Biochemical studies on the replication fork model with three DNA polymerases in human cells

• Project/Area Number: 25440011
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Molecular biology
• Research Institution: Kyushu University
• Principal Investigator: Tsurimoto Toshiki 九州大学, 理学研究院, 教授 (30163885)
• Project Period (FY): 2013-04-01 – 2017-03-31
• Project Status: Completed (Fiscal Year 2016)
• Budget Amount: ¥5,330,000 (Direct Cost: ¥4,100,000、Indirect Cost: ¥1,230,000); Fiscal Year 2015: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000); Fiscal Year 2014: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000); Fiscal Year 2013: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
• Keywords: 複製フォーク / DNAポリメラーゼ / in vitro複製 / in vitro 複製

Outline of Final Research Achievements

This research aimed to study the mechanism to proceed a replication fork with three functionally distinct DNA polymerases in eukaryotes. I especially focused on a biochemical background to distinguish DNA polymerases for syntheses of leading and lagging strands. Firstly, I reconstituted the replicative DNA helicase, human CMG complex and obtained the highly purified active CMG helicase. Secondly, I analyzed a functional significance of the interaction between the second PCNA loader Ctf18-RFC and DNA polymerase ε. I elucidated that Ctf18-RFC actively loads PCNA only when it complexes with DNA polymerase ε. This result indicated that Ctf18-RFC functions as a component of the leading-strand DNA polymerase complex and actively loads PCNA on the leading strands. Considering the role of RFC for loading of PCNA on the lagging strands, these two PCNA loaders will coordinate PCNA loading on both strands and play a role to distinguish DNA polymerases for syntheses of the two DNA strands.

Research Products

• [Journal Article] The dominant role of proofreading exonuclease activity of replicative polymerase ε in cellular tolerance to cytarabine (Ara-C). (2017)
  • Author(s): Tsuda M, Terada K, Ooka M, Kobayashi K, Sasanuma H, Fujisawa R, Tsurimoto T, Yamamoto J, Iwai S, Kadoda K, Akagawa R, Huang SN, Pommier Y, Sale JE, Takeda S, Hirota K.
  • Journal Title: Oncotarget Volume: in press Issue: 20 Pages: 33457-33474
  • DOI: 10.18632/oncotarget.16508
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Human CTF18-RFC clamp-loader complexed with non-synthesising DNA polymerase ε efficiently loads the PCNA sliding clamp. (2017)
  • Author(s): Fujisawa R, Ohashi E, Hirota K, Tsurimoto T
  • Journal Title: Nucleic Acids Res. Volume: 印刷中 Issue: 8 Pages: 4550-4563
  • DOI: 10.1093/nar/gkx096
  • Peer Reviewed / Open Access
• [Journal Article] MutSα maintains the mismatch repair capability by inhibiting PCNA unloading (2016)
  • Author(s): Kawasoe, Y., Tsurimoto, T., Nakagawa, T., Masukata, H., Takahashi, TS.
  • Journal Title: eLIFE Volume: 12;5
  • DOI: 10.7554/elife.15155
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Claspin recruits Cdc7 kinase for initiation of DNA replication in human cells. (2016)
  • Author(s): Yang CC, Suzuki M, Yamakawa S, Uno S, Ishii A, Yamazaki S, Fukatsu R, Fujisawa R, Sakimura K, Tsurimoto T, Masai H.
  • Journal Title: Nat Commun. Volume: 7 Issue: 1 Pages: 12135-12135
  • DOI: 10.1038/ncomms12135
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] In vivo evidence for translesion synthesis by the replicative DNA polymerase δ. (2016)
  • Author(s): Hirota K, Tsuda M, Mohiuddin, Tsurimoto T, Cohen IS, Livneh Z, Kobayashi K, Narita T, Nishihara K, Murai J, Iwai S, Guilbaud G, Sale JE, Takeda S.
  • Journal Title: Nucleic Acids Res Volume: 44(15) Pages: 7242-50
  • DOI: 10.1093/nar/gkw439
  • NAID: 120006338056
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Conserved interaction of Ctf18-RFC with DNA polymerase ε is critical for maintenance of genome stability in saccharomyces cerevisiae. (2016)
  • Author(s): Okimoto H, Tanaka S, Araki H, Ohashi E, Tsurimoto T.
  • Journal Title: Genes Cells. Volume: in press Issue: 5 Pages: 482-491
  • DOI: 10.1111/gtc.12356
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Rapid purification and characterization of mutant origin recognition complexes in Saccharomyces cerevisiae (2016)
  • Author(s): Kawakami, H. (corresponding author), Ohashi, E., Tsurimoto, T., and Katayama, T.
