1996 Fiscal Year Annual Research Report

細胞周期制御とDNA複製

Research Project

Project/Area Number	08265102
Research Category	Grant-in-Aid for Scientific Research on Priority Areas
Research Institution	Nara Institute of Science and Technology
Principal Investigator	釣本敏樹奈良先端科学技術大学院大学, バイオサイエンス研究科, 助教授 (30163885)
Co-Investigator(Kenkyū-buntansha)	藤田雅俊愛知県がんセンター研究所, 研究員 (30270713) 秋山昌広奈良先端科学技術大学院大学, バイオサイエンス研究科, 助教授 (80273837) 村上洋太京都大学, ウイルス研究所, 助教授 (20260622) 正井久雄東京大学, 医科学研究所, 助教授 (40229349) 加藤順也奈良先端科学技術大学院大学, バイオサイエンス研究科, 助教授 (00273839)
Keywords	蛋白質リン酸化 / E2F-1 / MCM / ORC / PCNA / 複製開始点結合因子 / S期導入因子 / ライセンシング因子
Research Abstract	細胞をS期に導入する機構、染色体を複製開始に導く因子群の機能、複製装置と細胞周期制御機構のつながりという3つの点に目標を絞り、複製反応の進行が細胞周期の進行とどのように協調しているかを明らかにし、これに基づいて細胞のがん化、がん抑制機構を説明することをめざしている。この目的に基づき、G1期の細胞がS期に導かれ、複製を行う諸過程の解析を行い以下の結果を得た。 1.細胞をS期に導入する過程で転写因子E2F-1によりサイクリンEの発現が誘導され、さらにE2F-1とサイクリンEが共同的に働いて細胞をS期に導くことを明らかにした。また核機能の制御に重要なRCC-RanサイクルがDNA複製に必要な核構造形成、およびS期進行過程に必要であることを明らかにした。 2.複製開始に直接係わる因子として、複製開始配列に結合するORC,ABP1およびライセンシング因子の候補であるMCM蛋白質の機能解析を行った。その結果、出芽酵母において、ORC-MCM間の相互作用があること、ヒトMCMがATP依存的にクロマチンへと相互作用すること、分裂酵母ABP1が複製開始領域へ結合し、そのDNA複製活性へ関与することが示された。 3.複製装置の機能制御の過程では、複製開始の活性化に係わるCDC7類似キナーゼ遺伝子がカエル、マウス、ヒトから単離された。また酵母のCDC7がMCM蛋白質リン酸化へ関与していることが示された。また細胞周期の制御因子であるp21(Waf1)によるDNA伸長過程の機能制御を解析し、この因子の複製因子PCNAへの結合様式とそれによる複製蛋白質複合体の形成過程の阻害を明らかにした。

Research Products

(18 results)

All Other

All Publications (18 results)

[Publications] Kitagawa,M.: "The consensus motif for phosphorylation by cyclin D1-Cdk4 is different fromthat for phosphorylation by cyclin A/E-Cdk2." EMBOJ. (in press).
[Publications] Azuma,Y.: "Conserved Histidine Residues of RCCI Are Essential for Nucleotide Exchange on Ran." J.Biochem.120. 82-91 (1996)
[Publications] Noguchi,E.: "Dis3,implicated in mitotic control,directly binds to Ran and enhances the GEF activity of RCC1." EMBO Journal. (in press).
[Publications] Fujita,M.: "hCDC47,a human member of the MCM family:dissociation of the nucleus-bound form during S phase." J.Biol.Chem.271. 4349-4354 (1996)
[Publications] Fujita,M.: "Inhibition of S phase entry of human fibroblasts by an antisense oligomer against hCDC47." Biochem.Biophys.Res.Commun.219. 604-607 (1996)
[Publications] Ohotoshi,A.: "Genetic interactions between CDC7 and CDC28:growth inhibition of cdc28-1N by cdc7 point mutants." Genes to Cells. 1. 895-904 (1996)
[Publications] Murakami,Y.: "Identification,purification and molecular cloning of autonomously replicating sequence-binding protein from fission yeast Schizosaccharomyces pombe." Proc.Natl.Acad.Sci.USA. 93. 502-507 (1996)
[Publications] Ishiai,M.: "Purification,gene cloning and reconstitution of the heterotrimeric single-stranded DNA binding protein from Schizosaccharomyces pombe." J.Biol.Chem.271. 20863-20878 (1996)
[Publications] Hori,Y.: "Characterization of a novel CDC gene(ORC1)partly homologous to CDC6 of Saccharomyces cerevisiae." Mol.Biol.Cell.7. 409-418 (1996)
[Publications] Ohba,T.: "Premature Chromatin Condensation Induced by Loss of RCC1 Is Inhibited by GTP-and GTPgS-Ran,but Not GDP-Ran." J.Biol.Chem.271. 14665-14667 (1996)
[Publications] Kiyono,T.: "Cloning of a cDNA encoding a human homologue of CDC47,a member of the MCM family." Biochim.Biophys.Acta.1307. 31-34 (1996)
[Publications] Hiraiwa,A.: "Immunolocalization of hCDC47 protein in normal and neoplastic human tissues and its relation to growth" Int.J.Cancer. (in press).
[Publications] Fujita,M.: "In vivo interaction of human MCM heterohexameric complexes with chromation:possible involvement of ATP." J.Biol.Chem.(in press).
[Publications] Ikeda,M.: "CTBP-1/RBP-1 of Saccharomyces cerevisiae protein which binds to T-rich single-stranded DNA containing the 1 base-pair core sequence of autonomously replicating sequence,is a polydeoxypyrimidine binding protein." Eur.J.Biochem.238. 38-47 (1996)
[Publications] Masai,M.: "DnaA-and PriA-dependent primosomes:two distinctreplication complexes for replication of Escherichia coli chromosome." Frontiers in Bioscience. 1. 49-59 (1996)
[Publications] Ikeda,M.: "A rapid and convenient method for screening of DNA binding and protein-interacting molecules by the use of GST fusion protein library." Gene. 181. 167-171 (1996)
[Publications] Masai,H.: "Mecahnisms of primer RNA synthesis andD-loop/R-loop-dependent DNA replication of Escherichia coli." Biochimie. (in press).
[Publications] Ito,K.: "c-Jun stimulates origin-dependent DNA unwinding by polyomavirus large T antigen." EMBOJ.15. 5636-5646 (1996)

