DNA損傷を乗り越えるDNAポリメラーゼζ/REV1複合体の構造機能解析

2011 Fiscal Year Annual Research Report

• Project/Area Number: 22770109
• Research Institution: Yokohama City University
• Principal Investigator: 橋本 博 横浜市立大学, 生命ナノシスラム科学研究科, 助教 (40336590)
• Keywords: DNA複製 / DNA損傷 / DNAポリメラーゼ / 損傷乘り越えDNA合成 / 突然変異 / 構造解析 / タンパク質間相互作用

Research Abstract

DNAポリメラーゼゼータ(Polζ)とREV1は損傷乗り越えDNA合成における中心的なDNA合成酵素であり,その機能欠失は胎性致死や高発がん性など,細胞機能に重大な変調を来す.PolζとREV1は複合体を形成するが,その立体構造、相互作用や損傷乗り越えDNA合成のメカニズムは不明である.本研究では,Polζ/REV1複合体の立体構造をX線結晶構造解析によって解明する.また,Polζ/REV1複合体(REV7-3-1複合体)の相互作用を生化学的に解析し,その構築原理を解明することを目指す.
これまで,ヒトREV1のC末端領域(REVICT)の安定性に問題があり,結晶化品質のREV7-3-1三者複合体を調製することが出来なかった.本年度は,ヒトREV1の発現条件を再検討し,結晶化に供することが可能なヒトREV7-3-1複合体の調製および結晶化に成功し,予備的なX線回折実験を行った.
REV7-REV3複合体は既に確立した方法で調製した.ヒトREVICTは大腸菌のコドンに最適化したcDNAをPCR法によって合成し,かつ様々な長さの発現系を構築した.ヒトREVICTをGST融合タンパク質として大腸菌で発現させ,その後GSTタグをプロテアーゼで切断し,イオン交換カラムクロマトグラフィー,ゲル濾過カラムクロマトグラフィーによって高純度に精製した.精製したREVICTをREV7-REV3複合体と混合することでREV7-3-1複合体を調製した.得られた試料を用いて,結晶化条件のスクリーニングを行ったところいくつかの結晶化条件で結晶を得ることに成功した.つくばフォトンファクトリーでX線回折実験を行ったところ,4-5A分解能程度の回折を確認し,およその格子定数を見積もることに成功した.

