Structural study on the clamp PCNA-loading mechanism by the clamp loader RFC

• Project/Area Number: 26440023
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Structural biochemistry
• Research Institution: University of Yamanashi
• Principal Investigator: OYAMA Takuji 山梨大学, 総合研究部, 准教授 (60423133)
• Co-Investigator(Renkei-kenkyūsha): ISHINO Yoshizumi 九州大学, 農学研究院, 教授 (30346837) ; SHIRAI Tsuyoshi 長浜バイオ大学, バイオサイエンス学部, 教授 (00262890) ; MAYANAGI Kouta 九州大学, 生体防御医学研究所, 助教 (50418571)
• Project Period (FY): 2014-04-01 – 2017-03-31
• Project Status: Completed (Fiscal Year 2016)
• Budget Amount: ¥5,070,000 (Direct Cost: ¥3,900,000、Indirect Cost: ¥1,170,000); Fiscal Year 2016: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2015: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2014: ¥2,470,000 (Direct Cost: ¥1,900,000、Indirect Cost: ¥570,000)
• Keywords: DNA複製 / タンパク質DNA複合体 / 結晶構造解析

Outline of Final Research Achievements

To understand the detailed loading mechanism of the clamp PCNA onto DNA by the clamp-loader RFC in an ATP-dependent manner, we aimed (1) preparation of selenomethionine-labeled (SeMet) PCNA, (2) high resolution structure determination of RFC, and (3) crystallization and structure determination of the RFC-PCNA-DNA ternary complex. We determined the crystal structure of SeMet PCNA at 2.5 angstrom resolution. We established a novel preparation method of the highly purified recombinant RFC, but did not succeed in the high-resolution structure determination of the RFC. We performed crystallization screening of the RFC-PCNA-DNA ternary complex in a wide range of conditions, but obtained no crystals during this research project.

