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ゲノム機能制御を支えるクロマチン分子動態のナノスケールイメージング解析

Research Project

Project/Area Number 09J05533
Research Category

Grant-in-Aid for JSPS Fellows

Allocation TypeSingle-year Grants
Section国内
Research Field Molecular biology
Research InstitutionKyoto University

Principal Investigator

鈴木 勇輝  京都大学, 生命科学研究科, 特別研究員(DC1)

Project Period (FY) 2009 – 2011
Project Status Completed (Fiscal Year 2011)
Budget Amount *help
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 2011: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2010: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2009: ¥700,000 (Direct Cost: ¥700,000)
KeywordsDNA / Holliday Junction / クロマチンリモデリング / 原子間力顕微鏡 / 高速液中原子間力顕微鏡 / リコンビナーゼ / Rad54 / 原子間力顕微鏡(AFM) / 液中高速原子間力顕微鏡 / RNAポリメラーゼ / ヌクレオソーム / 制限酵素 / ヒストン
Research Abstract

本年度はDNA修復,組換えに関与するDNA-タンパク質複合体構造とその機能動態解析を進めた.相同組換えはDNA二本鎖切断の修復における主要な経路である.相同組換えの過程では,相同性のある部分の検索が起こり,切断されたDNA分子の末端が相同な二本鎖DNA鎖に侵入することで,Holliday Junction(HJ)とよばれる鎖交換構造が形成される.B.subtilisのHJ解消過程においては,branch migrationを担うRuvBとHJ解消酵素RecUが主役となる.これらのタンパク質とHJ構造を有するDNAとの複合体構造を原子間力顕微鏡(AFM)により可視化解析することにより,(1)RecUの結合によってHJの構造に変化をもたらすこと,(2)RuvBは単体ではHJ構造へ結合せず,RecUがHJへ結合したのち,そこへリクルートされることを示すデータを得た.
真核生物における相同組換えにおいてはクロマチンリモデリング因子Swi2/Snf2タンパク質であるRad54がキータンパクと機能することが知られている.溶液中におけるRad54とDNA鎖との相互作用を高速液中AFMにより一分子レベルで経時観察・可視化解析することで.(1)ATP非存在下においてもRad54の単量体はDNA鎖の非特異的部位への解離と結合,DNA鎖上の一次元拡散運動を組み合わせながら,DNA上をランダムに移動しうること.(2)DNA鎖に結合したRad54は,更にオリゴマーを形成することで,DNAのループ構造をつくること.(3)このループ構造にATPが供給された場合,Rad54はDNA鎖を一方向へ引っ張り.DNA鎖のトランスロケーションを起こすことが示唆された.
これらの結果はDNA分子とタンパク質の相互作用が織り成す機能的な複合体構造の形成過程,およびDNA修復,組換え反応における分子の作用機序モデルの考察を可能とするものである.

Current Status of Research Progress
Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

平成21年度から平成23年度までの3年間において,再構成ヌクレオソーム構造や制限酵素,RNAポリメラーゼ,DNA修復酵素,クロマチンリモデリング因子などの溶液中機能動態観察を行い,それらの成果として,4報の学術論文を発表することができた.

Strategy for Future Research Activity

高速液中AFMで1分子の機能解析を行うためには,対象とする分子の機能と動きを維持しながらも,観察用のマイカ基板にそれらを吸着させなければならない.複数種の分子が担う反応を観察する際には,特定の分子を基板上に強く吸着させ,他の分子は弱く吸着させるなど,選択的な吸着が要求される.実験を進行していくうちに,再構成ヌクレオソーム上における酵素反応をAFMで観察するためには,観察試料を載せる基板そのものを工夫する必要があることがわかってきた.これまでは,マイカ基板のアミノ化などの化学修飾に頼ってきたが,ストレプトアビジンの二次元結晶や脂質の二重膜の導入を検討している.