  • Journal Title: Front. Microbiol. Volume: 7 Pages: 521-521
  • DOI: 10.3389/fmicb.2016.00521
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Rapid purification and characterization of mutant Origin Recognition Complexes in Saccharomyces cerevisiae. (2016)
  • Author(s): Kawakami H, Ohashi E, Tsurimoto T, and Katayama T.
  • Journal Title: Front. Microbiol. Volume: in press
  • Peer Reviewed
• [Journal Article] Specific binding of eukaryotic ORC to DNA replication origins depends on highly conserved basic residues. (2015)
  • Author(s): Kawakami H, Ohashi E, Kanamoto S, Tsurimoto T, Katayama T.
  • Journal Title: Sci Rep. Volume: 5 Issue: 1 Pages: 14929-14929
  • DOI: 10.1038/srep14929
  • Peer Reviewed / Open Access
• [Journal Article] Short CCG repeat in huntingtin gene is an obstacle for replicative DNA polymerases, potentially hampering progression of replication fork. (2015)
  • Author(s): Le HP, Masuda Y, Tsurimoto T, Maki S, Katayama T, Furukohri A, Maki H.
  • Journal Title: Genes Cells. Volume: 10 Issue: 10 Pages: 817-833
  • DOI: 10.1111/gtc.12275
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Intramolecular Binding of the Rad9 C Terminus in the Checkpoint Clamp Rad9-Hus1-Rad1 Is Closely Linked with Its DNA Binding. (2015)
  • Author(s): Takeishi Y, Iwaya-Omi R, Ohashi E, Tsurimoto T.
  • Journal Title: J Biol Chem. Volume: 290 Issue: 32 Pages: 19923-19932
  • DOI: 10.1074/jbc.m115.669002
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] The POLD3 subunit of DNA polymerase δ can promote translesion synthesis independently of DNA polymerase ζ. (2015)
  • Author(s): Hirota K, Yoshikiyo K, Guilbaud G, Tsurimoto T, Murai J, Tsuda M, Phillips L, Narita T, Nishihara K, Kobayashi K, Yamada K, Nakamura J, Pommier Y, Lehmann, A, Sale J, Takeda S
  • Journal Title: Nucleic Acids Res. Volume: 43 Issue: 3 Pages: 1671-1683
  • DOI: 10.1093/nar/gkv023
  • NAID: 120005825923
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Interaction between Rad9-Hus1-Rad1 and TopBP1 activates ATR-ATRIP and promotes TopBP1 recruitment to sites of UV-damage. (2014)
  • Author(s): Ohashi E, Takeishi Y, Ueda S, Tsurimoto T.
  • Journal Title: DNA Repair (Amst) Volume: 21 Pages: 1-11
  • DOI: 10.1016/j.dnarep.2014.05.001
  • Peer Reviewed
• [Journal Article] 3.Kaposi's sarcoma-associated herpesvirus LANA recruits the DNA polymerase clamp loader to mediate efficient replication and virus persistence. (2014)
  • Author(s): Sun Q, Tsurimoto T, Juillard F, Li L, Li S, De León Vázquez E, Chen S, Kaye K.
  • Journal Title: Proc Natl Acad Sci U S A. Volume: 111 Pages: 11816-11821
  • Peer Reviewed
• [Journal Article] RAD5a ubiquitin ligase is involved in ubiquitination of Arabidopsis thaliana proliferating cell nuclear antigen (2013)
  • Author(s): Strzalka W, Bartnicki F, Pels K, Jakubowska A, Tsurmoto T, Tanaka K.
