Structural studies of a novel mediator regulating homologous recombination

• Project/Area Number: 22370045
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Structural biochemistry
• Research Institution: The University of Tokyo
• Principal Investigator: SHIMIZU Toshiyuki 東京大学, 大学院・薬学系研究科, 教授 (30273858)
• Project Period (FY): 2010 – 2012
• Project Status: Completed (Fiscal Year 2012)
• Budget Amount: ¥18,590,000 (Direct Cost: ¥14,300,000、Indirect Cost: ¥4,290,000); Fiscal Year 2012: ¥5,330,000 (Direct Cost: ¥4,100,000、Indirect Cost: ¥1,230,000); Fiscal Year 2011: ¥5,590,000 (Direct Cost: ¥4,300,000、Indirect Cost: ¥1,290,000); Fiscal Year 2010: ¥7,670,000 (Direct Cost: ¥5,900,000、Indirect Cost: ¥1,770,000)
• Keywords: 相同組み換え / メディエーター / リコンビナーゼ / X線結晶構造解析 / Ｘ線結晶解析

Research Abstract

Rad51 has a central role in recombination, assembling onto single-stranded DNA as a nucleoprotein filament and catalyzing the invasion and exchange of homologous DNA sequences. Generally, Rad51 requires the assistance of mediators to overcome the inhibition by RPA. The Swi5-Sfr1 complex has been identified as a mediator, and recent studies have shown that it can activate recombinases directly, and thus this complex is the first molecule for the recombinase activator.We describe the crystal structure of the C-terminal half of Sfr1 in complex with Swi5 from S. pombe. Swi5 and Sfr1 form a parallel coiled-coil heterodimer, joined firmly together by formation of two newly identified leucine-zipper motifs and a bundle. Remarkably, the coiled-coil is sharply kinked, generating an elongated and curved shape. This molecular shape is suitable for binding within the reconstituted helical groove of the Rad51 filament, causing the activation of Rad51 filament by stabilizing it as an active form with a longer helical pitch. Biochemical analysis shows that the C-terminal region of Sfr1 complexed with Swi5 possesses the essential function of Swi5-Sfr1 for a recombination activator, while the N-terminal region of Sfr1 plays a role for the efficient recruitment of the Swi5-Sfr1 complex. Swi5-Sfr1 is conserved from yeast to humans, and therefore the phenomena presented here would be prevalent among all eukaryotic organisms.

