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.

酿酒酵母CDC7/DBF4蛋白激酶调节染色体DNA复制的起始，并从昆虫细胞中表达并纯化。发现纯化的CDC：7/DBF4磷酸化MCM2，这是MCM2-7复合物之一。此外，确定其磷酸化位点。鉴定了酿酒酵母CDC7/DBF4蛋白激酶和MCM10的异爪蟾同源物，并克隆其相应的基因。发现Xenopus cdc7/dbf4.蛋白激酶和MCM10是启动染色体DNA复制所必需的，如S. cerevisiae.s中所必需的。纯化了参与DNA破坏和S期检查点调节的酿酒酵母Rad53p，并纯化了其中一种底物为DBF4/CDC7蛋白激酶。发现RAD53P直接与CDC7/DBF4互动并磷酸化DBF4。与Rad53p的Cdc7/DBF4复合物的磷酸化使Cdc7/DBF4蛋白激酶活性失活。

项目成果

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Kamimura, Y.：“Sld3 与 Cdc45(Sld4) 相互作用，在酿酒酵母中发挥染色体 DNA 复制的作用”EMBO J.. 20. 2097-2107 (2001)

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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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