  • Journal Title: J. Exp. Bot. Volume: 64 Issue: 4 Pages: 859-869
  • DOI: 10.1093/jxb/ers368
  • Peer Reviewed
• [Presentation] Rad9 C末端による9-1-1クランプへの蛋白質結合の制御 (2017)
  • Author(s): 大橋 英治、岩田 直也、釣本 敏樹
  • Organizer: 第34回染色体ワークショップ・第15回核ダイナミクス研究会合同
  • Place of Presentation: 木更津市
  • Year and Date: 2017-01-11
• [Presentation] PCNA結合蛋白質のRad9-Hus1-Rad1リングとの結合メカニズム (2016)
  • Author(s): 大橋英治、岩田直也、釣本敏樹
  • Organizer: 第39回日本分子生物学会年会シンポジウム
  • Place of Presentation: 横浜市
  • Year and Date: 2016-12-02
  • Invited
• [Presentation] Polεを足場にしたCtf18-RFCによる動的PCNAローディング (2016)
  • Author(s): 釣本 敏樹 藤澤 遼 大橋英治
  • Organizer: 第39回日本分子生物学会年会シンポジウム
  • Place of Presentation: 横浜市
  • Year and Date: 2016-11-30
  • Invited
• [Presentation] Ctf18-RFC bound to Polε loads PCNA efficiently. (2016)
  • Author(s): Fujisawa R, Ohashi E, Hirota K, Tsurimoto T
  • Organizer: The 10th 3R Symposium
  • Place of Presentation: Matsue, Japan
  • Year and Date: 2016-11-13
  • Int'l Joint Research / Invited
• [Presentation] Intramolecular folding of Rad9 C-terminal tail in the Rad9-Hus1-Rad1 checkpoint clamp regulates its interaction with DNA damage response proteins. (2016)
  • Author(s): Ohashi E, Iwata N, Tsurimoto T
  • Organizer: The 10th 3R Symposium
  • Place of Presentation: Matsue, Japan
  • Year and Date: 2016-11-13
  • Int'l Joint Research
• [Presentation] クランプ・ローダー系による進行障害時の複製フォークの機能制御 (2015)
  • Author(s): Tsurimoto T, Ohashi E, Fujisawa R, Takeishi Y
  • Organizer: 第38回日本分子生物学会年会ワークショップ
  • Place of Presentation: 神戸
  • Year and Date: 2015-12-01
  • Invited
• [Presentation] Rad9 C末端によるチェックポイントクランプRad9-Hus1-Rad1の機能制御機構 (2015)
  • Author(s): Ohashi E, Takeishi Y, Tsurimoto T.
  • Organizer: 第38回日本分子生物学会年会ワークショップ
  • Place of Presentation: 神戸
  • Year and Date: 2015-12-01
  • Invited
• [Presentation] Rad9 C末端によるRad9-Hua1-Rad1(9-1-1)のDNA損傷応答制御機構 (2015)
  • Author(s): Ohashi E, Takeishi Y, and Tsurimoto T.
  • Organizer: 第23回DNA複製・組換え・修復ワークショップ
  • Place of Presentation: 焼津
  • Year and Date: 2015-10-19
• [Presentation] Polεに結合したCtf18-RFCのPCNAローディング過程の解析 (2015)
  • Author(s): Ryo Fujisawa, Ohashi E, Tsurimoto T.
  • Organizer: 第23回DNA複製・組換え・修復ワークショップ
  • Place of Presentation: 焼津
  • Year and Date: 2015-10-19
• [Presentation] Functional Significance of the interaction between the second PCNA loaderCtf18-RFC and Pol e (2015)
  • Author(s): Ryo Fujisawa, Ohashi E, Tsurimoto T.
  • Organizer: Eukaryotic DNA replication & genome maintenance Cold Spring Harbor Laboratory Meeting
  • Place of Presentation: New York USA
  • Year and Date: 2015-09-01
  • Int'l Joint Research
• [Presentation] Multiple roles of Rad9 C-tail in the checkpoint clamp Rad9-Hus1-Rad1 (2015)
  • Author(s): Ohashi E, Takeishi Y, Tsurimoto T.
