応機構

    発表年月: 2013年09月

  • DNA鎖間架橋の修復に働くFANCI-FANCD2複合体及びFAN1ヌクレアーゼの機能解析

    発表年月: 2013年09月

  • CENP-Aヌクレオソーム特異的なDNA構造の構造生物学的・生化学的解析

    発表年月: 2013年09月

  • ヒト精巣特異的に高発現するヒストンバリアントH3.5を含むヌクレオソームの構造解析および機能解析

    発表年月: 2013年09月

  • Stability of nucleosomes containing histone variants

    発表年月: 2013年09月

  • Stability of nucleosomes containing histone variants

    発表年月: 2013年09月

  • ヒトヒストンバリアントH2A.Bの機能解析

    発表年月: 2013年09月

  • シナプトネマ複合体構成因子ヒトSYCP3の生化学的機能解析

    発表年月: 2013年09月

  • 紫外線損傷DNAを含むヌクレオソームの生化学的解析

    発表年月: 2013年09月

  • リンカーヒストンH1バリアントの生化学的解析

    発表年月: 2013年09月

  • 相同組換えにおける高次クロマチンの動的な制御

    発表年月: 2013年09月

  • テロメアリピート配列はin vivoでのヌクレオソーム形成を阻害する

    発表年月: 2013年09月

  • リンカーヒストンH1-ヌクレオソーム複合体(クロマトソーム)の生化学および構造生物学的解析

    発表年月: 2013年09月

  • ヒストン修飾特異的scFv抗体の機能解析

    発表年月: 2013年09月

  • 染色体DNA相同組換え修復の細胞核内局在

    発表年月: 2013年09月

  • ヘテロクロマチンタンパク質HP1による高次クロマチン構造形成のメカニズム

    発表年月: 2013年09月

  • High order chromatin regulation in skeletal muscle differentiation

    発表年月: 2013年09月

  • G-clusters play crucial roles in the nucleosome-disfavoring properties of telomeric repeat sequences in vivo

    発表年月: 2013年09月

  • Analysis of the molecular function and identification of target genes of ∆ 1stTAD-p53, an isoform of p53

    発表年月: 2013年09月

  • 再構成ヌクレオソームを用いた動的クロマチン構造の解明

    発表年月: 2013年08月

  • ヒト精巣特異的クロマチンの構造と性質の解析

    発表年月: 2013年07月

  • ヒストンバリアントH2A.Bによるヌクレオソーム形成とその構造・機能解析

    発表年月: 2013年05月

  • エピゲノムを担う機能的クロマチンにおけるヒストンバリアントの構造的役割

    発表年月: 2012年12月

  • DNA修復タンパク質Rad52と一本鎖DNAの複合体のX線結晶構造解析

    発表年月: 2012年12月

  • FANCD2のヒストンシャペロン活性がDNA修復機能に果たす役割

    発表年月: 2012年12月

  • FANCD2のモノユビキチン化機構の解析

    発表年月: 2012年12月

  • CENP-CによるCENP-Aヌクレオソーム認識機構の生化学的・構造生物学的解析

    発表年月: 2012年12月

  • 出芽酵母ミニ染色体においてヒトテロメア配列およびテロメア結合因子がヌクレオソーム形成に及ぼす影響

    発表年月: 2012年12月

  • RAD51によるヌクレオソーム構造上での相同鎖検索機構の解析

    発表年月: 2012年12月

  • クロマチン相同組換えにおけるPSFの機能解析

    発表年月: 2012年12月

  • DNA鎖間架橋応答におけるファンコニ貧血経路によるCtIPの調節

    発表年月: 2012年12月

  • H2A.Zを含むヌクレオソームの構造生物学的および生化学的解析

    発表年月: 2012年12月

  • Cdt1結合蛋白質GRWD1は複製ライセンシングおよび細胞増殖に関わる新規ヒストンシャペロンである

    発表年月: 2012年12月

  • クロマチン構造上におけるDMC1及びRAD51の機能的解析

    発表年月: 2012年12月

  • 固体NMRを用いたクロマチン構造におけるヒストンの構造解析

    発表年月: 2012年12月

  • 出芽酵母のUme6のactivatorとrepressorとしての機能に関与する天然変性領域の解析

    発表年月: 2012年12月

  • ATTCTとCTGリピートによるヌクレオソーム形成の促進とその転写に及ぼす影響

    発表年月: 2012年12月

  • リンカーヒストンH1サブタイプを含むクロマトソームの生化学的解析

    発表年月: 2012年12月

  • DNA損傷による人Rad52タンパク質のアセチル化はDNA二重鎖切断部位への集積に必要である

    発表年月: 2012年12月

  • ヒト間葉系幹細胞における人Rad52タンパク質のDNA損傷によるアセチル化誘導

    発表年月: 2012年12月

  • クロマチン高次構造上における相同組換え反応の解析

    発表年月: 2012年12月

  • ヒストンとヒストンシャペロンによるクロマチンダイナミクス機構

    発表年月: 2012年12月

  • 再構成系によるクロマチンでの相同組換え機構の解析

    発表年月: 2012年12月

  • 相同組換え修復におけるFANCD2の機能解析

    発表年月: 2012年12月

  • 新規ヒストン相互作用因子hsSpt2の核小体クロマチンダイナミクスにおける機能

    発表年月: 2012年12月

  • 質量分析を用いたヌクレオソームのアセチル化に伴う構造変化の解析

    発表年月: 2012年12月

  • ヘキサソームの構造生物学的・生化学的解析

    発表年月: 2012年12月

  • 高次クロマチン構造上における相同組換え修復機構

    発表年月: 2012年12月

  • ヒトH2A.Zを含むヌクレオソームの機能解析

    発表年月: 2012年12月

  • 動的なヌクレオソーム構造によるクロマチン制御機構

    発表年月: 2012年12月

  • Human telomeric repeat sequences cause destabilization of nucleosome positioning in yeast cells

