DNA Replication Apparatus and S-Phase Checkpoint Control

DNA 复制装置和 S 期检查点控制

• 批准号: 11694277
• 负责人: SUGINO Akio
• 金额: $ 12.23万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11694277/
• 关键词: Chromosomal DNA replication; Mcm2-7 protein complex; Mcm10 protein; S-phase checkpoint; Rad53p; Cdc7; Dbf4 protein kinase complex; Protein kinase; Xenopus egg in vitro replication system; MCM10蛋白; Cdc7p; Dbf4p複合体; in vitro 複製系; Xenopus egg

Saccharomyces cerevisiae Cdc7/Dbf4 protein kinase, which regulates the initiation of chromosomal DNA replication, was expressed and purified from insect cells. It was found that the purified Cdc:7/Dbf4 phosphorylates Mcm2, which is one of Mcm2-7 complex. Furthermore, its phosphorylation sites were determined. Xenopus homologs of S. cerevisiae Cdc7/Dbf4 protein Kinase and Mcm10 were identified and their corresponding genes were cloned. it was found that Xenopus Cdc7/Dbf4.protein kinase and Mcm10 are required for the initiation of chromosomal DNA replication as in S. cerevisiae.S. cerevisiae Rad53p, which involved in both DNA-damage-and S-phase checkpoint regulation, was purified and one of the substrates was identified to be Dbf4/Cdc7 protein kinase. It was found that Rad53p directly intercts with Cdc7/Dbf4 and phosphorylates Dbf4. Phosphorylation of Cdc7/Dbf4 complex with Rad53p inactivates Cdc7/Dbf4 protein kinase activity.

从昆虫细胞中表达并纯化了调控染色体DNA复制起始的酿酒酵母Cdc7/Dbf4蛋白激酶。纯化后的Cdc:7/Dbf4磷酸化Mcm2，是Mcm2-7复合体之一。进一步测定了其磷酸化位点。鉴定了酿酒葡萄球菌Cdc7/Dbf4蛋白激酶和Mcm10的爪蟾同源物，并克隆了相应的基因。发现非洲爪蟾Cdc7/Dbf4。在酿酒葡萄球菌中，蛋白激酶和Mcm10是启动染色体DNA复制所必需的。对参与dna损伤和s期检查点调控的cerevisiae Rad53p进行了纯化，其中一个底物被鉴定为Dbf4/Cdc7蛋白激酶。发现Rad53p与Cdc7/Dbf4直接相交，使Dbf4磷酸化。Cdc7/Dbf4复合物与Rad53p的磷酸化使Cdc7/Dbf4蛋白激酶活性失活。

项目成果

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Shimizu, K：“酿酒酵母中第五种必需的 DNA 聚合酶 Φ 定位于核仁，在核糖体 RNA 的合成中发挥重要作用”（出版中）。

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