1999 Fiscal Year Annual Research Report

真核生物染色体DNA複製装置とS-期チェックポイント制御

Research Project

Project/Area Number	11694277
Research Category	Grant-in-Aid for Scientific Research (A)
Research Institution	Osaka University
Principal Investigator	杉野明雄大阪大学, 微生物病研究所, 教授 (90231737)
Keywords	染色体DNA複製開始機構 / DNAポリメラーゼ / Cdc7p / Dbf4p複合体 / 蛋白リン酸化酵素 / Mcm2-7蛋白群 / Rad53蛋白 / Mcm10蛋白 / Sー期チェックポイント
Research Abstract	a)Mcm2-7蛋白、Mcm10蛋白の精製と染色体DNA複製における役割:現在までに、分子遺伝学的、生化学的研究より、Mcm2-7蛋白は細胞周期G_2/M期において細胞質から核に移行し、染色体DNA複製をS期に1回だけ保障する"Licensing Factor"として機能していると考えられている。これらの蛋白群の染色体DNA複製開始または鎖伸長反応にどうかかわっているかを解明するため、これらの複合体と相互作用している複製蛋白因子の解明を行った。その結果、Mcm10蛋白とMcm2、Mcm5、Dna2、Slm2、やPolε subunitであるDpb3 Dpb4 Dpb11やPolδ のsubunitであるPol32、Hys2と遺伝的な相互作用があることが明らかになった。又、Mcm2蛋白が複製開始に必須なCdc7p/Dbf4pリン酸化酵素の基質になっていることを明らかにした。しかしMcm2蛋白がCdc7p/Dbf4p複合体の基質になるためには、あらかじめ、別のキナーゼのよってリン酸化されていなければならないことを明らかにした。 b)Rad53蛋白による複製開始の制御機構: 染色体の上に多数ある複製開始部位はS-期に一斉に活性化されるのではない。この複製開始部位の活性化の制御に、S期やDNA損傷チェックポイント制御に関与するRad53 protein kinaseが関与していることが明らかになりつつある。このRad53 蛋白の複製開始反応における機能を解析する目的で、まずRad53 蛋白をHydroxyurea 未処理と処理した出芽酵母細胞から大量に精製した。こうして精製したRad53 蛋白を用い試験管内で、以前に精製した、Cdc7p/Dbf4p キナーゼ複合体をリン酸化し、Cdc7p/Dbf4p 複合体が有する、Mcm2リン酸化酵素活性を阻害することを明らかにした。これらの結果を基に、Rad53 蛋白による複製開始部位の活性化制御に関する新しいモデルを提唱した。

Research Products

(17 results)

All Other

All Publications (17 results)

[Publications] Kamimura,Y.: "DNA helicase III of Saccharomyces cerevisiae, encoded by YER176w(HEL1), highly unwinds covalently closed, circular DNA in the presence of a DNA topoisomerase and yRF-A"Journal of Biochemistry. 125. 236-244 (1999)
[Publications] Johnston,L.H.: "First the CDKs, now the DDKs"Tends in Cell Biology. 9. 249-252 (1999)
[Publications] Masumoto,H.: "Dpb11 controls the association between DNA polymerase α and ε, and the ARS region of budding yeast"Molecular and Cellular Biology. in press. (2000)
[Publications] Kihara,M.: "The Cdc7p/Dbf4p ARS-binding and portein kinase activities are both required for entry to S phase and are regulated by Rad53p in Saccharomyces cerevisiae"Molecular Cell. in press. (2000)
[Publications] Gary R.: "A novel role in DNA metabolism for the binding of Fenl/Red27 to PCNA and implications for genetic risk"Mol Cell Biol. 19. 5373-5382 (1999)
[Publications] Tran H.T.: "Genetic factors affecting the impact of DNA polymerase delta proofreading activity on mutation avoidance in yeast"Genetics. 152. 47-59 (1999)
[Publications] Tran H.T.: "The 3'-->5' exonucleases of DNA polymerases delta and epsilon and the 5'-->3' exonuclease Exo1 have major roles in postreplication mutation avoidance in Saccharomyces cerevisiae"Mol Cell Biol.. 19. 2000-2007 (1999)
[Publications] Kroutil L.C.: "Deletion errors generated during replication of CAG repeats"Nucleic Acids Res.. 27. 3481-3486 (1999)
[Publications] Labib K.: "G1-phase and B-type cyclins exclude the DNA-replication factor Mcm4 from the nucleus"Nat. Cell Biol.. 1. 415-422 (1999)
[Publications] Santocanale C.: "Activation of dormant origins of DNA replication in budding yeast"Genes Dev.. 13. 2360-2364 (1999)
[Publications] Noton E.: "CDK inactivation is the only essential function of the APC/C and the mitotic exit network proteins for origin resetting during mitosis"Mol.Cell.. 5. 85-95 (2000)
[Publications] Ferreira M.F.: "Dbf4p, an essential S phase-promoting factor, is targeted for degradation by the anaphase-promoting complex"Mol Cell Biol.. 20. 242-248 (2000)
[Publications] Neecke H.: "Cell cycle progression in the presence of irreparable DNA damage is controlled by a Mec1-and Rad53-dependent checkpoint in budding yeast"EMBO J.. 18. 4485-4497 (1999)
[Publications] Weinreich M.: "Cdc7p-Dbf4p kinase binds to chromatin during S phase and is regulated by both the APC and the RAD53 checkpoint pathway"EMBO J.. 18. 5334-5346 (1999)
[Publications] Weinreich M.: "The Cdc6p nucleotide-binding motif is required for loading mcm proteins onto chromatin"Proc.Natl.Acad.Sci.USA. 96. 441-446 (1999)
[Publications] Chong J.P.: "A double-hexamer archaeal minichromosome maintenance protein is an ATP-dependent DNA helicase"Proc.Natl.Acad.Sci.USA. 97. 1530-1535 (2000)
[Publications] Johnston,L.H.: "Progress in Cell Cycle Reseach (Meijier,L.,Jezequel,A.,and Ducommun,B.,eds.) Plenum Press,New York,USA Vol.4"A Cdc7-Dbf4 kinase activity is conserved from yeast to human. 8 (199)

