DNA Replication Apparatus and S-Phase Checkpoint Control

从昆虫细胞中表达并纯化了调节染色体DNA复制起始的酿酒酵母Cdc 7/Dbf 4蛋白激酶。发现纯化的Cdc：7/Dbf 4磷酸化Mcm 2，这是Mcm 2 -7复合物之一。并对其磷酸化位点进行了测定。S.对酿酒酵母Cdc 7/Dbf 4蛋白激酶和Mcm 10进行了鉴定，并克隆了相应的基因。发现非洲爪蟾Cdc 7/Dbf 4蛋白激酶和Mcm 10是启动染色体DNA复制所必需，如在S.党产会本研究纯化了参与DNA损伤和S期检查点调控的酿酒酵母Rad 53 p，并鉴定其底物之一为Dbf 4/Cdc 7蛋白激酶。研究发现Rad 53 p直接与Cdc 7/Dbf 4相互作用并磷酸化Dbf 4。Cdc 7/Dbf 4复合物与Rad 53 p的磷酸化使Cdc 7/Dbf 4蛋白激酶活性失活。

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.