    発表年月: 2012年11月

  • Histone chaperon activity of Fanconi anemia proteins, FANCD2 and FANCI, is required for DNA crosslink repair

    発表年月: 2012年11月

  • DNA damage induced-acetylation of human Rad52 protein

    発表年月: 2012年11月

  • Biochemical analysis of the FANCD2 monoubiquitination

    発表年月: 2012年11月

  • Biochemical studies of homologous pairing on chromatinized DNA

    発表年月: 2012年11月

  • Acetylation of human Rad52 protein by CBP/p300

    発表年月: 2012年11月

  • Nucleosome structures during transcription and DNA repair

    発表年月: 2012年11月

  • クロマチンにおける相同組換え反応の解析

    発表年月: 2012年09月

  • Cdt1結合タンパク質GRWD1はライセンシングおよび細胞増殖に関与している新規のヒストンシャペロンである

    発表年月: 2012年09月

  • ファンコーニ貧血原因遺伝子産物FANCD2のヒストンシャペロン活性

    発表年月: 2012年09月

  • ヌクレオソームにおけるヒストンH2A.Zバリアント交換機構の構造生物学的解析

    発表年月: 2012年05月

  • ヒストン修飾抗体scFvを用いたH3K9アセチル化レベルの生細胞イメージング

    発表年月: 2012年05月

  • 転写制御に重要なヘキサソームの構造生物学的解析

    発表年月: 2012年05月

  • ヒト精巣特異的ヒストンバリアントを含むヌクレオソームの構造解析

    発表年月: 2012年05月

  • 新規ヒストン相互作用因子hSPT2の同定および核小体における機能解析

    発表年月: 2012年05月

  • エピジェネティクスのクロマチン構造基盤

    発表年月: 2012年03月

  • 二重差切断損傷修復タンパク質RAD52によるDNAアニーリングの分子機構の構造生物学的解析

    発表年月: 2012年01月

  • クロマチンでの相同組換えにおけるヒストンバリアント及びリンカーヒストンの影響

    発表年月: 2012年01月

  • クロマチンにおけるヒト相同組換え因子の生化学的解析

    発表年月: 2012年01月

  • 出芽酵母ミニ染色体におけるヌクレオソームの形成にヒトテロメア配列が及ぼす影響

    発表年月: 2012年01月

  • ヒトH2A.Z-1、H2A.Z-2を含むヌクレオソームの生化学的および構造生物学的解析

    発表年月: 2012年01月

  • ヘキサソームおよびH2A.Bbdを含むヌクレオソームの構造生物学的解析

    発表年月: 2012年01月

  • セントロメアに特異的なCENP-T-W-S-Xヒストンフォールド複合体は正のスーパーコイルを導入する活性を持つ

    発表年月: 2012年01月

  • FANCD2-FANCI複合体のモノユビキチン化機構

    発表年月: 2012年01月

  • CENP-Aヌクレオソームの高次クロマチン構造形成における影響

    発表年月: 2012年01月

  • クロマチン構造変換と細胞核機能におけるアクチンファミリーの寄与

    発表年月: 2012年01月

  • Chd2-dependent deposition of H3.3 is crucial for Brg1 recruitment in myogenesis

    発表年月: 2011年12月

  • Structural analysis of human nucleosomes containing H2A.Z-1 and H2A.Z-2

    発表年月: 2011年12月

  • Biochemical and structural analyses of the nucleosomes in transcriptionally active region

    発表年月: 2011年12月

  • CENP-T-W-S-X 複合体は、動原体タンパク質群の集合機構に重要な役割を担う

    発表年月: 2011年12月

  • The effect of human telomeric repeat sequences on nucleosome positioning in yeast cells

    発表年月: 2011年12月

  • Promotion of nucleosome assembly with DNA repeat sequences associated with hereditary neurological diseases

    発表年月: 2011年12月

  • Histone chaperone activity of a novel histone interacting factor SPT2

    発表年月: 2011年12月

  • Biochemical analyses of the formation of the centromere-specific CENP-A nucleosome bound to the CENP-B DNA-binding domain

    発表年月: 2011年12月

  • Reconstitution and biochemical analyses of the centromere specific chromatin

    発表年月: 2011年12月

  • ファンコニ貧血蛋白FANCD2によるNF-kappaB転写制御機構の解析

    発表年月: 2011年12月

  • Robust stimulation of the FANCD2 monoubiquitination by three-way branched DNAs

    発表年月: 2011年12月

  • Histone chaperone activity of the FANCI-FANCD2 complex and its importance in repair of inter-strand DNA crosslinks by the Fanconi anemia pathway

    発表年月: 2011年12月

  • ATR-ATRIP kinase complex is responsible for triggering activation of the FA pathway

    発表年月: 2011年12月

  • Homologous pairing by RAD51 and RAD54 in the nucleosomal DNA

    発表年月: 2011年12月

  • Crystal structure of the DNA repair protein RAD52 complexed with single-stranded DNA

    発表年月: 2011年12月

  • 出芽酵母Rad52-Rad51によるD-loop 形成におけるRad52のC末端ドメインの必要性

    発表年月: 2011年12月

  • ADP accelerates the dissociation of RAD51-DNA complexes: possible role of ATP hydrolysis for the DNA strand exchange reaction

