Molecular Mechanism of Chromosomal DNA Replication Apparatus

为了了解真核细胞染色体DNA复制的分子机制，本项目一直致力于阐明各种真核系统（包括酵母、爪蟾卵、果蝇、小鼠和人类细胞）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。在这些研究的过程中，我们发现了几个新的复制蛋白与DNA聚合酶的相互作用，这可能是一个领先的链聚合酶。此外，我们还纯化了参与染色体DNA复制起始和延伸步骤的酵母Cdc 7/Dbf 4蛋白激酶和人Mcm 2 -7蛋白复合物。通过这些研究，我们发现Cdc 7/Dbf 4蛋白激酶的活性受Rad 53蛋白激酶的调节，Rad 53蛋白激酶参与了S-和DNA损伤检查点，并且人Mcm 2 -7蛋白的亚复合物具有DNA解旋酶活性，表明该复合物是一种在复制叉处起作用的DNA解旋酶。

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.