1999 Fiscal Year Annual Research Report

真核生物染色体DNA複製装置とS-期チェックポイント制御

Principal Investigator

杉野 明雄 大阪大学, 微生物病研究所, 教授 (90231737)

Research Products

[Publications] Kamimura,Y.: "DNA helicase III of Saccharomyces cerevisiae, encoded by YER176w(HEL1), highly unwinds covalently closed, circular DNA in the presence of a DNA topoisomerase and yRF-A"Journal of Biochemistry. 125. 236-244 (1999)

[Publications] Johnston,L.H.: "First the CDKs, now the DDKs"Tends in Cell Biology. 9. 249-252 (1999)

[Publications] Masumoto,H.: "Dpb11 controls the association between DNA polymerase α and ε, and the ARS region of budding yeast"Molecular and Cellular Biology. in press. (2000)

[Publications] Kihara,M.: "The Cdc7p/Dbf4p ARS-binding and portein kinase activities are both required for entry to S phase and are regulated by Rad53p in Saccharomyces cerevisiae"Molecular Cell. in press. (2000)

[Publications] Gary R.: "A novel role in DNA metabolism for the binding of Fenl/Red27 to PCNA and implications for genetic risk"Mol Cell Biol. 19. 5373-5382 (1999)

[Publications] Tran H.T.: "Genetic factors affecting the impact of DNA polymerase delta proofreading activity on mutation avoidance in yeast"Genetics. 152. 47-59 (1999)

[Publications] Tran H.T.: "The 3'-->5' exonucleases of DNA polymerases delta and epsilon and the 5'-->3' exonuclease Exo1 have major roles in postreplication mutation avoidance in Saccharomyces cerevisiae"Mol Cell Biol.. 19. 2000-2007 (1999)

[Publications] Kroutil L.C.: "Deletion errors generated during replication of CAG repeats"Nucleic Acids Res.. 27. 3481-3486 (1999)

[Publications] Labib K.: "G1-phase and B-type cyclins exclude the DNA-replication factor Mcm4 from the nucleus"Nat. Cell Biol.. 1. 415-422 (1999)

[Publications] Santocanale C.: "Activation of dormant origins of DNA replication in budding yeast"Genes Dev.. 13. 2360-2364 (1999)

[Publications] Noton E.: "CDK inactivation is the only essential function of the APC/C and the mitotic exit network proteins for origin resetting during mitosis"Mol.Cell.. 5. 85-95 (2000)

[Publications] Ferreira M.F.: "Dbf4p, an essential S phase-promoting factor, is targeted for degradation by the anaphase-promoting complex"Mol Cell Biol.. 20. 242-248 (2000)

[Publications] Neecke H.: "Cell cycle progression in the presence of irreparable DNA damage is controlled by a Mec1-and Rad53-dependent checkpoint in budding yeast"EMBO J.. 18. 4485-4497 (1999)

[Publications] Weinreich M.: "Cdc7p-Dbf4p kinase binds to chromatin during S phase and is regulated by both the APC and the RAD53 checkpoint pathway"EMBO J.. 18. 5334-5346 (1999)

[Publications] Weinreich M.: "The Cdc6p nucleotide-binding motif is required for loading mcm proteins onto chromatin"Proc.Natl.Acad.Sci.USA. 96. 441-446 (1999)

[Publications] Chong J.P.: "A double-hexamer archaeal minichromosome maintenance protein is an ATP-dependent DNA helicase"Proc.Natl.Acad.Sci.USA. 97. 1530-1535 (2000)

[Publications] Johnston,L.H.: "Progress in Cell Cycle Reseach (Meijier,L.,Jezequel,A.,and Ducommun,B.,eds.) Plenum Press,New York,USA Vol.4"A Cdc7-Dbf4 kinase activity is conserved from yeast to human. 8 (199)

杉野明雄大阪大学, 微生物病研究所, 教授 (90231737)