    発表年月: 2011年12月

  • SMN-GEMIN2 complex stimulates the RAD51-mediated recombination reactions

    発表年月: 2011年12月

  • PSF stimulates the recombination reactions mediated by DMC1 in vivo

    発表年月: 2011年12月

  • Recombination activities of DMC1 on chromatinized DNA

    発表年月: 2011年12月

  • Biochemical analysis of the yeast Rad51-Rad52 complex

    発表年月: 2011年12月

  • Biochemical analysis of the interactions between human RAD51 and RAD52

    発表年月: 2011年12月

  • Analysis of molecular mechanisms underlying regulation of RAD52 function by acetylation

    発表年月: 2011年12月

  • Molecular Mechanism of the Mediator Activity of Yeast Repair Protein Rad52

    発表年月: 2011年12月

  • Crystal structure and biochemical property of the human centromere specific nucleosome

    発表年月: 2011年12月

  • The Cdt1-binding protein GRWD1 is a novel histone chaperone involved in replication licensing

    発表年月: 2011年12月

  • Structural basis for formation of functional chromatin architecture

    発表年月: 2011年12月

  • PLEKHF1 IS A NOVEL FACTOR THAT INTERACTS WITH THE MEIOSIS-SPECIFIC RECOMBINASE, HUMAN DMC1

    発表年月: 2011年10月

  • HUMAN PSF INTERACTS WITH THE MEIOTIC DMCI RECOMBINASE AND STIMULATES THE DMC1-MEDIATED RECOMBINATION REACTIONS IN VITRO

    発表年月: 2011年10月

  • INSTABILITY AND STRUCTURAL PROPERTY OF A HUMAN TESTIS-SPECIFIC HISTONE H3T NUCLEOSOME

    発表年月: 2011年10月

  • STRUCTURAL AND FUNCTIONAL ANALYSES OF HUMAN NUCLEOSOMES CONTAINING TESTIS-SPECIFIC HISTONE VARIANTS

    発表年月: 2011年10月

  • An Absolute Requirement of ATPIP-ATR Kinase in Replication Stressinduced Triggering of the FA Pathway Activation

    発表年月: 2011年10月

  • Histone chaperone Activity of FANCD2-FANCI Complex, the Key Proteins for the Fanconi Anemia Pathway

    発表年月: 2011年10月

  • セントロメア特異的なヌクレオソームの構造・生化学・細胞生物学的解析

    発表年月: 2011年09月

  • PSFによるDMC1依存的な相同組換え反応の促進

    発表年月: 2011年09月

  • RAD51活性制御化合物の探索とその生化学的解析

    発表年月: 2011年09月

  • ヒストンバリアントによるヌクレオソーム構造の多様性と機能発現

    発表年月: 2011年09月

  • DNAリピート配列が形成するヌクレオソームの構造と機能

    発表年月: 2011年09月

  • The structural and biochemical analyses of centromere specific nucleosome

    発表年月: 2011年06月

  • Identification and functional analyses of a novel histone interacting factor, human SPT2

    発表年月: 2011年06月

  • Structural similarity and versatility of the human nucleosomes

    発表年月: 2011年06月

  • 出芽酵母Rad51によるD-loop形成促進のためのRad52第二DNA結合部位の機能

    発表年月: 2011年01月

  • ヒストンH3バリアントCENP-Aを含むヌクレオソームのX線結晶構造解析

    発表年月: 2011年01月

  • H2A.Bbdを含むヌクレオソームの生化学的解析

    発表年月: 2011年01月

  • Atomic Resolution Structures of the Human Nucleosomes Containing Histone Variants.

    発表年月: 2011年01月

  • SLX4 links the Fanconi anemia pathway to structure-specific endonucleases, SLX1, XPF and MUS81 in interstrand crosslink repair.

    発表年月: 2011年01月

  • Roles of the N-terminal and C-terminal domains of the Rad52 protein in homologous recombination repair.

    発表年月: 2011年01月

  • Monitoring Histone Modifications in Single Cells using FabLEM(Fab-based Live Endogenous Modification Labeling).

    発表年月: 2011年01月

  • Biochemical and cell biological analyses of a novel histone interacting factor, SPT2.

    発表年月: 2011年01月

  • Functional analyses of the Fanconi anemia protein, FANCD2.

    発表年月: 2011年01月

  • C-terminal Tail of Histone H2AX Reduces Intra-nucleosome Array Interaction.

    発表年月: 2011年01月

  • The Crystal STructure of Centromere Specific Nucleosome.

    発表年月: 2011年01月

  • A novel homologous recomination factor, human EVL forms single-stranded DNA catemers with a type I topoisomerase.

    発表年月: 2011年01月

  • Regulation of Radiation-induced focus formation by SUMOylation.

    発表年月: 2011年01月

  • ヒストンバリアントを含むヌクレオソームの構造多様性と機能

    発表年月: 2010年12月

  • Chd2 interacts with H3.3 to determine cell fate in myogenesis.