Research Products

• [Journal Article] 細胞内の巨大な分子モーター「ダイニン」の構造解析 (2016)
  • Author(s): 大山拓次、栗栖源嗣、昆隆英
  • Journal Title: 放射光 Volume: 29 Pages: 82-89
  • NAID: 40020850525
  • Peer Reviewed
• [Journal Article] Atomic structure of an archaeal GAN suggests its dual roles as an exonuclease in DNA repair and a CMG component in DNA replication. (2016)
  • Author(s): Oyama, T., Ishino, S., Shirai, T., Yamagami, T., Nagata, M., Ogino, H., Kusunoki, M., and Ishino, Y.
  • Journal Title: Nucleic Acids, Res. Volume: 44 Issue: 19 Pages: 9509-9517
  • DOI: 10.1093/nar/gkw789
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Integrated molecular mechanism directing nucleosome reorganization by human FACT (2015)
  • Author(s): Tsunaka Y, Fujiwara Y, Oyama T, Hirose S, Morikawa K
  • Journal Title: Genes Dev Volume: 30(6) Issue: 6 Pages: 673-686
  • DOI: 10.1101/gad.274183.115
  • Peer Reviewed / Open Access
• [Journal Article] Different structures of the two peroxisome proliferator-activated receptor gamma (PPARγ) ligand-binding domains in homodimeric complex with partial agonist, but not full agonist (2015)
  • Author(s): M. Ohashi, T. Oyama, H. Miyachi
  • Journal Title: Bioorganic & Medicinal Chemistry Letters Volume: - Issue: 13 Pages: 2639-2644
  • DOI: 10.1016/j.bmcl.2015.04.076
  • Peer Reviewed
• [Journal Article] Peroxisome proliferator-activated receptor gamma (PPARgamma) has multiple binding points that accommodate ligands in various conformations: Structurally similar PPARgamma partial agonists bind to PPARgamma LBD in different conformations (2015)
  • Author(s): Ohashi M, Gamo K, Oyama T, Miyachi H
  • Journal Title: Bioorg Med Chem Lett Volume: 25 Issue: 14 Pages: 2758-2762
  • DOI: 10.1016/j.bmcl.2015.05.025
  • Peer Reviewed
• [Journal Article] Structural design and synthesis of arylalkynyl amide-type peroxisome proliferator-activated receptor γ (PPARγ)-selective antagonists based on the helix12-folding inhibition hypothesis. (2015)
  • Author(s): M. Ohashi, K. Gamo, Y. Tanaka, M. Waki, Y. Beniyama, K. Matsuno, J. Wada, M. Tenta, J. Eguchi, M. Makishima, N. Matsuura, T. Oyama, H. Miyachi.
  • Journal Title: European Journal of Medicinal Chemistry Volume: 90 Pages: 53-67
  • DOI: 10.1016/j.ejmech.2014.11.017
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Modification of Chimeric (2S, 3S)-butanediol dehydrogenase based on structural information. (2015)
  • Author(s): Shimegi T, Mochizuki K, Oyama T, Ohtsuki T, Kusunoki M, Ui S
  • Journal Title: Protein Pept Lett Volume: 22 Pages: 226-233
  • Peer Reviewed
• [Journal Article] Crystallization and preliminary X-ray diffraction analysis of domain chimeric L-(2S, 3S)-butanediol dehydrogenase (2014)
  • Author(s): Shimegi T, Oyama T, Ohtsuki T, Kurisu G, Kusunoki M, Ui S
  • Journal Title: Acta Cryst F Volume: 70 Issue: 4 Pages: 461-463
  • DOI: 10.1107/s2053230x13032755
  • Peer Reviewed
• [Journal Article] Purification and characterization of a protease from Isaria cicadae (2014)
  • Author(s): Takekawa C, Nakamura K, Homma H, Oyama T
  • Journal Title: Mushroom Science and Biotechnology Volume: 22 Pages: 74-78
  • NAID: 110009863113
  • Peer Reviewed
• [Journal Article] Molecular dynamics study-guided identification of cyclic amine structures as novel hydrophobic tail components of hPPARγ agonists. (2014)
  • Author(s): Y. Tanaka, K. Gamo, T. Oyama, M. Ohashi, M. Waki, K. Matsuno, N. Matsuura, H. Tokiwa, H. Miyachi.
  • Journal Title: Bioorganic & Medicinal Chemistry Letters Volume: 24 Issue: 16 Pages: 4001-4005
  • DOI: 10.1016/j.bmcl.2014.06.023
  • Peer Reviewed
• [Journal Article] Structure of the entire stalk region of the dynein motor domain (2014)
  • Author(s): Yosuke Nishikawa, Takuji Oyama, Narutoshi Kamiya, Takahide Kon, Yoko Y. Toyoshima, Haruki Nakamura, Genji Kurisu
  • Journal Title: Journal of Molecular Biology Volume: 426 Issue: 19 Pages: 3232-3245
  • DOI: 10.1016/j.jmb.2014.06.023
  • Peer Reviewed
• [Journal Article] Disordered interdomain region of Gins is important for functional tetramer formation to stimulate MCM helicase in Thermoplasma acidophilum (2014)
  • Author(s): Ogino H, Ishino S, Oyama T, Kohda D, Ishino Y
  • Journal Title: Biosci Biotechnol Biochem Volume: 79 Issue: 3 Pages: 432-438
  • DOI: 10.1080/09168451.2014.982503
  • Peer Reviewed
• [Presentation] 結晶構造から明らかになった古細菌GANのDNA複製でのCMG因子および修復でのヌクレアーゼとしての2つの役割 (2016)
  • Author(s): 大山拓次、石野園子、白井剛、山上健、永田麻梨子、尾木野弘実、楠木正巳、石野良純
  • Organizer: 第39回日本分子生物学会年会
  • Place of Presentation: パシフィコ横浜（横浜市西区）
  • Year and Date: 2016-11-30
• [Presentation] 大豆イソフラボングルコシルトランスフェラーゼGmIF7GTの結晶構造解析 (2014)
  • Author(s): 桑名美貴子、楠木正巳、大山拓次、中山亨
  • Organizer: 平成26年度日本結晶学会年会
  • Place of Presentation: 東京大学農学部（東京都文京区）
  • Year and Date: 2014-11-01 – 2014-11-03
• [Presentation] 大豆イソフラボングルコシルトランスフェラーゼGmIF7GTの構造生物学 (2014)
  • Author(s): 桑名美貴子、楠木正巳、大山拓次、中山亨
  • Organizer: 第87回日本生化学会年会
  • Place of Presentation: 国立京都国際会館（京都市左京区）
  • Year and Date: 2014-10-15 – 2014-10-18
• [Remarks] 山梨大学 研究者総覧 大山拓次
  • URL: http://erdb.yamanashi.ac.jp/rdb/A_DispInfo.Scholar/0/32B8A9CF314A20D1.html
• [Remarks] 研究成果掲載記事（山梨大学生命環境学部HPトピックス）
  • URL: http://www.les.yamanashi.ac.jp/modules/news/index.php?page=article&storyid=204
• [Remarks] 研究成果掲載記事（山梨大学生命環境学部HPトピックス）
  • URL: http://www.les.yamanashi.ac.jp/modules/news/index.php?page=article&storyid=232
• [Remarks] 研究成果掲載記事（山梨大学NEWS）
  • URL: http://www.yamanashi.ac.jp/6595
• [Remarks] 山梨大学 研究者総覧 大山拓次
  • URL: http://erdb.yamanashi.ac.jp/rdb/A_DispInfo.Scholar/0/32B8A9CF314A20D1.html