Molecular Mechanism of Chromosomal DNA Replication Apparatus

染色体DNA复制仪的分子机制

• 批准号: 08277102
• 负责人: SUGINO Akio
• 金额: $ 158.08万
• 依托单位: Research Institute for Microbial Diseases, Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research on Priority Areas
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-08277102/
• 关键词: Eukaryotic chromosomal DNA replication; Leading strand synthesis apparatus; Lagging strand synthesis apparatus; DNA helicase; Mcm2-7 complex; Cdc7; Dbf4 protein kinase; Checkpoint control; Bloom症候群原因遺伝子産物; ヘリカーゼ; Mcm蛋白複合体; Dbf4複合体; 真核生物染色体DNA複

In order to understand the molecular mechanism of eukaryotic chromosomal DNA replication, this project has been focussing on elucidating the molecular mechanism of formation of the replication apparatus in various eukaryotic systems including yeast, Xenopus eggs, Drosophila, mouse and human cells. In particular, we extensively studied how both the lagging strand and leading strand synthesis complexes are formed. In the course of these studies, we identified several new replication proteins interacting with DNA polymerase epsilon which might be a leading strand polymerase. Furthermore, we purified yeast Cdc7/Dbf4 protein kinase and human Mcm2-7 protein complex both of which participates in the initiation and elongation step of chromosomal DNA replication. From these studies, we found that Cdc7/Dbf4 protein kinase activity is regulated by Rad53 protein kinase that participates in both S- and DNA damage checkpoint and the subcomplex of human Mcm2-7 proteins has a DNA helicase activity, suggesting that the complex is a DNA helicase which functions at the replication fork.

为了了解真核染色体DNA复制的分子机制，该项目一直致力于阐明酵母、非洲爪蟾卵、果蝇、小鼠和人类细胞等各种真核系统中复制装置形成的分子机制。特别是，我们广泛研究了滞后链和前导链合成复合物是如何形成的。在这些研究过程中，我们鉴定了几种与 DNA 聚合酶 epsilon 相互作用的新复制蛋白，DNA 聚合酶 epsilon 可能是一条前导链聚合酶。此外，我们纯化了酵母Cdc7/Dbf4蛋白激酶和人Mcm2-7蛋白复合物，它们都参与染色体DNA复制的起始和延伸步骤。从这些研究中，我们发现Cdc7/Dbf4蛋白激酶活性受到参与S-和DNA损伤检查点的Rad53蛋白激酶的调节，并且人Mcm2-7蛋白的亚复合物具有DNA解旋酶活性，表明该复合物是在复制叉上起作用的DNA解旋酶。

项目成果

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Shiratori,M., S.Sakamoto, et al.: "Detection by epitope-defined monoclonal antibodies of Werner DNA helicases in the nucleoplasm and their upregulation by cell transformation and immortalization"J Cell Biol. 144. 1-9 (1999)

Shiratori，M.，S.Sakamoto 等人：“通过表位定义的单克隆抗体检测核质中的 Werner DNA 解旋酶及其通过细胞转化和永生化的上调”J Cell Biol。

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Mizuno,T., N.Ito 等人：“小鼠 DNA 聚合酶 α-引物酶复合物的第二大亚基促进 DNA 聚合酶 α 催化亚基的产生和核转位。”Mol Cell Biol。

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Nakayabu，M.，S.Miwa 等人：“错配的核苷酸可能促进遗传疾病中三核苷酸重复的扩展。”《核酸研究》。

Kawabe, Y., M.Seki, T.Seki, W.S.Wang, O.Imamura, Y.Furuichi, H.Saitoh and T.Enomoto: "Covalent modification of the Werner's syndrome gene product with the ubiquitinrelated protein, SUMO-1."J Biol Chem. 275. 20963-20966 (2000)

Kawabe, Y.、M.Seki、T.Seki、W.S.Wang、O.Imamura、Y.Furuichi、H.Saitoh 和 T.Enomoto：“用泛素相关蛋白 SUMO-1 对维尔纳综合征基因产物进行共价修饰。