    発表年月: 2010年12月

  • 生細胞におけるメチル化およびアセチル化修飾ヒストンの可視化

    発表年月: 2010年12月

  • RAD51によるparanemic joint molecule形成過程へのtopoisomerase � αの役割

    発表年月: 2010年12月

  • 新規RAD51組換え活性制御化合物の同定とその作用機序

    発表年月: 2010年12月

  • PSFによるDNA組換え酵素DMC1の活性化

    発表年月: 2010年12月

  • 出芽酵母Rad52の第二DNA結合部位も相同組換えに必要である

    発表年月: 2010年12月

  • DNA修復タンパク質RAD52の翻訳後修飾による機能制御機構の解析

    発表年月: 2010年12月

  • 酵母とヒトDNA修復タンパク質Rad52の機能ドメイン解析

    発表年月: 2010年12月

  • Chd2 interacts with H3.3 to determine cell fate in myogenesis.

    発表年月: 2010年12月

  • DNA結合活性を有するCCANタンパク質群の機能解析

    発表年月: 2010年12月

  • ファンコニ貧血原因遺伝子産物FANCD2およびFANCIの複合体形成と機能解析

    発表年月: 2010年12月

  • セントロメア領域に特異的なヌクレオソームのX線結晶構造解析

    発表年月: 2010年12月

  • CENP-Aターゲッティングドメインを含むH3変異体が形成するヌクレオソームのX線結晶構造解析

    発表年月: 2010年12月

  • 新規ヒストン結合因子ヒトSPT2のDNA損傷修復における機能解析

    発表年月: 2010年12月

  • 質量分析を用いたヒストンH2A-H2Bのシトルリン化の解析

    発表年月: 2010年12月

  • NMRによるヒストンヌクレオソームコアの立体構造解析

    発表年月: 2010年12月

  • ヌクレオソームの立体構造からみたエピジェネティクスの分子機構

    発表年月: 2010年11月

  • Structual and biochemical studies of DMC1 polymorphisms in the human population.

    発表年月: 2010年09月

  • ヒストンバリアントH3Tを含むヌクレオソームの構造解析および生化学的解析

    発表年月: 2010年05月

  • 新規RAD51活性化因子GEMIN2の相同組換え修復における機能

    発表年月: 2010年05月

  • SUMO修飾システムによるRAD51核内ドメイン形成の制御

    発表年月: 2010年05月

  • ヒストンシャペロンsNASPの生化学的解析

    発表年月: 2010年05月

  • Fanconi貧血原因遺伝子FANCD2及びFANCIの機能解析

    発表年月: 2010年05月

  • 低分子化合物によるRAD51依存的相同組換え反応の制御

    発表年月: 2010年05月

  • シトルリン化タンパク質PAD4のSNP変異体の解析

    発表年月: 2010年05月

  • ブーケ配向形成因子S. japonicus Bqt1-Bqt2複合体の生化学的解析

    発表年月: 2010年05月

  • ヒトと酵母のDNA修復タンパク質Rad52の機能比較

    発表年月: 2010年05月

  • 高等動物のセントロメアクロマチン構造

    発表年月: 2010年01月

  • セントロメア特異的なCENP-Aヌクレオソームの生化学的解析

    発表年月: 2010年01月

  • SPTY2D1のヒストン結合および機能解析

    発表年月: 2010年01月

  • 相同DNA組換えにおける2種の相同DNA対合蛋白質の役割分担

    発表年月: 2010年01月

  • ブーケ配向形成因子S. japonicus Bqt1-Bqt2複合体の精製および機能解析

    発表年月: 2010年01月

  • ヒトSPF45の相同組換えにおける機能解析

    発表年月: 2010年01月

  • クロマチンの基盤構造であるヌクレオソームの機能・構造解析

    発表年月: 2009年12月

  • Structural feature of the CENP-A nucleosome in the vertebrate centromere

    発表年月: 2009年12月

  • ブーケ配向形成因子S. japonicus Bqt1-Bqt2複合体の大量精製および生化学的解析

    発表年月: 2009年12月

  • Fanconi貧血原因遺伝子FANCD2およびFANCIの生化学的機能解析

    発表年月: 2009年12月

  • セントロメア特異的H3バリアントCENP-Aを含むヌクレオソームの構造解析

    発表年月: 2009年12月

  • セントロメアDNA結合タンパク質CENP-T/CENP-W複合体の生化学的解析

    発表年月: 2009年12月

  • 出芽酵母におけるヒト由来ヒストンH3バリアントの発現と機能解析

    発表年月: 2009年12月

  • ヒト相同組換えタンパク質DMC1と相互作用する新規タンパク質PLEKHF1の生化学的機能解析

    発表年月: 2009年12月

  • GEMIN2によるRAD51組換え活性の制御

    発表年月: 2009年12月

  • ヒトRAD51と相互作用する新規タンパク質PSFの機能解析

    発表年月: 2009年12月

  • 出芽酵母Rad52-Rad51複合体によるD-loop形成におけるRAD52の二本鎖DNAへの結合の影響D-loop形成においてRad52は二本鎖DNAの結合に働く

    発表年月: 2009年12月

  • DNA配列を改変したヌクレオソーム複合体の生産技術開発

    発表年月: 2009年12月

  • 新規ヒストン結合因子SPTY2D1の機能解析

    発表年月: 2009年12月

  • 大腸菌染色体分配に関わる候補因子MigP(YncE)の構造生物学的解析

    発表年月: 2009年12月

  • HP1とPOGZの生化学的および構造生物学的解析

    発表年月: 2009年12月

  • RAD51と相互作用するSMN-GEMIN2複合体の生化学的解析

    発表年月: 2009年12月

  • DNA修復タンパク質RAD52のヒトと酵母における機能比較

    発表年月: 2009年12月

  • 新規相同組換え修復タンパク質ヒトSPF45の生化学的解析

    発表年月: 2009年12月

  • 低分子化合物を用いた組換え修復タンパク質RAD51の活性制御

    発表年月: 2009年12月

  • 相同DNA組換えにおける天然変性領域の機能

    発表年月: 2009年10月

  • シトルリン化によるヒストンH2A/HBの構造変化

    発表年月: 2009年10月

  • ヒストンバリアントH3tを含むヌクレオソームの立体構造とその機能解析

    発表年月: 2009年10月

  • Fanconi貧血原因遺伝子産物FANCD2のヌクレオソーム形成活性

    発表年月: 2009年10月

  • 新規RAD51結合タンパク質EVLの機能解析

    発表年月: 2009年10月

  • Stractual and functional analyses of the nucleosome containing a testis-specific histone variant, human H3T