Research Products

• [Journal Article] Identification of a small molecule PCNA inhibitor that disrupts interactions with PIP-Box proteins and inhibits DNA replication (2012)
  • Author(s): Chandanamali Punchihewa, Akira Inoue, Asami Hishiki, Yoshihiro Fujikawa, Michele Connelly, Benjamin Evison, Youming Shao, Richard Heath, Isao Kuraoka, Patrick Rodrigues, Hiroshi Hashimoto, Masanobu Kawanishi, Mamoru Sato, Takashi Yagi, Naoaki Fujii
  • Journal Title: J Biol Chem Volume: 287 Pages: 14289-14300
  • DOI: 10.1074/jbc.M112.353201
  • Peer Reviewed
• [Journal Article] Relationship between heat-induced fibrillogenicity and hemolytic activity of thermostable direct hemolysin and a related hemolysin of Vibrio parahaemolyticus (2011)
  • Author(s): Kiyouhisa Ohnishi, Kumiko Nakahira, Satoru Unzai, Kouta Mayanagi, Hiroshi Hashimoto, Kentaro Shiraki, Takeshi Honda, Itaru Yanagihara
  • Journal Title: FEMS Microbiol Lett Volume: 318 Pages: 10-17
  • DOI: 10.1111/j.1574-6968.2011.02233.x
  • Peer Reviewed
• [Journal Article] A novel heterotetrameric structure of the crenarchaeal PCNA2-PCNA3 complex (2011)
  • Author(s): Akito Kawai, Hiroshi Hashimoto, Shigesada Higuchi, Masaru Tsunoda, Mamoru Sato, Kazuo T Nakamura, Shuichi Miyamoto
  • Journal Title: J Struct Biol Volume: 174 Pages: 443-450
  • DOI: 10.1016/j.jsb.2011.02.006
  • Peer Reviewed
• [Journal Article] Crystal structure of bacis 7S globurin, a xyloglucan-specific endo-beta-1, 4-glucanase inhibitor protein-like protein from soybean lacking inhibitory activity against endo-beta-glucanase (2011)
  • Author(s): Takuya Yoshizawa, Toshiyuki Shimizu, Mayuki Yamabe, Misako Taichi, Yuji Nishiuchi, Naoki Shichijo, Satoru Unzai, Hisashi Hirano, Mamoru Sato, Hiroshi Hashimoto
  • Journal Title: FEBS J Volume: 278 Pages: 1944-1954
  • DOI: 10.1111/J.1742-4658.2011.08111.x
  • Peer Reviewed
• [Journal Article] Purification, crystallization and X-ray diffraction study of extracellular dermal glycoprotein from carrot and the inhibition complex it forms with an endo-beta-glucanase from Aspergillus aculeatus (2011)
  • Author(s): Takuya Yoshizawa, Toshiyuki Shimizu, Hisashi Hirano, Mamoru Sato, Hiroshi Hashimoto
  • Journal Title: Acta Cryatallogr Sect F Struct Biol Cryst Commun Volume: 67 Pages: 830-832
  • DOI: 10.1107/S1744309111020045
  • Peer Reviewed
• [Journal Article] Structural basis for matrix metalloproteinase-2 (MMP-2)-selective inhibitory action of beta-amyloid precursor protein-derived inhibitor (2011)
  • Author(s): Hiroshi Hashimoto, Tomoka Takeuchi, Kyoko Komatsu, Kaoru Miyazaki, Mamoru Sato, Shouichi Higashi
  • Journal Title: J Biol Chem Volume: 286 Pages: 33236-33243
  • DOI: 10.1074/jbc.M111.264176
  • Peer Reviewed
• [Journal Article] The Development of N-α-(2-Carboxyl)benzoyl-N5-(2-fluoro-1-iminoethyl)-L-ornithine Amide (o-F-amidine) and N-α-(2-Carboxyl)benzoyl-N5-(2-chloro-1-iminoethyl)-L-ornithine Amide (o-Cl-amidine) As Second Generation Protein Arginine Deiminase (PAD) Inhibitors (2011)
  • Author(s): Corey P.Causey, Justin E.Jones, Jessica L.Slack, Daisuke Kamei, Larry E.Jones, Jr., Venkataraman Subramanian, Bryan Knuckley, Pedram Ebrahimi, Alexander A.Chumanevich, Yuan Luo, Hiroshi Hashimoto, Mamoru Sato, Lorne J.Hofseth, Paul R.Thompson
  • Journal Title: J Med Chem Volume: 54 Pages: 6919-6935
  • DOI: 10.1021/jm2008985
  • Peer Reviewed
• [Journal Article] Mechanistic insights into the activation of rad51-mediated strand exchange from the structure of a recombination activator, the swi5-sfr1 complex (2011)
  • Author(s): Naoyuki Kuwabara, Yasuto Murayama, Hiroshi Hashimoto, Yuuichi Kokabu, Mitsunori Ikeguchi, Mamoru Sato, Kouta Mayanagi, Yasuhiro Tsutsui, Hiroshi Iwasaki, Toshiyuki Shimizu
  • Journal Title: Structure Volume: 20 Pages: 440-449
  • DOI: 10.1016/j.str.2012.01.005
  • Peer Reviewed
• [Journal Article] Fission yeast Swi5-Sfr1 protein complex, an activator of Ead51 recombinase, forms an extremely elongated dogleg-shaped structure (2011)