  • Organizer: Eukaryotic DNA replication & genome maintenance Cold Spring Harbor Laboratory Meeting
  • Place of Presentation: New York USA
  • Year and Date: 2015-09-01
  • Int'l Joint Research
• [Presentation] DNA損傷応答因子ATR-ATRIP, Rad9-Hus1-Rad1, TopBP1の3者間結合によるATR活性化機構 (2014)
  • Author(s): Ohashi E, Takeishi Y, Ueda S, Tsurimoto T
  • Organizer: 第37回日本分子生物学会年会ワークショップ
  • Place of Presentation: 横浜
  • Year and Date: 2014-11-27
  • Invited
• [Presentation] ヒトCtf18-RFCはPolεと複合体となって機能的にPCNAをロードする (2014)
  • Author(s): Fujisawa R, Tsrurimoto T
  • Organizer: 第37回日本分子生物学会年会ワークショップ
  • Place of Presentation: 横浜
  • Year and Date: 2014-11-27
• [Presentation] 出芽酵母ORC複合体の分子内外クロストークの同定 (2014)
  • Author(s): Kawakami H, Kawanishi T, Ohashi E, Tsrurimoto T, Katayama T
  • Organizer: 第37回日本分子生物学会年会ワークショップ
  • Place of Presentation: 横浜
  • Year and Date: 2014-11-27
• [Presentation] The C-terminal of Rad9 associates to the core ring structure of the checkpoint clamp, Rad9-Hus1-Rad1, and regulates its DNA binding activity (2014)
  • Author(s): Takeishi Y, Ohashi E, Tsrurimoto T
  • Organizer: 第37回日本分子生物学会年会
  • Place of Presentation: 横浜
  • Year and Date: 2014-11-25
• [Presentation] Novel mechanisms of PCNA loader complexes to specify their functions. (2014)
  • Author(s): Tsurimoto T, Fujisawa R, Ohashi E, Tanaka S, Araki H, Sun Q, Kaye K
  • Organizer: The 9th 3R Symposium
  • Place of Presentation: Gotemba, Shizuoka Japan
  • Year and Date: 2014-11-20
  • Invited
• [Presentation] The ATR activation through the accumulation of the checkpoint factors and their interactions at the site of DNA damage (2014)
  • Author(s): Ohashi E, Takeishi Y, Tsrurimoto T
  • Organizer: The 9th 3R Symposium
  • Place of Presentation: Gotemba, Shizuoka Japan
  • Year and Date: 2014-11-20
• [Presentation] Interaction of Ctf18-RFC with DNA polymerase ε specifies their functional sites for the active PCNA loading (2014)
  • Author(s): Fujisawa R, Tsrurimoto T
  • Organizer: The 9th 3R Symposium
  • Place of Presentation: Gotemba, Shizuoka Japan
  • Year and Date: 2014-11-19
• [Presentation] Polε/Ctf18-RFC複合体による複製フォーク進行障害への応答 (2014)
  • Author(s): 沖本寛子、藤澤遼、田栗蘭、大橋英治、田中誠司、荒木弘之、釣本敏樹
  • Organizer: 「ゲノムを支る非コードDNA領域の機能」 公開シンポジウム インターメアによる染色体制御機構
  • Place of Presentation: 東京
• [Presentation] ヒトから酵母まで保存されたPolε/Ctf18-RFC間相互作用 (2013)
  • Author(s): 沖本寛子、藤澤遼、田栗蘭、大橋英治、田中誠司、荒木弘之、釣本敏樹
  • Organizer: DNA複製・組換え・修復ワークショップ
  • Place of Presentation: 仙台
• [Presentation] DNAポリメラーゼεとPCNAローダーCtf18-RFCのホロ複合体形成とその機能
  • Author(s): Tsurimoto T, Okimoto H, Ryo Fujisawa, Ran Taguri, Ohashi E, Tanaka S, Araki H
  • Organizer: 第36回日本分子生物学会年会ワークショップ
  • Place of Presentation: 神戸
  • Invited
• [Presentation] 出芽酵母Ctf18のC末端はヒトと同様にDNA ポリメラーセεとの結合に関与し、ケノム安定性に重要である
  • Author(s): 沖本 寛子、田栗 蘭、田中 誠司、荒木 弘之、釣本 敏樹
  • Organizer: 第36回日本分子生物学会年会
  • Place of Presentation: 神戸
• [Presentation] Roles of loader proteins for coupling between replication and chromosome maintenance.
  • Author(s): Okimoto H, Tanaka S, Araki H, Ohashi E, Tsurimoto T.
  • Organizer: International Conference "Coupling of replication, repair and transcription, and their common mechanism of chromatin remodeling"
  • Place of Presentation: 京都
  • Invited
• [Presentation] Functions of the C-terminal of the chromosome cohesion PCNA loader, Ctf18-RFC
  • Author(s): Okimoto H, Tanaka S, Araki H, Ohashi E, Tsurimoto T.
  • Organizer: Cold Spring Harbor Lab. Eukaryotic DNA Replication & Genome Maintenanceワークショップ
  • Place of Presentation: 米国、NewYork
• [Book] Functions of multiple clamp and clamp-loader complexes in eukaryotic DNA replication in "DNA Replication: From Old Principles to New Discovery” Advances in Experimental Medicine and Biology, (2017)
  • Author(s): Ohashi E, Tsurimoto T
  • Publisher: Springer
• [Book] 分子遺伝学 in 遺伝学（遺伝子から見た生物）鷲谷いずみ、桂勳編 (2017)
  • Author(s): 釣本敏樹
  • Total Pages: 32
  • Publisher: 培風館
• [Remarks] 染色体機能学研究室複製グループ
  • URL: http://seibutsu.biology.kyushu-u.ac.jp/~chromosome/