1999 年度実績報告書

染色体DNA鎖合成装置の分子機構

研究課題

研究課題/領域番号	08277102
研究機関	大阪大学
研究代表者	杉野明雄大阪大学, 微生物病研究所, 教授 (90231737)
研究分担者	釣本敏樹奈良先端科学技術大学院大学, 助教授 (30163885) 榎本武美東北大学, 薬学部, 教授 (80107383) 吉田松年名古屋大学, 医学部, 教授 (70090420) 大森治夫京都大学, ウイルス研究所, 助教授 (10127061) 石見幸男三菱化学生命科学研究所, 主任研究員
キーワード	真核生物染色体複製機構 / Bloom症候群原因遺伝子産物 / Leading鎖複製装置 / Lagging鎖複製装置 / ヘリカーゼ / Mcm蛋白複合体 / Cdc7 / Dbf4複合体 / チェックポイント制御
研究概要	複製開始に必要なマウスとヒトMcm4,6,7複合体に、DNAヘリカーゼ活性が存在することが明らし、変異Mcm4,6,7複合体の解析から、その活性がMcmに内在し、また、4,6,7のそれぞれのMcmがヘリカーゼ活性に対し異なる生化学的役割を果たすことが明らかになった。又、複製開始反応に重要な働きをしている出芽酵母Cdc7/Dbf4複合体を精製し、Mcm2蛋白が特異的にリン酸化されること、又、このCdc7p/Dbf4P複合体がDNA修復チェックやS/Mチェックポイント制御に関与しているRad53pによってリン酸化され、cdc7p/Dbf4P複合体が持っているリン酸化酵素活性が阻害されることを明らかにした。一方、Leading鎖及びLagging鎖複製装置の複製開始部位への呼込みにCdc45p蛋白やDpb11-Sld2蛋白複合体が重要な働きをしていることを明らかにした。マウスのDNAポーリメラーゼα/プライマーゼ複合体を使って、遺伝性神経変性疾患の原因と考えられているトリヌクレオチド反復配列の異常増幅を再現した。Lagging鎖複製装置の中心であるヒトDNAポリメラーゼδは出芽酵母と同じく3つのサブユニットからなることを明らかにし、PCNAが第三番目のサブユニットと結合することを明らかにした。出芽酵母で、Lagging鎖複製装置の一部であるRF-C複合体とRad24蛋白が複合体を形成することによって、DNA修復装置によるシグナルを染色体分裂装置に伝える役目をしていることを明らかにした。DNA複製への関与が示唆されているBloom症侯群原因遺伝子産物(Blm)の機能の解析を行い、Blmの機能が欠損するとDNA複製中にDNAの二本鎖切断が生じること、この二本鎖切断はDNAの組換えによって修復されることを明らかにした。最後に、大腸菌の突然変異誘発蛋白であるDinBのヒトにおけるホモログであるDINB1蛋白を過剰生産させて部分精製し、それが確かにnon-processiveなDNA polymerase活性を持ち、様々なDNA損傷部位を乗り越える際にerror-prone bypassをすることを証明した。

研究成果
(25件)

すべてその他

すべて文献書誌 (25件)