Research Products

• [Journal Article] Characterisation of an intrinsically disordered protein complex of Swi5-Sfr1 by ion mobility mass spectrometry and small-angle X-ray scattering (2013)
  • Author(s): Saikusa K, Kuwabara N, Kokabu Y, Inoue Y, Sato M, Iwasaki H, Shimizu T, Ikeguchi M, Akashi S
  • Journal Title: Analyst Volume: 138 Pages: 1441-1449
  • 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 (2013)
  • Author(s): N.Kuwabara, Y.Murayama, H.Hashimoto, Y.Kokabu, M.Ikeguchi, M.Sato, K.Mayanagi, Y.Tsutsui, H.Iwasaki,and T.Shimizu
  • Journal Title: Structure Volume: 20 Issue: 3 Pages: 440-449
  • DOI: 10.1016/j.str.2012.01.005
  • 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 (2012)
  • Author(s): Kuwabara N, Murayama Y, Hashimoto H, Kokabu Y, Ikeguchi M, Sato M, Mayanagi K, Tsutsui Y, Iwasaki H, Shimizu T
  • Journal Title: Structure Volume: 20 Pages: 440-449
  • Peer Reviewed
• [Journal Article] Crystal Structure of Human β-Galactosidase: The Structural Basis of GM1 Gangliosidosis and Morquio B Diseases (2012)
  • Author(s): Ohto U, Usui K, , Ochi T, ,Yuki K, Satow Y andShimizu T
  • Journal Title: J. Biol. Chem Volume: 287 Pages: 1801-1812
  • Peer Reviewed
• [Journal Article] Crystal Structure of Human β-Galactosidase: The Structural Basis of GM1 Gangliosidosis and Morquio B Diseases (2012)
  • Author(s): Ohto U, et al.
  • Journal Title: J.Biol.Chem. Volume: 287 Issue: 3 Pages: 1801-1812
  • DOI: 10.1074/jbc.m111.293795
  • Peer Reviewed
• [Journal Article] Structural analyses of human Toll-like receptor polymorphisms D299G and T399I (2012)
  • Author(s): Ohto U, Yamakawa N, Akashi-Takamura S, Miyake K, Shimizu T
  • Journal Title: J Biol Chem. Volume: 287 Issue: 48 Pages: 40611-40617
  • DOI: 10.1074/jbc.m112.404608
  • Peer Reviewed
• [Journal Article] The fission yeast Swi5-Sfr1 complex, an activator of Rad51 recombinase, forms an extremely elongated Dogleg-shaped structure (2011)
  • Author(s): Kokabu Y, Murayama Y, Kuwabara N, Oroguchi T, Hashimoto H, Tsutsui Y, Nozaki N, Akashi S, Unzai S, Shimizu T, Iwasaki H, Sato M, Ikeguchi M
  • Journal Title: J Biol Chem Volume: 286 Pages: 43569-76
  • Peer Reviewed
• [Journal Article] Crystal Structures of Mouse and Human RP105/MD-1 Complexes Reveal Unique Dimer Organization of the Toll-Like Receptor Family (2011)
  • Author(s): Ohto U, Miyake K, Shimizu T
  • Journal Title: J Mol Biol Volume: 413 Pages: 815-825
  • Peer Reviewed
• [Journal Article] Fission yeast Swi5-Sfr1 protein complex, an activator of Rad51 recombinase, forms an extremely elongated dogleg-shaped structure (2011)
  • Author(s): Kokabu Y, Murayama Y, Kuwabara N, Oroguchi T, Hashimoto H, Tsutsui Y, Nozaki N, Akashi S, Unzai S, Shimizu T, Iwasaki H, Sato M, Ikeguchi M
  • Journal Title: J Biol Chem Volume: 286 Issue: 50 Pages: 43569-43576
  • DOI: 10.1074/jbc.m111.303339
  • Peer Reviewed
• [Journal Article] Crystal Structures of Mouse and Human RP105/MD-1 Complexes Reveal Unique Dimer Organization of the Toll-Like Receptor Family (2011)
  • Author(s): Ohto, et al.
  • Journal Title: J.Mol.Biol. Volume: 413 Pages: 815-825
  • 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): T.Yoshizawa, T.Shimizu, H.Hirano, M.Sato, H.Hashimoto
  • Journal Title: Acta Cryst. Volume: F67 Issue: 7 Pages: 830-832
  • DOI: 10.1107/s1744309111020045
  • Peer Reviewed
• [Journal Article] Expression, Purification and crystallization of Swi5 and the Swi5-Sfr1 complex from fission yeast (2010)
  • Author(s): Kokabu Y, Murayama Y, Kuwabara N, Oroguchi T, Hashimoto H, Tsutsui Y, Nozaki N, Akashi S, Unzai S, Shimizu T, Iwasaki H, Sato M, Ikeguchi M
  • Journal Title: J Biol Chem Volume: 286 Pages: 43569-76
  • Peer Reviewed
• [Journal Article] The structure of the N-terminal regulatory domain of a plant NADPH oxidase and its functional implications (2010)
  • Author(s): Kuwabara, N., Hashimoto, H., Yamada, N., Unzai, S., Ikeguchi, M., Sato, M., Murayama, Y., Iwasaki, H. and Shimizu, T.
  • Journal Title: Acta Crystallogr Volume: F66 Pages: 1124-1126
  • Peer Reviewed
