Mcm蛋白質ならびにMcmと相互作用する蛋白質複合体による複製モニター機構

• Project/Area Number: 11241201
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: Osaka University
• Principal Investigator: 升方 久夫 大阪大学, 大学院・理学研究科, 教授 (00199689)
• Co-Investigator(Kenkyū-buntansha): 石見 幸男 三菱化学生命, 科学研究所, 主任研究員 ; 滝澤 温彦 (滝沢 温彦) 大阪大学, 大学院・理学研究科, 教授 (60154944)
• Project Period (FY): 1999 – 2003
• Project Status: Completed (Fiscal Year 2003)
• Budget Amount: ¥131,500,000 (Direct Cost: ¥131,500,000); Fiscal Year 2003: ¥27,900,000 (Direct Cost: ¥27,900,000); Fiscal Year 2002: ¥29,200,000 (Direct Cost: ¥29,200,000); Fiscal Year 2001: ¥30,600,000 (Direct Cost: ¥30,600,000); Fiscal Year 2000: ¥23,800,000 (Direct Cost: ¥23,800,000); Fiscal Year 1999: ¥20,000,000 (Direct Cost: ¥20,000,000)
• Keywords: 複製開始複合体 / 細胞周期 / 複製チェックポイント / DNAヘリカーゼ / 染色体 / リン酸化制御 / 複製開始点 / 複製開始前複合体 / リン酸化 / 複製開始点認識 / チェックポイント制御 / 細胞周期制御 / 複製開始点認識複合体 / チェックポイント

Research Abstract

1.複製開始モニター機構におけるMCMの機能制御に関して以下の成果を得た。
(1)細胞周期でのMCM4リン酸化について:マウスMcm4のアミノ末端領域の6箇所のCDKリン酸化は、MCM4/6/7ヘリカーゼ機能を抑制する。各リン酸化はクロマチン結合性、結合するクロマチン領域、細胞周期でのリン酸化レベルなど異なるため、特異的なゲノム複製抑制機能に関与する可能性か示唆された。
(2)DNA複製チェックポイント系でのMCM4リン酸化の促進:ヒトHeLa細胞や正常繊維芽細胞のMcm4は紫外線、ヒドロキシ尿素によって活性化されるATR/ATMに依存して、CDKを介してリン酸化される。よって、チェックポイント系でのゲノム複製抑制反応に重要な役割を担うことが示唆される。
2.MCM複合体と相互作用する因子の機能に関して以下の知見を得た。
(1)分裂酵母Mcm5変異株の解析から、複製前複合体から複製開始複合体への移行段階に、Cdc45が結合せずS1d3が結合する新たな中間体を見いだした。
(2)分裂酵母MCMとCdc45は、複製フォーク停止に伴う複製チェックポイント因子Cds1の活性化に必要であり、また停止した複製フォークの再構築にも関与することを見いだした。
(2)アフリカツメガエル卵無細胞複製系を用いて複製開始に必要な新規因子として4種類の蛋白質(S1d5-Psf1-Psf2-Psf3)からなる新規複合体GINS(go-ichi-ni-san)を同定した。
(3)複製開始とチェックポイントに働いている酵母Dpb11/Cut5のカエルホモログを同定した。これらカエルホモログは卵抽出液の系で複製開始に必要であり複製開始複合体の形成に働いている事を明らかにした。