[文献書誌] Kamimura, Y.: "DNA helicase III of Saccharomyces cerevisiae, encoded by YER176w (HEL1), highlyun winds covalently closed, circular DNA in the presence of a DNA topoisomerase and yRF-A"Journal of Biochemistry. 125. 236-244 (1999)
[文献書誌] Johnston, L. H.: "First the CDKs, now the DDKs"Trends in Cell Biology. 9. 249-252 (1999)
[文献書誌] 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)
[文献書誌] Kihara, M.: "The Cdc7p/Dbf4p ARS-binding and protein kinase activities are both required for entry to S phase and are regulated by Rad53p in Saccharomyces cerevisiae"Molecular Cell. (in press). (2000)
[文献書誌] Ogi, T.: "Mutation enhancement by DINB1, a mammalian homologue of the Escherichia coli mutagenesis protein DinB"Genec to Cells. 4. 607-618 (1999)
[文献書誌] Femabdedez de Henestrosa, A. R.: "Identification of additional genes belonging to the LexA regulon in Escherichia coli"Molecular Microbiology. 120 (in press). (2000)
[文献書誌] Kondo, T.: "Role of a complex containing Rad17, Mec3, and Ddc1 in the yeast DNA damage checkpoint pathway"Molecular and Cellular Biology. 19. 1136-1143 (1999)
[文献書誌] Mizuno, T.: "Molecular architecture of the mouse DNA polymerase alpha-primase complex"Molecular and Cellular Biology. 19. 7886-7896 (1999)
[文献書誌] Onoda, F.: "Elevation of sister chromatid exchange in Saccharomyces cerevisiae sgs1 disruptants and the relevance of the disruptant as system to evaluate mutations in Bloom's syndrome gene"Mutation Research. (in press). (2000)
[文献書誌] Siratori, M.: "Detection by epitope-defined monoclonal antibodies of Werner DNA helicases in nucleoplasm and their upregulation by cell transformation and immortalization"Journal of Cell Biology. 144. 1-9 (1999)
[文献書誌] Hayakawa, N.: "Isothiazolone derivatives selectively inhibit telomerase form human and rat cancer cells in vitro"Biochemistry. 38. 11501-11507 (1999)
[文献書誌] Nozawa, K.: "Up-regulation of telomerase in primary cultured rat hepatocytes"Journal of Biochemistry. 126. 361-367 (1999)
[文献書誌] Kurumiya, Y.: "Active form of human hepatocyte growth factor is excreted in to bile after hepatobiliary resection."Journal of Hepatology. 30. 22-28 (1999)
[文献書誌] Takemura, M.: "Nuclear protein B23.1 binds to retinoblastoma protein and synergistically stimulates DNA polymerase a activity"Journal of Biochemistry. 125. 904-909 (1999)
[文献書誌] Tsugane, K.: "A possible role of nuclear ceramide and sphinogosine in hepatocyte apoptosis in rat liver"Journal of Hepatology. 31. 8-17 (1999)
[文献書誌] Ohta, K.: "Action of new mammalian DNA polymerase inhibotors, sulfoquinovosy-ldiacylaglycerol"Biological and Pharmacological. Bull.. 22. 111-116 (1999)
[文献書誌] Hayata, A.: "Hepatocyte growth factor concentration in rat bile is affected by hepatic resection volume and external bilially drainage"Journal of Surgical Research. 85. 71-76 (1999)
[文献書誌] Kurumiya, Y.: "Differential suppression of liver-specific genes in regenerating rat liver induced by extended hepatectomy"Journal of Hepaatology. (in press). (2000)
[文献書誌] You, Z.: "Biochemical analysis of the intrinsic Mcm4-Mcm6-Mcm7 DNA helicase activity"Molecular and Cellular Biology. 19. 8003-8015 (1999)
[文献書誌] Saito S.: "Functional domains of Template Activating Factor-I as a protein phosphatase 2A inhibitor"Biochem. and Biophys. Res. Commun.. 259. 471-475 (1999)
[文献書誌] Matsumoto K.: "Sperm chromatin decondensation by Template Activating Factor-I through direct interaction with basic proteins"Molecular and Cell. ular Biology. 19. 6940-6952 (1999)
[文献書誌] Miyaji-Yamaguchi M.: "Coiled-coil structure-mediated dimerization of Template Activating Factor-I is critical for its chromatin remodeling activity"Journal of Molecular Biology. 290. 547-557 (1999)
[文献書誌] Matsumoto K.: "Histone- and chromatin-binding activity of Template Activating Factor-I"FEBS Letters. 463. 285-288 (1999)
[文献書誌] Nagata K.: "Acidic molecular chaperones : their involvement in viral genome replication and transcription"Recent Researches in Developmental Virology. 1. 559-597 (1999)
[文献書誌] Johnston, L. H.: "Progress in Cell Cycle Research, Vol. 4 (Meijer, L., Jezequel, A., and Ducommun, B., eds.) Pleum Press, New York"A Cdc7-Dbf4 kinase activity is conserved from yeast to human. 8 (1999)

1999 年度 実績報告書

染色体DNA鎖合成装置の分子機構

研究代表者

杉野 明雄 大阪大学, 微生物病研究所, 教授 (90231737)

研究成果

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

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

[文献書誌] 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)

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

[文献書誌] Ogi, T.: "Mutation enhancement by DINB1, a mammalian homologue of the Escherichia coli mutagenesis protein DinB"Genec to Cells. 4. 607-618 (1999)