    発表年月: 2009年09月

  • Rad52の立体構造に基づく相同組換え反応機構の解析

    発表年月: 2009年06月

  • ヒトSUMO化Rad52の生化学的および物理化学的解析

    発表年月: 2009年06月

  • 新規RAD51相互作用タンパク質PSFの相同組換えにおける機能

    発表年月: 2009年06月

  • リコンビナントヒストンによるin vitroヌクレオソーム再構築

    発表年月: 2009年06月

  • 精巣特異的に高発現するH3バリアントH3tを含むヌクレオソームの構造解析

    発表年月: 2009年01月

  • ヒストンH3バリアントと相互作用する因子群の同定および生化学的解析

    発表年月: 2009年01月

  • Fanconi貧血原因遺伝子FANCD2の機能解析

    発表年月: 2009年01月

  • ゲノム配列情報の安定維持と多様化に重要な相同組換え因子群の機能解析

    発表年月: 2008年12月

  • 大腸菌染色体分配に働くMigPタンパク質のX線結晶構造解析

    発表年月: 2008年12月

  • ヒストンH3バリアントと相互作用する因子のプロテオーム解析

    発表年月: 2008年12月

  • セントロメア特異的H3バリアントCENP-Aを含むヌクレオソームの結晶化

    発表年月: 2008年12月

  • H2AXを含むヌクレオソームの再構成とX線結晶構造解析

    発表年月: 2008年12月

  • S. japonicus Bqt1-Bqt2複合体の生化学的解析

    発表年月: 2008年12月

  • ヒストンH3バリアントH3tを含むヌクレオソームの構造解析

    発表年月: 2008年12月

  • 出芽酵母におけるヒト由来ヒストンH3バリアントの発現

    発表年月: 2008年12月

  • Fanconi貧血相補性群D2原因遺伝子,FANCD2の生化学的解析

    発表年月: 2008年12月

  • ヒトGEMIN2とRAD51の相互作用

    発表年月: 2008年12月

  • 新規ヒトRAD51相互作用タンパク質PSFの生化学的解析

    発表年月: 2008年12月

  • 相同組換え修復タンパク質RAD51Bと相互作用するEVLの単鎖DNAアニーリング活性

    発表年月: 2008年12月

  • 新規RAD51B相互作用タンパク質EVLの相同組換えにおける機能

    発表年月: 2008年12月

  • 低分子化合物によるRAD51鎖交換活性の制御

    発表年月: 2008年12月

  • ヒトDMC1タンパク質およびDMC1バリアントの機能・構造解析

    発表年月: 2008年12月

  • ヒトDMC1-F89C変異体の8量体リング構造不安定化メカニズム

    発表年月: 2008年12月

  • 減数分裂特異的組換えタンパク質イネDMC1の精製と生化学的機能解析

    発表年月: 2008年12月

  • DNA修復タンパク質RAD52の変異体の機能解析

    発表年月: 2008年12月

  • RAD54B N末端ドメインのDNA分岐構造結合基質特異性の解析

    発表年月: 2008年12月

  • 出芽酵母Rad52-Rad51複合体によるD-loop形成におけるRAD52の二本鎖DNAへの結合の影響

    発表年月: 2008年12月

  • Gemin2 promotes the assembly of Rad51 at resected single-stranded tails of DSBs in Homologous recombination

    発表年月: 2008年12月

  • Identification of the chemical compounds that regulate the recombinase activity of the human RAD51 protein.

    発表年月: 2008年07月

  • Biochemical study of a novel RAD51B-interacting protein, EVL (Ena/Vasp-like)

    発表年月: 2008年07月

  • Rad52の立体構造に基づく相同DNA組換え反応機構の解析

    発表年月: 2008年06月

  • 新規因子DiaA蛋白質を含む、複製開始複合体の構造とメカニズム

    発表年月: 2008年06月

  • 真核生物DNA組換え酵素の機能・構造解析

    発表年月: 2008年06月

  • FRET法によるRecombinaseの組換え活性測定

    発表年月: 2008年06月

  • 生命情報学的解析手法によって推定したRecAタンパク質の機能解析

    発表年月: 2008年06月

  • Analyses of the polymorphic Dmc1-M200V variant

    発表年月: 2008年06月

  • The functional differences between HsRad51 ad its isoform

    発表年月: 2008年06月

  • In vitro recombinase activities of the rice Dmc1A and Dmc1B proteins

    発表年月: 2008年06月

  • The human PSF protein is a novel Rad51-interacting protein

    発表年月: 2008年06月

  • Identification of a novel Rad51B-interacting protein

    発表年月: 2008年06月

  • Identification and biochemical analysis of the novel Rad51-binding protein

    発表年月: 2008年06月

  • Biochemical analyses of human histone H3 variant, H3t

    発表年月: 2008年06月

  • Structural and Biochemical Studies of Homologous Recombination toward Drug Design and Discovery

