Molecular Mechanism of Initiation of DNA Replication

在酿酒酵母（Saccharomycescerevisiae）和其它真核细胞中，Cdc 7/Dbf 4蛋白激酶复合物是启动染色体DNA复制所必需的。为了阐明Cdc 7/Dbf 4在细胞周期中的功能，将该复合物在昆虫细胞中表达并纯化至其均一性。纯化的复合物特异性磷酸化酵母Mcm 2蛋白，表明Cdc 7/Dbf 4蛋白激酶的一个体内靶点是Mcm 2 -7蛋白复合物，这种磷酸化导致Mcm 2 -7蛋白复合物在细胞周期中的功能改变。McmlO蛋白也是已知的在S. cereuisiae中启动染色体DNA复制所需的。已经表明，McmlOp与ORC（起始识别复合物）物理相互作用，ORC在整个细胞周期中在起始处结合，并且也是染色体DNA复制起始所需的，这表明McmlOp在染色体DNA复制起始处起形成起始复合物的锚作用。 ...更多信息 n S.parae.粟酒裂殖酵母cdc 23 ^+互补了酿酒酵母的mcm 10突变，表明Cdc 23 p是McmlOp的功能同源物。DPB 11基因与DNA聚合酶II（DNA polymerase II，DNA polymerase II）相互作用，DNA聚合酶II是酿酒酵母中DNA复制和S期检查点所必需的三种复制型DNA聚合酶之一。为了鉴定与Dpbllp相互作用的因子，我们分离了sld（与dpbll-1合成致死）突变，其落入六个互补组（sld至-6）。SLD 1与编码DNA聚合酶II第三亚基的DPB 3相同，SLD 4与启动染色体DNA复制所需的0 DC 45相同，SLD 6与DNA损伤和S期检查点所需的RAD 53相同，而SLD 2、-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在酿酒酵母DNA复制的起始步骤以及染色体DNA复制的延伸步骤中应发挥关键作用。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