• [Journal Article] Expression, Purification and crystallization of Swi5 and the Swi5-Sfr1 complex from fission yeast (2010)
  • Author(s): Kuwabara, et al
  • Journal Title: Acta Crystallogr. Volume: F66 Pages: 1124-1126
  • Peer Reviewed
• [Journal Article] The structure of the N-terminal regulatory domain of a plant NADPH oxidase and its functional implications (2010)
  • Author(s): Oda, et al
  • Journal Title: J.Biol.Chem. Volume: 285 Pages: 1435-1445
  • Peer Reviewed
• [Journal Article] Crystal structure of human REV7 in complex with a human REV3 fragment and structural implication of the interaction between DNA polymerase ζ and REV1 (2010)
  • Author(s): Hara, et al
  • Journal Title: J.Biol.Chem. Volume: 285 Pages: 12299-122307
  • Peer Reviewed
• [Journal Article] Crystal structure and functional implication of zinc-finger domain of Nanos (2010)
  • Author(s): Hashimoto, et al
  • Journal Title: EMBO reports Volume: 11 Pages: 848-853
  • Peer Reviewed
• [Journal Article] Structure and functional characterization of Vibrio parahaemolyticus thermostable direct hemolysin (TDH) (2010)
  • Author(s): Yanagihara, et al
  • Journal Title: J.Biol.Chem. Volume: 285 Pages: 16267-16274
  • Peer Reviewed
• [Journal Article]
  • Author(s): Oda, T., Hashimoto, H., Kuwabara, N., Akashi, S., Hayashi, K., Kojima, C., Wong, H.L., Kawasaki, T., Shimamoto, K., Sato, M., and Shimizu, T
  • Journal Title: J.Biol.Chem Volume: 285 Pages: 1435-1445
  • Peer Reviewed
• [Presentation] Program for Young Researcher Symposium inEurope'SGC Oxford (2012)
  • Author(s): Toshiyuki Shimizu
  • Organizer: Mechanistic insights into the activation of the Rad51-mediated strand exchange from the structure of recombination activator, Swi5-Sfr1 complex
  • Place of Presentation: Oxford, UK
  • Year and Date: 2012-06-08
• [Presentation] New frontier of the research in RecAfamily recombinases and their accessory proteins (RecA family 2012) (2012)
  • Author(s): Toshiyuki Shimizu
  • Organizer: Structural study of recombination activator, Swi5-Sfr1 complex
  • Place of Presentation: Kyoto Royal Hotel & Spa, Kyoto, Japan
• [Presentation] Structural study of recombination activator, Swi5-Sfr1 complex (2012)
  • Author(s): Shimizu T
  • Organizer: New frontier of the research in RecAfamily recombinases and their accessory proteins (RecA family 2012)
  • Place of Presentation: Kyoto Royal Hotel & Spa, Kyoto, Japan
  • Invited
• [Presentation] Symposium on Structure and Folding of Disease Related Proteins (2011)
  • Author(s): Toshiyuki Shimizu
  • Organizer: Mechanistic insights into the activation of the Rad51-mediated strand exchange from the structure of a recombination activator, the Swi5-Sfr1 complex
  • Place of Presentation: Seoul National University, Korea
  • Year and Date: 2011-07-08
• [Presentation] Mechanistic insights into the activation of the Rad51-mediated strand exchange from the structure of a recombination activator, the Swi5-Sfr1 comple (2011)
  • Author(s): Shimizu T
  • Organizer: Symposium on Structure and Folding of Disease Related Proteins
  • Place of Presentation: Seoul National University, Korea(招待講演)
  • Year and Date: 2011-07-08
• [Presentation] New frontier of the research in Rad51 recombinase and its accessory proteinsInstitut de Biologie Physico Chimique (2011)
  • Author(s): Toshiyuki Shimizu
  • Organizer: Crystal structure of recombination activator, Swi5-Sfr1 complex, from fission yeast
  • Place of Presentation: France
• [Presentation] 関節リウマチ関連タンパク質PAD4の構造科学的研究 (2010)
  • Author(s): 清水敏之
  • Organizer: 構造生物社会連携シンポジウム
  • Place of Presentation: 東京大学
  • Year and Date: 2010-11-20
• [Presentation] Mechanistic insights into the activation of the Rad51-mediated strand exchange from the structure of recombination activator, Swi5-Sfr1 complex
  • Author(s): Shimizu T
  • Organizer: Program for Young Researcher Symposium in Europe
  • Place of Presentation: Oxford, UK
  • Invited
• [Book] 「入門構造生物学」(「転写)部分を担当) (2010)
  • Author(s): 清水敏之
  • Total Pages: 199
  • Publisher: 共立出版
• [Remarks]
  • URL: http://www.f.u-tokyo.ac.jp/~kouzou/