    発表年月: 2008年02月

  • ヒト由来ヒストンH3バリアントの機能解析

    発表年月: 2008年01月

  • ヒストンH3バリアントの生化学的・物理化学的解析

    発表年月: 2008年01月

  • 進化的に高度に保存されたヒトDmc1およびRad51の機能・構造解析

    発表年月: 2007年12月

  • DnaAによる複製開始複合体の形成と2重鎖開裂機構モデル

    発表年月: 2007年12月

  • ヒストン交換と修飾のダイナミクス

    発表年月: 2007年12月

  • ヒトRad52タンパク質の第二のDNA結合領域

    発表年月: 2007年12月

  • 大腸菌染色体分配に働くMigPタンパク質の生化学的機能解析

    発表年月: 2007年12月

  • 大腸菌染色体の複製開始制御因子DiaAの立体構造解析

    発表年月: 2007年12月

  • 大腸菌染色体の複製開始促進因子DiaAはATP-DnaA特異的な開裂複合体形成を促進する

    発表年月: 2007年12月

  • ヒストンH3バリアントの生化学的解析

    発表年月: 2007年12月

  • シャペロン依存的なヒストンH3バリアントのヌクレオソーム形成機構

    発表年月: 2007年12月

  • ヒトGemin2タンパク質の相同DNA組換えにおける役割

    発表年月: 2007年12月

  • 新規Rad51結合タンパク質Gemin2の欠失変異体解析

    発表年月: 2007年12月

  • 相同組換え修復タンパク質Rad51と相互作用する新規タンパク質PSFの解析

    発表年月: 2007年12月

  • ヒトRad51タンパク質およびisoformの生化学的解析

    発表年月: 2007年12月

  • Rad51Bと相互作用する新規タンパク質Evlの単鎖DNAカテネーション活性

    発表年月: 2007年12月

  • 新規Rad51B相互作用タンパク質Evlのドメイン解析

    発表年月: 2007年12月

  • ヒト不妊症で検出されたDmc1遺伝子多型の機能解析

    発表年月: 2007年12月

  • イネの二つのDmc1バリアントの生化学的解析

    発表年月: 2007年12月

  • イネWRKY DNA結合ドメインDNA配列特異性の向上

    発表年月: 2007年12月

  • ヒトSUMO化Rad52の生化学的解析

    発表年月: 2007年12月

  • S. pombe Rap1のS. pombe Bqt1-Bqt2複合体との結合領域同定

    発表年月: 2007年12月

  • Analyses of the human proteins involved in double strand break repair

    発表年月: 2007年05月

  • ヒトDmc1 F89D変異体の会合状態の解析

    発表年月: 2007年05月

  • イネWRKY DNA結合ドメインの特異的DNA結合能の改良

    発表年月: 2007年05月

  • Rad51と相互作用する新規タンパク質Gemin2の解析

    発表年月: 2007年05月

  • In vitro analyses of human nucleosomes

    発表年月: 2007年01月

  • ヒストンH3バリアントのシャペロン依存的なヌクレオソーム形成機構

    発表年月: 2007年01月

  • 二重鎖切断修復に重要な相同組換え因子群の機能解析

    発表年月: 2006年12月

  • DnaA蛋白質の特異的AAA+領域を介した複製開始点開裂のメカニズム

    発表年月: 2006年12月

  • 新規ヒトRad51相互作用タンパク質Gemin2の生化学的解析

    発表年月: 2006年12月

  • HIV-1アクセサリー遺伝子産物VprはATM依存的なDNA傷害シグナルを活性化する

    発表年月: 2006年12月

  • HIV-1 Vpr causes aberrant sister chromatid separation

    発表年月: 2006年06月

  • Biochemical analysis of the human Rad51 variants, Rad51-Q313 and Rad51-K313

    発表年月: 2006年06月

  • Biochemical analyses of the monomeric human Dmc1 mutant

    発表年月: 2006年06月

  • Function of the N-terminal domain of the human Dmc1 protein

    発表年月: 2006年06月

  • Gemin2 is a novel Rad51-interacting factor that is involved in repair of double strand DNA breaks

    発表年月: 2006年06月

  • The human Rad52 protein introduces positive supercoils into DNA

    発表年月: 2006年06月

  • The human RNB6 protein is a novel Rad51B-interacting protein

    発表年月: 2006年06月

  • The DNA-binding activity of Fanconi anemia protein FancD2

    発表年月: 2006年06月

  • ATP-dependent activation of an initiation complex in the minichromosomal replication of hyperthermophile Thermotoga maritima in vitro