1996 Fiscal Year Annual Research Report

細胞周期制御とDNA複製

Principal Investigator

釣本 敏樹 奈良先端科学技術大学院大学, バイオサイエンス研究科, 助教授 (30163885)

Research Products

[Publications] Kitagawa,M.: "The consensus motif for phosphorylation by cyclin D1-Cdk4 is different fromthat for phosphorylation by cyclin A/E-Cdk2." EMBOJ. (in press).

[Publications] Azuma,Y.: "Conserved Histidine Residues of RCCI Are Essential for Nucleotide Exchange on Ran." J.Biochem.120. 82-91 (1996)

[Publications] Noguchi,E.: "Dis3,implicated in mitotic control,directly binds to Ran and enhances the GEF activity of RCC1." EMBO Journal. (in press).

[Publications] Fujita,M.: "hCDC47,a human member of the MCM family:dissociation of the nucleus-bound form during S phase." J.Biol.Chem.271. 4349-4354 (1996)

[Publications] Fujita,M.: "Inhibition of S phase entry of human fibroblasts by an antisense oligomer against hCDC47." Biochem.Biophys.Res.Commun.219. 604-607 (1996)

[Publications] Ohotoshi,A.: "Genetic interactions between CDC7 and CDC28:growth inhibition of cdc28-1N by cdc7 point mutants." Genes to Cells. 1. 895-904 (1996)

[Publications] Murakami,Y.: "Identification,purification and molecular cloning of autonomously replicating sequence-binding protein from fission yeast Schizosaccharomyces pombe." Proc.Natl.Acad.Sci.USA. 93. 502-507 (1996)

[Publications] Ishiai,M.: "Purification,gene cloning and reconstitution of the heterotrimeric single-stranded DNA binding protein from Schizosaccharomyces pombe." J.Biol.Chem.271. 20863-20878 (1996)

[Publications] Hori,Y.: "Characterization of a novel CDC gene(ORC1)partly homologous to CDC6 of Saccharomyces cerevisiae." Mol.Biol.Cell.7. 409-418 (1996)

[Publications] Ohba,T.: "Premature Chromatin Condensation Induced by Loss of RCC1 Is Inhibited by GTP-and GTPgS-Ran,but Not GDP-Ran." J.Biol.Chem.271. 14665-14667 (1996)

[Publications] Kiyono,T.: "Cloning of a cDNA encoding a human homologue of CDC47,a member of the MCM family." Biochim.Biophys.Acta.1307. 31-34 (1996)

[Publications] Hiraiwa,A.: "Immunolocalization of hCDC47 protein in normal and neoplastic human tissues and its relation to growth" Int.J.Cancer. (in press).

[Publications] Fujita,M.: "In vivo interaction of human MCM heterohexameric complexes with chromation:possible involvement of ATP." J.Biol.Chem.(in press).

[Publications] Ikeda,M.: "CTBP-1/RBP-1 of Saccharomyces cerevisiae protein which binds to T-rich single-stranded DNA containing the 1 base-pair core sequence of autonomously replicating sequence,is a polydeoxypyrimidine binding protein." Eur.J.Biochem.238. 38-47 (1996)

[Publications] Masai,M.: "DnaA-and PriA-dependent primosomes:two distinctreplication complexes for replication of Escherichia coli chromosome." Frontiers in Bioscience. 1. 49-59 (1996)

[Publications] Ikeda,M.: "A rapid and convenient method for screening of DNA binding and protein-interacting molecules by the use of GST fusion protein library." Gene. 181. 167-171 (1996)

[Publications] Masai,H.: "Mecahnisms of primer RNA synthesis andD-loop/R-loop-dependent DNA replication of Escherichia coli." Biochimie. (in press).

[Publications] Ito,K.: "c-Jun stimulates origin-dependent DNA unwinding by polyomavirus large T antigen." EMBOJ.15. 5636-5646 (1996)

釣本敏樹奈良先端科学技術大学院大学, バイオサイエンス研究科, 助教授 (30163885)