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Molecular Mechanism of Initiation of DNA Replication

DNA复制起始的分子机制

基本信息

批准号：
08044204
负责人：
SUGINO Akio
金额：
$ 8.51万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08044204/
关键词：
Cdc7 Dbf4 protein kinase S.cerevisiae S.pombe Mcm2-7 proteins Mcm10 DNA polymeraseII (epsilon)Dpb11 Sld

项目摘要

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

在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体，并对其进行了纯化。纯化的复合体能特异性地磷酸化酵母Mcm2蛋白，提示CDC7/Dbf4蛋白激酶的作用靶点之一是Mcm2-7蛋白复合体，这种磷酸化导致了Mcm2-7蛋白复合体在细胞周期中的功能变化。已知McmlO蛋白也是启动酿酒酵母染色体DNA复制所必需的。已经证明mcmlop在物理上与ORC相互作用，ORC是一种起源识别复合体，它结合在整个细胞周期的各个器官上，也是启动染色体dna复制所必需的，这表明mcmlOp在染色体dna复制起始点I…处起着形成起始复合体的锚作用。更多的是酿酒酵母。裂殖酵母cdc23^+证实了酿酒酵母的mcmlO突变，提示该基因是Mcmlop的功能同源物。DPB11基因在基因上与DNA聚合酶II(Epsilon)相互作用，是酿酒酵母复制所必需的三种复制聚合酶之一，也是酿酒酵母的S阶段检查点。为了确定与Dpbllp1相互作用的因素，我们分离了SLD(与Dpbl1-1合成致死)突变，这些突变分为六个互补组(sld1至-6)。SLD1与编码DNA聚合酶II第三亚单位的DPB3相同，SLD4与0DC45相同，0DC45是启动染色体DNA复制所必需的；SLD6与RAD53相同，RAD53是DNA损伤和S阶段检查点所必需的；而SLD2、-3和-5是新发现的基因。这些新发现的基因产物也是启动DNA复制所必需的。因此，在酿酒酵母S中，DNA聚合酶II在DNA复制的起始阶段和染色体复制的延伸阶段都可能发挥关键作用。较少