    発表年月: 2006年06月

  • Chromosome abnormality and double strand DNA breaks caused by HIV-1 Vpr

    発表年月: 2006年06月

  • 新規Rad51結合因子Gemin2の染色体修復における役割

    発表年月: 2006年01月

  • HIV-1 Vprによる核崩壊の分子機序

    発表年月: 2006年01月

  • ヒトRad51 Tyr-315残基の機能解析

    発表年月: 2005年12月

  • HIV-1 Vprが誘導する宿主染色体の形態異常

    発表年月: 2005年12月

  • ヒトDmc1のダブルリング構造

    発表年月: 2005年12月

  • マウス及びヒトTBPIP/Hop2の機能解析

    発表年月: 2005年12月

  • ヒトRad51N末端ドメイン結合タンパク質Sip1の機能解析

    発表年月: 2005年12月

  • HIV-1アクセサリ-遺伝子産物Vprによるp53の機能修飾

    発表年月: 2005年12月

  • HIV-1 Vprタンパク質によるヒト染色体の形成異常

    発表年月: 2005年12月

  • Analysis on Structure and Regulation of a Bacterial Initiation Complex

    発表年月: 2005年12月

  • DnaAタンパク質の立体構造と複製開始のメカニズム

    発表年月: 2005年12月

  • 新規ヒトRad51相互作用タンパク質Sip1の機能解析

    発表年月: 2005年12月

  • HIV-1, Vprによるヘテロクロマチン蛋白消失と姉妹染色分体の早期分離

    発表年月: 2005年12月

  • ファンコーニ貧血責任遺伝子産物FancD2のDNA結合活性

    発表年月: 2005年12月

  • ヒトRad54Bタンパク質の機能解析

    発表年月: 2005年12月

  • ヒトArp8の生化学的解析

    発表年月: 2005年12月

  • ヒトRad52タンパク質のhomologous pairingのメカニズム

    発表年月: 2005年12月

  • Mutational analysis of the human Rad51 L1 and L2 loops

    発表年月: 2005年12月

  • ヒトRad51Bタンパク質と相互作用する新規タンパク質RNB6の解析

    発表年月: 2005年12月

  • ヒトRad51とマウスRad51におけるDNA結合親和性の差異

    発表年月: 2005年12月

  • ヒトTBPIP/Hop2-Mnd1複合体によるDNA鎖交換反応の活性化

    発表年月: 2005年12月

  • イネWRKYドメインの特異的DNA配列との相互作用の解析

    発表年月: 2005年12月

  • ヒト由来ヒストンH2AXを用いたヌクレオソームの再構成

    発表年月: 2005年12月

  • HIV アクセサリー遺伝子産物Vprの機能を阻害する因子の探索

    発表年月: 2005年12月

  • 大腸菌DNA複製制御因子SeqAおよびDnaAの複製開始点DNA認識機構

    発表年月: 2005年03月

  • ヒトセントロメア特異的クロマチン構造の形成機構の解析

    発表年月: 2005年01月

  • HIV-1 Vprによる早期娘染色体分離

    発表年月: 2005年01月

  • HIV-1 Vprが誘導する宿主染色体の形態異常

    発表年月: 2005年01月

  • Structural and functional analyses of the human homologous-pairing protein, Dmc1

    発表年月: 2005年01月

  • ヒト相同組換えタンパク質の構造・機能解析

    発表年月: 2004年12月

  • 大腸菌の複製開始を制御する タンパク質DnaAおよびSeqAのDNA認識機構

    発表年月: 2004年06月

  • 減数分裂期の相同染色体組換えに働くヒトDmc1タンパク質の分子構造

    発表年月: 2004年01月

  • V(D)J組換え酵素RAG1のNMRによる構造解析

    発表年月: 2003年12月

  • 大腸菌DnaA domain IVとDnaA boxを含むDNAの複合体の立体構造解析

    発表年月: 2003年12月

  • ヒトXrcc2・Rad51B・Rad51C・Rad51Dタンパク質複合体の機能解析

    発表年月: 2003年12月

  • ヒトDmc1の立体構造解析

    発表年月: 2003年12月

  • マウスTBPIPの機能ドメインの解析

    発表年月: 2003年12月

  • ヒトRad51-Tyr315残基の機能解析

    発表年月: 2003年12月

  • ヒトCENP-B dimerization domainのX線構造解析

    発表年月: 2003年12月

  • ヒト相同組換え因子群の機能および構造解析

    発表年月: 2003年12月

  • Structure and activity of the human recombination proteins, Rad51, Dmc1, Rad52, and Rad51 paralogs

    発表年月: 2003年10月

  • ヒトDNA二重鎖切断修復酵素Rad52の生化学的および構造生物学的解析

    発表年月: 2003年10月

  • Biochemical and structural analyses of the human centromere-specific nucleosome

    発表年月: 2003年06月

  • ヒトRad51N末端ドメインと相互作用する新規タンパク質の探索

  • ヒトDmc1変異体の生化学的解析

  • HIV-1 Vprによるクロマチン二重鎖切断の導入

▼全件表示

特定課題研究

  • 多様なクロマチン構造を介した相同組換えの制御機構の解明

    2016年  

     概要を見る

    本年度は、RAD51およびDMC1のクロマチン上での相同鎖検索の制御メカニズムに着目し、研究を行なった。再構成クロマチンを用いて、両者の相同鎖検索過程を解析した結果、RAD51とは異なり、DMC1はヌクレオソームが形成されていない領域で優先的に相同鎖検索を行うことが明らかになった。実際に、このようなヌクレオソームが形成されていない特殊なクロマチン構造は、減数分裂期の組換えホットスポットに存在する。これらの事実は、クロマチン上におけるRAD51とDMC1の機能差異を示すと共に、クロマチン構造が両者の組換え反応を制御することを示唆している。