Report

(3 results)
  • 2011 Annual Research Report
  • 2010 Annual Research Report
  • 2009 Annual Research Report
  • Research Products

    (18 results)

All 2011 2010 2009

All Journal Article (11 results) (of which Peer Reviewed: 11 results) Presentation (5 results) Book (2 results)

  • [Journal Article] Protein-DNA interactions in high speed AFM : direct observation of hopping, sliding and ATP driven translocation of human RAD542011

    • Author(s)
      Humberto Sanchez, Yuki Suzuki, Masatoshi Yokokawa, Kunio Takeyasu, Claire Wyman
    • Journal Title

      Integrative Biology

      Volume: 3 Issue: 11 Pages: 1127-1134

    • DOI

      10.1039/c1ib00039j

    • Related Report
      2011 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Visual analysis of concerted cleavage by Type IIF restriction enzyme SfiI in subsecond time region2011

    • Author(s)
      Yuki Suzuki, Jamie L.Gilmore, Shige H.Yoshimura, Robert M.Henderson, Yuri L.Lyubchenko, Kunio Takeyasu
    • Journal Title

      Biophysical Journal

      Volume: 101 Issue: 12 Pages: 2992-2998

    • DOI

      10.1016/j.bpj.2011.09.064

    • Related Report
      2011 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Critical behavior of megabase-size DNA toward the transition into a compact state2011

    • Author(s)
      Yuko Yoshikawa, Yuki Suzuki, Kozo Yamada, Wakao Fukuda, Kenichi Yoshikawa, Kunio Takeyasu, Tadayuki Imanaka
    • Journal Title

      Journal of Chemical Physics

      Volume: 135 Issue: 22 Pages: 225101-225107

    • DOI

      10.1063/1.3666845

    • Related Report
      2011 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Opposing role of condensing hinge against replication protein A in mitosis and interphase through promoting DNA annealing2011

    • Author(s)
      Yuko Akai, Yumiko Kurokawa, Norihiko Nakazawa, Yuko Tonami-Murakami, Yuki Suzuki, Shige H.Yoshimura, Hiroshi Iwasaki, Yoshiharu Shiroiwa, Takahiro Nakamura, Eri Shibata, Mitsuhiro Yanagida
    • Journal Title

      Open Biology

      Volume: 1 Issue: 4 Pages: 110023-110023

    • DOI

      10.1098/rsob.110023

    • Related Report
      2011 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Unraveling DNA dynamics using atomic force microscopy (AFM)2011

    • Author(s)
      Yuki Suzuki, Yuko Yoshikawa, Shige H.Yoshimura, Kenichi Yoshikawa, Kunio Takeyasu
    • Journal Title

      Wiley Interdisciplinary Reviews : Nanomedicine and Nanobiotechnology

      Volume: 3 Issue: 6 Pages: 574-588

    • DOI

      10.1002/wnan.150

    • Related Report
      2011 Annual Research Report
    • Peer Reviewed
  • [Journal Article] RecO-mediated DNA homology search and annealing is facilitated by SsbA2010

    • Author(s)
      Candela Manfredi, Yuki Suzuki, Tribhuwan Yadav, Kunio Takeyasu, Juan C, Alonso.
    • Journal Title

      Nucleic Acids Research

      Volume: 38 Pages: 6920-6929

    • Related Report
      2010 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Evolutionary dynamics of spliceosomal intron revealed by in silico analyses of the P-Type ATPase superfamily genes2010

    • Author(s)
      Toshiyuki Oda, Ryosuke L.Ohniwa, Yuki Suzuki, Masatsugu Denawa, Masahiro Kumeta, Hideyuki Okamura, Kunio Takeyasu
    • Journal Title

      Molecular Biology Reports

      Volume: 38 Pages: 2285-2293

    • Related Report
      2010 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Molecular dynamics of DNA and nucleosomes in solution studied by fast-scanning atomic force microscopy2010

    • Author(s)
      Yuki Suzuki, Yuji Higuchi, Kohji Hizume, Masatoshi Yokokawa, Shige H.Yoshimura, Kenichi Yoshikawa, Kunio Takeyasu
    • Journal Title

      Ultramicroscopy (Epub ahead of print)

    • Related Report
      2009 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Nucleosomal arrays reconstituted from ring and linear DNA2009

    • Author(s)
      Sumiko Araki, Kohji Hizume, Takafumi Iwaki, Yuki Suzuki, Kunio Takeyasu, Kenichi Yoshikawa
    • Journal Title