[文献書誌] Femabdedez de Henestrosa, A. R.: "Identification of additional genes belonging to the LexA regulon in Escherichia coli"Molecular Microbiology. 120 (in press). (2000)

[文献書誌] Kondo, T.: "Role of a complex containing Rad17, Mec3, and Ddc1 in the yeast DNA damage checkpoint pathway"Molecular and Cellular Biology. 19. 1136-1143 (1999)

[文献書誌] Mizuno, T.: "Molecular architecture of the mouse DNA polymerase alpha-primase complex"Molecular and Cellular Biology. 19. 7886-7896 (1999)

[文献書誌] Onoda, F.: "Elevation of sister chromatid exchange in Saccharomyces cerevisiae sgs1 disruptants and the relevance of the disruptant as system to evaluate mutations in Bloom's syndrome gene"Mutation Research. (in press). (2000)

[文献書誌] Siratori, M.: "Detection by epitope-defined monoclonal antibodies of Werner DNA helicases in nucleoplasm and their upregulation by cell transformation and immortalization"Journal of Cell Biology. 144. 1-9 (1999)

[文献書誌] Hayakawa, N.: "Isothiazolone derivatives selectively inhibit telomerase form human and rat cancer cells in vitro"Biochemistry. 38. 11501-11507 (1999)

[文献書誌] Nozawa, K.: "Up-regulation of telomerase in primary cultured rat hepatocytes"Journal of Biochemistry. 126. 361-367 (1999)

[文献書誌] Kurumiya, Y.: "Active form of human hepatocyte growth factor is excreted in to bile after hepatobiliary resection."Journal of Hepatology. 30. 22-28 (1999)

[文献書誌] Takemura, M.: "Nuclear protein B23.1 binds to retinoblastoma protein and synergistically stimulates DNA polymerase a activity"Journal of Biochemistry. 125. 904-909 (1999)

[文献書誌] Tsugane, K.: "A possible role of nuclear ceramide and sphinogosine in hepatocyte apoptosis in rat liver"Journal of Hepatology. 31. 8-17 (1999)

[文献書誌] Ohta, K.: "Action of new mammalian DNA polymerase inhibotors, sulfoquinovosy-ldiacylaglycerol"Biological and Pharmacological. Bull.. 22. 111-116 (1999)

[文献書誌] Hayata, A.: "Hepatocyte growth factor concentration in rat bile is affected by hepatic resection volume and external bilially drainage"Journal of Surgical Research. 85. 71-76 (1999)

[文献書誌] Kurumiya, Y.: "Differential suppression of liver-specific genes in regenerating rat liver induced by extended hepatectomy"Journal of Hepaatology. (in press). (2000)

[文献書誌] You, Z.: "Biochemical analysis of the intrinsic Mcm4-Mcm6-Mcm7 DNA helicase activity"Molecular and Cellular Biology. 19. 8003-8015 (1999)

[文献書誌] Saito S.: "Functional domains of Template Activating Factor-I as a protein phosphatase 2A inhibitor"Biochem. and Biophys. Res. Commun.. 259. 471-475 (1999)

[文献書誌] Matsumoto K.: "Sperm chromatin decondensation by Template Activating Factor-I through direct interaction with basic proteins"Molecular and Cell. ular Biology. 19. 6940-6952 (1999)

[文献書誌] Miyaji-Yamaguchi M.: "Coiled-coil structure-mediated dimerization of Template Activating Factor-I is critical for its chromatin remodeling activity"Journal of Molecular Biology. 290. 547-557 (1999)

[文献書誌] Matsumoto K.: "Histone- and chromatin-binding activity of Template Activating Factor-I"FEBS Letters. 463. 285-288 (1999)

[文献書誌] Nagata K.: "Acidic molecular chaperones : their involvement in viral genome replication and transcription"Recent Researches in Developmental Virology. 1. 559-597 (1999)

[文献書誌] Johnston, L. H.: "Progress in Cell Cycle Research, Vol. 4 (Meijer, L., Jezequel, A., and Ducommun, B., eds.) Pleum Press, New York"A Cdc7-Dbf4 kinase activity is conserved from yeast to human. 8 (1999)

1999 年度実績報告書

杉野明雄大阪大学, 微生物病研究所, 教授 (90231737)