  • Author(s): Yuichi Kokabu, Yasuto Murayama, Naoyuki Kuwabara, Tomotaka Oroguchi, Hiroshi Hashimoto, Yasuhiro Tsutsui, Naohito Nozaki, Satoko Akashi, Satoru Unzai, Toshiyuki Shimizu, Hiroshi Iwasaki, Mamoru Sato, Mitsunori Ikeguchi
  • Journal Title: J.Biol.Chem. Volume: 286 Pages: 43569-43576
  • DOI: 10.1074/jbc.M111.303339
  • Peer Reviewed
• [Presentation] ニンジンEDGPの麹菌キシログルカン特異的エンドグルカナーゼ阻害機構 (2012)
  • Author(s): 吉澤拓也, 清水敏之, 平野久, 橋本博, 佐藤衛
  • Organizer: 第29回PFシンポジウム
  • Place of Presentation: つくば国際会議場
  • Year and Date: 20120315-20120316
• [Presentation] ニンジンEDGPによる麹菌XEGの阻害複合体構造 (2011)
  • Author(s): 吉澤拓也, 橋本博, 清水敏之, 平野久, 佐藤衛
  • Organizer: 平成23年度日本結晶学会年会
  • Place of Presentation: 北海道大学学術交流会館
  • Year and Date: 20111124-20111125
• [Presentation] APP由来インヒビターペプチドによるMMP-2選択的阻害機構の解明 (2011)
  • Author(s): 竹内友香, 橋本博, 宮崎香, 佐藤衛, 東昌市
  • Organizer: 第84回日本生化学
  • Place of Presentation: 京都国際会館
  • Year and Date: 20110921-20110924
• [Presentation] Analysis on dynamical structure of intrinsically disordered protein Hef, using MD-SAXS method (2011)
  • Author(s): Tomotaka Oroguchi, Takashi Oda, Hiroshi Hashimoto, Yoshizumi Ishino, Mitsunori Ikeguchi, Mamoru Sato
  • Organizer: 第49回日本生物物理学会年会
  • Place of Presentation: 兵庫県立大学姫路書写キャンパス
  • Year and Date: 20110916-20110918
• [Presentation] Crystal structure of zinc-finger domain of Nanos and its functional implications (2011)
  • Author(s): Hiroshi Hashimoto, Kodai Hara, Asami Hishiki, Shigeta Kawaguchi, Takuya Yoshizawa, Satoru Unzai, Yutaka Tamaru, Toshiyuki Shimizu, Mamoru Sato
  • Organizer: XXII Congress IUCr
  • Place of Presentation: Municipal Conference Center, Madrid, Spain
  • Year and Date: 20110822-20110830
• [Presentation] Structural basis of inhibition mechanism by carrot EDGP against GH12 The inhibitory structure of carrot EDGP against endoglucanase from A.aculeatus (2011)
  • Author(s): Takuya Yoshizawa, Toshiyuki Shimizu, Hisashi Hirano, Mamoru Sato, Hiroshi Hashimoto
  • Organizer: XXII Congress IUCr
  • Place of Presentation: Municipal Conference Center, Madrid, Spain
  • Year and Date: 20110822-20110830
• [Presentation] MMP-2とAPP由来インヒビターペプチドとの複合体の結晶構造解析 (2011)
  • Author(s): 竹内友香, 橋本博, 宮崎香, 佐藤衛, 東昌市
  • Organizer: 第16回日本病態プロテアーゼ学会学術集会
  • Place of Presentation: 千里ライフサイエンスセンタ
  • Year and Date: 20110816-20110827
• [Presentation] 始原生殖細胞の形成に関わるNanosの構造生物学的研究 (2011)
  • Author(s): 橋本博, 原幸大, 菱木麻美, 川口茂太, 七條直記, 中村敬至, 雲財悟, 田丸浩, 清水敏之, 佐藤衛
  • Organizer: 第28回PFシンポジウム
  • Place of Presentation: つくば国際会議場
  • Year and Date: 20110712-20110713
• [Presentation] 腸炎ビブリオ由来耐熱性溶血毒TDHの構造 (2011)
  • Author(s): 橋本博, 中平久美子, 山根努, 福井貴史, 大西紀陽久, 清水敏之, 本田武司, 佐藤衛, 池口満徳, 柳原格
  • Organizer: 第28回PFシンポジウム
  • Place of Presentation: つくば国際会議場
  • Year and Date: 20110712-20110713
• [Presentation] 制限酵素EcoO109IのDNA結合に関する構造研究 (2011)
  • Author(s): 渡部みなみ, 橋本博, 喜多恵子, 清水敏之, 佐藤衛
  • Organizer: 第28回PFシンポジウム
  • Place of Presentation: つくば国際会議場
  • Year and Date: 20110712-20110713
• [Presentation] 始原生殖細胞の維持に関わるNanosの構造生物学的研究 (2011)
  • Author(s): 橋本博, 原幸大, 菱木麻美, 川口茂太, 吉澤拓也, 雲財悟, 田丸浩, 清水敏之, 佐藤衛
  • Organizer: 第11回日本蛋白質科学会
  • Place of Presentation: ホテル阪急エキスポパーク
  • Year and Date: 20110607-20110609
• [Presentation] Structure of REV7 in complex with a REV3 fragment and structural implication of the interaction between DNA polymerase ζ and REV1 (2011)
  • Author(s): Hiroshi Hashimoto, Kodai Hara, Yoshiki Murakumo, Shunsuke Kobayashi, Toshiaki Kogame, Satoru Unzai, Satoko Akashi, Shunichi Takeda, Toshiyuki Shimizu, Mamoru Sato
  • Organizer: Current Opinion in Structural Biology & DNA Repair
  • Place of Presentation: NH Grand Hotel Krasnapolsky, Amsterdam, Netherlands
  • Year and Date: 20110607-20110609
• [Presentation] Structure of REV7 in complex with a REV3 fragment and structural implication of the interaction between DNA polymerase ζ and REV1 (2011)
  • Author(s): Hiroshi Hashimoto
  • Organizer: SNY-YCU Bilateral Symposium on Structural Biology
  • Place of Presentation: RIEKN Yokohama Institute, Yokohama, Japan
  • Year and Date: 2011-11-14