      Chemical Physics Letters 479

      Pages: 284-289

    • Related Report
      2009 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Single-molecule analysis of protein-DNA complexes formed during partition of newly replicated plasmid molecules in Streptococcus pyogenes2009

    • Author(s)
      Florencia Pratto, Yuki Suzuki, Kunio Takeyasu, Juan C.Alonso
    • Journal Title

      The Journal of Biological Chemistry 284

      Pages: 30298-306

    • Related Report
      2009 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Single-Molecule Dynamics of the DNA-EcoRII Protein Complexes Revealed with High-Speed Atomic Force Microscopy2009

    • Author(s)
      Jamie L.Gilmore, Yuki Suzuki, Gintautas Tamulaitis, Virginijus Siksnys, Kunio Takeyasu, Yuri L.Lyubchenko
    • Journal Title

      Biochemistry 48

      Pages: 10492-8

    • Related Report
      2009 Annual Research Report
    • Peer Reviewed
  • [Presentation] Catching the scene of transcription in sub-second time region by fast-scanning atomic force microscopy2011

    • Author(s)
      Yuki Suzuki, Minsang Shin, Aiko Yoshida, Shige H.Yoshimura, Kunio
    • Organizer
      XIII International Scanning Probe Microscopy Conference
    • Place of Presentation
      Munich, Germany
    • Related Report
      2011 Annual Research Report
  • [Presentation] Fast-scanning AFM analysis of glutamate-induced height transition of NR1/NR2A receptor2011

    • Author(s)
      Yuki Suzuki, Tom Alexander Goetze, David Stroebel, Shige H.Yoshimura, Pierre Paoletti, Robert M.Henderson, Kunio Takeyasu, J.Michael
    • Organizer
      The American Society for Cell Biology 51th Annual Meeting
    • Place of Presentation
      Denver, USA
    • Related Report
      2011 Annual Research Report
  • [Presentation] Direct imaging of transcription by E.coli RNA Polymerase in sub-second time region2011

    • Author(s)
      Yuki Suzuki, Minsang Shin, Kunio Takeyasu
    • Organizer
      XIII.Annual Linz Winter Workshop
    • Place of Presentation
      Linz, Austria
    • Related Report
      2010 Annual Research Report
  • [Presentation] Nucleosome fluctuation in solution revealed by fast-scanning AFM2009

    • Author(s)
      Yuki Suzuki, Kohji Hizume, Masatoshi Yokokawa, Kunio Takeyasu
    • Organizer
      XI International Scanning Probe Microscopy Conference
    • Place of Presentation
      Madrid, Spain
    • Related Report
      2009 Annual Research Report
  • [Presentation] 高速原子間力顕微鏡によるヌクレオソーム動態のナノスケールイメージング解析2009

    • Author(s)
      鈴木勇輝, 横川雅俊, 日詰光治, 吉村成弘, 竹安邦夫
    • Organizer
      第82回日本生化学会大会
    • Place of Presentation
      神戸
    • Related Report
      2009 Annual Research Report
  • [Book] Scanning probe Microscopy in Nanoscience and Nanotechnology 2 (Biological Application of Fast-Scanning Atomic Force Microscopy.)2011

    • Author(s)
      Bhushan, Bharat (EDT) Yuki Suzuki, Masatoshi Yokokawa, Shige.H.Yoshimura, Kunio Takeyasu
    • Publisher
      Springer-Verlag Berlin Heidelberg
    • Related Report
      2010 Annual Research Report
  • [Book] Scanning probe Microscopy in Nanoscience and Nanotechnology(内"Modern Atomic Force Microscopy and Its Application to the Study of Genome Architecture."担当)2010

    • Author(s)
      Bhushan,Bharat(EDT), Kunio Takeyasu, Hugo Maruyama, Yuki Suzuki, Kohji Hizume, Shige. H.Yoshimura.
    • Total Pages
      1170
    • Publisher
      Springer-Verlag Berlin Heidelberg
    • Related Report
      2009 Annual Research Report

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Published: 2009-04-01   Modified: 2024-03-26  

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