Molecular Mechanism of Initiation of DNA Replication

在酵母、酿酒酵母和其他真核细胞中，Cdc7/Dbf4蛋白激酶复合体是启动染色体DNA复制所必需的。为了阐明Cdc7/Dbf4在细胞周期中的功能，我们在昆虫细胞中表达了该复合物，并对其进行了纯化。纯化的复合物在子宫内特异性磷酸化酵母Mcm2蛋白，表明Cdc7/Dbf4蛋白激酶的两个靶点之一是Mcm2-7蛋白复合物，这种磷酸化导致Mcm2-7蛋白复合物在细胞周期内的功能改变。McmlO蛋白也被认为是酿酒酵母染色体DNA复制启动所必需的。研究表明，McmlOp与ORC （Origin Recognition Complex）相互作用，ORC在整个细胞周期的起点结合，也是染色体DNA复制起始所必需的，这表明McmlOp在染色体DNA复制起点形成起始复合物中起着锚定作用。Schizosaccharomyces pombe cdc23^+对S.cerevisiae的mcmlO突变进行了补充，表明Cdc23p是McmlOp的功能同源物。DPB11基因在遗传上与DNA聚合酶II （epsilon）相互作用，epsilon是三种复制DNA聚合酶之一，是s.e cerevistae DNA复制和S期检查点所必需的。为了确定与dpbll-1相互作用的因子，我们分离出了6个互补组（sldl至-6）的sld（与dpbll-1合成致死）突变。研究发现SLD1与编码DNA聚合酶II全酶第三亚基的DPB3相同，SLD4与启动染色体DNA复制所需的0DC45相同，SLD6与DNA损伤和S期检查点所需的RAD53相同，SLD2、-3和-5是新发现的基因。这些新发现的基因产物也是DNA复制起始所必需的。因此，DNA聚合酶II在DNA复制的起始阶段和染色体DNA复制的延伸阶段发挥了关键作用。少

In the yeast Saccharomyces cerevisiae as well as other eukaryotic cells, Cdc7/Dbf4 protein kinase complex is required for intiation of chromosomal DNA replication. To elucidate the function of Cdc7/Dbf4 in the cell cycle the complex was expressed in insect cells and purified to its homogeniety. The purified complex specifically phosphorylated yeast Mcm2 protein in uttro, suggesting that one of in utuo targets of Cdc7/Dbf4 protein kinase is Mcm2-7 protein complex and this phosphorylation leads to functional change of Mcm2-7 protein complex during the cell cycle. McmlO protein is also known to be required for intiation of chromosomal DNA replication in S.cereuisiae. It has been shown that Mcm lOp physically interacts with ORC, Origin Recognition Complex, which bind at orgins thoughout the cell cycle and is also required for initiation of chromosomal DNA replication, suggesting that McmlOp plays as an anchor for the formation of initiation cornplex at chromosomal DNA replication origins i … More n S.cerevisiae. Schizosaccharomyces pombe cdc23^+ complimented the mcmlO mutations of S.cerevisiae, suggesting that Cdc23p is a functional homolog of McmlOp.The DPB11 gene, which genetically interacts with DNA polymerase II (epsilon), one of three replicative DNA polymerases is required for DNA replication and the S phase checkpoint in S.cerevistae. To identify factors interacting with Dpbllpwe have isolated sld (synthetically lethal with dpbll-1) mutations which fall into six complementation groups (sldl to -6). It has been found that SLD1 is identical to DPB3, which encodes the third subunit of DNA polymerase II holoenzyme SLD4 is the same as 0DC45, which is required for initiation of chrornsomal DNA replication, and SLD6 is identical to RAD53, which is required for DNA damage as wellas S phase checkpoint, while SLD2, -3, and -5 are newly identified genes. These newly identified gene products are also required for initiation of DNA replication. Thus, DNA polymerase II should play an crutial role during the intiation step of DNA replication as well as during the elongation step of chromsomal DNA replication in S, cerevisiae. Less