Research Products

• [Publications] Takahashi, T.: "Multiple ORC-binding sites are required for efficient MCM loading and origin firing in fission yeast."EMBO J.. 22. 964-974 (2003)
• [Publications] Ono, Y.: "A novel allele of fission yeast rad11 that causes defects in DNA repair and telomere length regulation."Nucl.Acids Res.. 31. 7141-7149 (2003)
• [Publications] Masuda, T.: "CDK-and Cdc45-dependent priming of the MCM complex on chromatin during S-phase in Xenopus egg extracts."Genes Cells. 8. 145-161 (2003)
• [Publications] Kubota, Y.: "A novel ring-like complex of Xenopus proteins essential for the initiation of DNA replication."Genes Dev.. 17. 1141-1152 (2003)
• [Publications] Hashimoto, Y.: "Xenopus Cut5 is essential for a CDK-dependent process in the initiation of DMA replication."EMBO J.. 22. 2526-2535 (2003)
• [Publications] Kubota, Y.: "Block to DNA replication in meiotic maturation : a unified view for a robust arrest of cell cycle in oocytes and somatic cells"BioEssays. 25. 313-316 (2003)
• [Publications] You, Z.: "Roles of Mcm7 and Mcm4 subunits in the DNA helicase activity of the mouse Mcm4/6/7 complex."J.Biol.Chem.. 277. 42471-42479 (2003)
• [Publications] Yabuta, N.: "Mamalian Mcm2/4/6/7 complex forms a toroidal structure"Genes Cells. 8. 413-421 (2003)
• [Publications] Ishimi, Y.: "Enhanced expression of Mcm proteins in cancer cells derived from uterine cervix."Eur.J.Biochem.. 270. 1089-1101 (2003)
• [Publications] Ishimi, Y.: "Identification of MCM4 as a target of the DNA replication block checkpoint system."J.Biol.Chem.. 278. 24644-24650 (2003)
• [Publications] You, Z.: "Thymidine-rich single-stranded DNA sequences specifically activate mouse Mcm4/6/7 helicase on Y-fork and bubble-like substrates."EMBO J.. 22. 6148-6160 (2003)
• [Publications] Ishimi, Y.: "Levels of MCM4 phosphorylation and DNA synthesis in DNA replication block checkpoint control."J.Struc.Biol.. (in press). (2004)
• [Publications] 升方 久男(柳田充弘他編): "生命科学"東京化学同人. 228 (2004)
• [Publications] Nakajima, R.: "SpSld3 is required for loading and maintenance of SpCdc45 on chromatin in DNA replication in fission yeast"Mol. Biol. Cell.. 13. 1462-1472 (2002)
• [Publications] You, Z.: "Roles of Mcm7 and Mcm4 subunits in the DNA helicase activity of the mouse Mcm4/6/7 complex"J. Biol. Chem.. 277. 42471-42479 (2002)
• [Publications] Fujita, M.: "Nuclear organization of DNA replication initiation proteins in mammalian cells"J. Biol. Chem.. 277. 10354-10361 (2002)
• [Publications] Takahashi, T.: "Multiple ORC-binding sites are required for efficient MCM loading and origin firing in fission yeast"EMBO J.. 22. 964-974 (2003)
• [Publications] Masuda, T.: "CDK-and Cdc45-dependent priming of the MCM complex on chiomatin during S-phase in Xenopus egg extracts"Genes Cells. 8. 145-161 (2003)
• [Publications] Kubota, Y.: "A novel ring-like complex of Xenopus proteins essential for the initiation of DNA replication"Genes Dev.. (in press). (2003)
• [Publications] Ishimi, Y.: "Enhanced expression of Mcm proteins in cancer cells derived from uterine cervix"Eur. J. Biochem.. (in press). (2003)
• [Publications] Ishimi, Y.: "Regulation of eukaryotic DNA replication and the role of Mcm helicase"Recent Res. Devel. Mcl. Cell. Biol.. 3. 47-57 (2002)
• [Publications] Kubota, Y.: "Block to DNA replication in meiotic maturation"Bio Essays.. (in press). (2003)
• [Publications] Yabuta, N.: "Mamalian Mcm2/4/6/7 complex forms a toroidal structure"Genes Cells.. (in press). (2003)
• [Publications] Masukata, H.: "Condensations and commentaries on "S-Cdk-dependent phosphorylation of Sld2 essential for chromosomal DNA replication in budding yeast""Chemtracts, Biochemistry and Molecular Biology. 15. 513-519 (2002)
• [Publications] Ishimi, Y.: "Condensations and commentaries on "Cyclin-dependent kinases prevent DNA replication through multiple mechanisms""Chemtracts, Biochemistry and Molecular Biology. 15. 565-571 (2002)
• [Publications] 升方久夫: "「ゲノムの複製と分配」松影昭夫、正井久雄編"シュプリンガーフェアラーク東京. 40-49/234 (2002)
• [Publications] 石見幸男: "「ゲノムの複製と分配」松影昭夫、正井久雄編"シュプリンガーフェアラーク東京. 87-92/234 (2002)
• [Publications] Y.Okuno: "Clustered adenine/thymine stretches are essential for the function of a fission yeast replication origin"Mol.Cell.Biol.. 19・10. 6699-6709 (1999)
• [Publications] Y.Ogawa: "Association of fission yeast. Orp1 and Mcm6 proteins with chromosomal replication origins"Mol.Cell.Biol.. 19・10. 7228-7236 (1999)
• [Publications] Z.You: "Biochemical analysis of the intrinsic Mcm4-Mcm6-Mcm7 DNA helicase activity"Mol.Cell.Biol.. 19・12. 8003-8015 (1999)