  • 遺伝的組換えの分子機構に関する研究

    2008年  

     概要を見る

    相同DNA組換えは、減数分裂の第一分裂期におこり(減数分裂期組換え)、相同染色体間を物理的に連結するキアズマの形成に必須である。その結果として遺伝情報の再編成が行われ、この遺伝子再編成が現代の多大な生物種を生み出す原動力となった。またゲノムDNAは、担体として優れた性質を持っているが、環境からの外的要因(紫外線や放射線など)や細胞自身による内的要因(DNA複製のエラーなど)によって日常的に損傷を受けている。なかでも、二重鎖切断などの重篤なDNA損傷は、相同DNA組換えを経由したDNA修復経路(体細胞分裂期組換え)によって速やかに修復されることが近年明らかにされてきた。このように相同DNA組換えは、減数分裂期と体細胞分裂期での両方のDNA組換えにおいて機能している。しかし、そのメカニズムの詳細に関してはいまだ明らかにされていない。そこで本研究では、相同DNA組換えの分子機構を明らかにすることを目的として、相同DNA組換えにおいて働くタンパク質に着目し、それらの生化学的解析を行った。まず、リアルタイムで相同DNA組換え反応を検出するin vitro系を確立するために、蛍光標識したDNAを利用したFRET法によるDNA組換え検出系を確立した。本法では、DNA配列の中央部に蛍光色素を有するDNAを使用しており、正確に相同DNA組換え反応が触媒されたときのみ検出できる系である。この新たに確立した系を用いて、大腸菌RecA、ヒトRAD51およびDMC1、イネDMC1AおよびDMC1Bなどの組換え活性をリアルタイムで評価することに成功した。また、ヒトRAD51およびDMC1をアガロースビーズに固定し、RAD51ビーズおよびDMC1ビーズを作製した。これらのRAD51ビーズおよびDMC1ビーズを用いて、培養細胞の抽出液からRAD51およびDMC1と結合する因子群候補を、プロテオミクス法によって多数同定した。

  • 相同DNA組換え装置の分子機構解明のための構造・生化学的アプローチ

    2007年  

     概要を見る

    相同DNA組換えは減数分裂の第一分裂期におこる(遺伝的組換え)。その結果として遺伝情報の再編成が行われ、現代の300万種を超えるとも言われている生物種を生み出す原動力となった。またゲノムDNAは、環境からの外的要因(紫外線や放射線など)や細胞自身による内的要因(DNA複製のエラーなど)によって日常的に損傷を受けている。なかでも、二重鎖切断などの重篤なDNA損傷は、相同DNA組換えを経由したDNA修復経路によって速やかに修復されることが明らかにされてきた。このように相同DNA組換えは、ゲノムDNAの進化と保護の両面において重要であるが、その分子機構についてはいまだ十分に理解されていない。本研究では、ヒト由来の相同DNA組換え装置およびゲノムDNAを収容する染色体に着目し、これらの生体超分子複合体がの構成成分をリコンビナント蛋白質として精製した。具体的には、Rad51やDmc1などの相同DNA組換え装置の活性中心酵素、染色体の主要構成蛋白質であるヒストンH2A、H2B、H3、H4などである。これらの蛋白質が、生体内でどのような超分子複合体を形成しているのかを解析するために、これらの蛋白質を共有結合によってリンクしたビーズの作製を行った。そして、それらの蛋白質が結合したビーズを用いて、ヒト培養細胞から得た抽出液より、それぞれの蛋白質と特異的に結合する因子群を、プロテオミクス解析により同定することに成功した。本プロテオミクス解析および蛋白質分析は、北海道大学の小布施研究室と、大阪大学の木村准教授研究室において行った。得られた新規相同DNA組換え酵素群および新規染色体構成蛋白質群の遺伝子のクローニングを現在行っており、これらの蛋白質をリコンビナントとして精製し、それらの機能および構造解析を行う予定である。また、Rad51およびDmc1の解析においては、それらの天然に存在するバリアントの精製に成功した。そしてそれらの活性の異同について生化学的解析によって明らかにした。

  • DNA組換えタンパク質の機能的分子進化に関する研究

    2003年  

     概要を見る

    DNA組換えタンパク質は、ウイルス、バクテリアからヒトに至るまで普遍的に存在している。このように、進化的に高度に保存されたタンパク質は、生命体の生存にとって中心的な役割を果たす例が多く、DNA組換えタンパク質も、遺伝情報を担う染色体DNAに日常的に起こる二重鎖切断を修復するために重要な役割を果たしている。実際にがん細胞において、DNA組換えに関係した遺伝子上での変異やSNPが頻繁に見出され、このことは、DNA組換え修復の破綻が、発がんの主要な原因の1つであることを示唆している。DNA組換え反応のうち、特に相同組換えに関わるタンパク質が進化上高度に保存されている。相同組換えの要の反応は相同的対合であるが、この反応を直接触媒することが知られているバクテリアRecAタンパク質は、ヒトでは7種類のホモログが存在する。それらは、Rad51、Dmc1、そしてRad51バラログであるXrcc2、Xrcc3、Rad51B、Rad51C、Rad51Dである。この相同的対合タンパク質であるRecAの機能的分子進化を明らかにする目的で、これらヒトのRecAホモログとバクテリアのRecAの比較を生化学的解析により行った。その結果、ヒトのRecAホモログは7種類いずれも、単体もしくは複合体としてRecA様の相同的対合活性を有するが、その活性はバクテリアRecAとは比較にならないほど弱いことが明らかになった。また、白血病細胞で特異的に見られるRad51のリン酸化部位(Tyr315)が、Rad51のポリマー形成に重要な役割を果たすことを、部位特異的変異体作製法により作製した6種類の変異Rad51の生化学的および分光学的解析により明らかにした。Tyr315はバクテリアRecAには保存されておらず、Rad51が進化により獲得した新機能を担うと考えられる。また、ヒトRad51パラログであるXrcc3とRad51Cの複合体形成機構を明らかにするために、欠失変異体および点変異体を用いた生化学的解析により、それぞれの複合体形成に必要なドメイン領域を決定した。