• [Publications] Ishimi, Y.: "Inhibition of Mcm4,6,7 helicase activity by phosphorylation with cyclinA/Cdk2"J.Biol.Chem.. 275. 16235-16241 (2000)
• [Publications] Masai, H.: "Human Cdc7-related kinase complex. In vitro phosphorylation of MCM by concerted actions of Cdks and Cdc7 and that of a critical threonine residue of Cdc7"J.Mol.Biol.. 275. 29042-29052 (2000)
• [Publications] Sato, M.: "Electron microscopic observation and single-stranded DNA binding activity of Mcm4,6,7 complex"J.Mol.Biol.. 300. 421-431 (2000)
• [Publications] Mimura, S.: "Central role for Cdc45 in establishing an initiation complex of DNA replication in Xenopus egg extracts"Genes Cells. 5. 439-452 (2000)
• [Publications] Takisawa, H.: "Eukaryotic DNA replication : from pre-replication complex to initiation complex"Curr.Opin.Cell Biol.. 12. 690-696 (2000)
• [Publications] Waga, S.: "DNA polymerasee is required for coordinated and efficient chromosomal DNA replication in Xenopus egg extracts"Proc.Natl.Acad.Sci.USA. 98. 4978-4983 (2001)
• [Publications] Gotoh, T.: "Inactivation of the checkpoint kinase Cds1 is dependent on cyclin B-Cdc2 kinase activation at the meiotic G2/M-phase transition in Xenopus oocytes"J.Cell Sci.. 114. 3397-3406 (2001)
• [Publications] Takahashi, T.: "Interaction of fission yeast ORC with essential adenine/thymine stretches in replication origins"Genes to Cells. 6. 837-849 (2001)
• [Publications] Ishimi, Y.: "Phosphorylation of Mcm4 at specific sites by cyclin-dependent kinase leads to loss of Mcm4,6,7helicase activity. J. Biol. Chem."J.Biol.Chem.. 276. 34428-34433 (2001)
• [Publications] Ishimi, Y.: "Biochemical activities associated with mouse Mcm2 protein"J.Biol.Chem.. 276. 42744-42752 (2001)
• [Publications] Tada, S.: "Molecular cloning of a cDNA encoding mouse DNA helicase B, which has homology to Escherichia coli RecD protein, and identification of a mutation in the DNA helicase B from tsFT848 temperature-sensitive DNA replication mutant cells"Nucleic Acids Res.. 29. 3835-3840 (2001)
• [Publications] Nakajima, R.: "SpSld3 is required for loading and maintenance of SpCdc45 on chromatin in DNA replication in fission yeast"Mol.Biol.Cell. (IN PRESS). (2002)
• [Publications] Okuno,Y.: "Clustered adenine/thymine stretches are essential for the function of a fission yeast replication origin."Mol.Cell.Biol.. 19. 6699-6709 (1999)
• [Publications] Ogawa,Y.: "Association of fission yeast ORC and Mcm proteins with chromosomal replication origins."Mol.Cell.Biol.. 19. 7228-7236 (1999)
• [Publications] You,Z.: "Biochemical analysis of the intrinsic Mcm4-Mcm6-Mcm7 DNA helicase activity."Mol.Cell.Biol.. 19. 8003-8015 (1999)
• [Publications] Ishimi,Y.: "Inhibition of-Mcm4,6,7 helicase activity by phosphorylation with cyclinA/Cdk2."J.Biol.Chem.. 275. 16235-16241 (2000)
• [Publications] Masai,H.: "Human Cdc7-related kinase complex. In vitro phosphorylation of MCM by concerted actions of Cdks and Cdc7 and that of a critical threonine residue of Cdc7."J.Biol.Chem.. 275. 29042-29052 (2000)
• [Publications] Sato,M.: "Electron microscopic observation and single-stranded DNA binding activity of Mcm4,6,7 complex."J.Mol.Biol.. 300. 421-431 (2000)
• [Publications] Mimura,S.: "Central role for Cdc45 in establishing an initiation complex of DNAreplication in Xenopus egg extracts."Genes Cells. 5. 439-452 (2000)
• [Publications] Takisawa,H.: "Eukaryotic DNA replication : from pre-replication complex to initiation complex."Curr.Opin.Cell Biol.. 12. 690-696 (2000)
• [Publications] Waga,S.: "DNA polymerase ε is required for co-ordinated and efficient chromosomal DNA replication in Xenopus egg extracts."Proc.Natl.Acad.Sci.USA. (in press). (2001)
• [Publications] Y. Okuno: "Clustered adenine/thymine stretches are essential for the function of a fission yeast replication origin"Mol. Cell. Biol.. 19・10. 6699-6709 (1999)
• [Publications] Y. Ogawa: "Association of fission yeast Orp1 and Mcm6 proteins with chromosomal replication origins"Mol. Cell. Biol.. 19・10. 7228-7236 (1999)
• [Publications] Z. You: "Biochemical analysis of the intrinsic Mcm4-mcm6-Mcm7 DNA helicase activity"Mol. Cell. Biol.. 19・12. 8003-8015 (1999)
• [Publications] S. Mimura: "Central role for Cdc45 in establishing an initiation complex of DNA replication in Xenopus egg extracts"Genes Cells (in press).
• [Publications] 高橋達郎: "DNA複製開始に機能する蛋白質合体"蛋白質核酸酵素. 44. 1804-1812 (1999)
• [Publications] 三村覚: "複製のライセンス化と、S期の進行を制御するCDK"生化学. 71. 255-268 (1999)