Molecular dynamism of replication machinary in eukaryotic chromosomal DNA replication

真核染色体DNA复制中复制机器的分子动力学

• 批准号: 10680652
• 负责人: KAWASAKI Yasuo
• 金额: $ 1.92万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10680652/
• 关键词: DNA replication; Mcm10; Saccharomyces cerevisiae; Mcm2-7; ORC; Mcm10; DNAポリメラーゼII; Dpb4; 複製開始複合体

Mcm1Op is an essential factor for the initiation of DNA replication in Saccharomyces cerevisiae. Mcm10p interacts physically and genetically with Mcm2-7 protein family and a lesion in MCM10 results in the pausing of elongation forks at replication origins which failed to be activated, suggesting that in addition to mediating the activation of replication initiation at replication origins, Mcm10p is required for the disassembly of the pre-replicative complex, which allows smooth passage of replication forks. Mcm10p is structurally conserved among eukaryotes like other replicatgion proteins.We found that an mcm10 mutation causes loss fo chrommosome integrity during S phase and prevents entry into mitosis despite completion of chromosomal DNA replication. Furthermore, Mcm10p interacts genetically with origin recognition complex (ORC) and various replication elongation factors, including a subunit of DNA polymerase e and d.We also screened mutants that are synthetically lethal with mcm10. We obtained not only mutations in intiation factors, CDC45, MCM2, and MCM7, but also DNA2 which encodes an essential DNA helicase required for processing of Okazaki fragment.Mcm10p is an abundant protein (approximately 4 x 10^4 copies per haploid cell) that is almost exclusively localized in the chromatin and/or nuclear matrix fraction during all phases of the cell cycle. When visualized by chromosome-spreading method followed by immunostaining, Mcm10p formed punctated foci on chromatin throughout the cell cycle and these foci mostly overlapped with those of Orc1p, a component of ORC.Furthermore, we found that Mcm10p physically associated with ORC by immunoprecipitation.These results suggest that Mcm10p, like Mcm2-7 proteins, is a critical component of the pre-replication chromatin and acts together with ORC during the initiation of chromosomal DNA replication ; in addition, Mcm10p plays an essentail role during elongation of DNA replication.

MCM1OP是酿酒酵母中DNA复制启动的重要因素。 Mcm10p interacts physically and genetically with Mcm2-7 protein family and a lesion in MCM10 results in the pausing of elongation forks at replication origins which failed to be activated, suggesting that in addition to mediating the activation of replication initiation at replication origins, Mcm10p is required for the disassembly of the pre-replicative complex, which allows smooth passage of replication forks. MCM10P像其他复制蛋白一样在真核生物中是在结构上保守的。我们发现，MCM10突变在S期间导致成铬体完整性损失，并防止尽管完成了染色体DNA复制，但仍会进入有丝分裂。此外，MCM10P与原始识别复合物（ORC）和各种复制伸长因子（包括DNA聚合酶E的亚基）和D。我们还筛选了与MCM10合成致死的突变体。我们不仅获得了Intiation因子CDC45，MCM2和MCM7的突变，而且还获得了编码Okazaki碎片处理所需的必需DNA解旋酶的DNA2。mcm10p是一种丰富的蛋白质（大约4 x 10^4个单倍型细胞），几乎是在chratrix和/或necation cyrix cyrix cyrix cyrix cyrix cyrix bercome copers of Chartrix berc的均几乎被排除的。当通过染色体膨胀的方法进行免疫染色的方法观察时，MCM10P在整个细胞周期中形成了对染色质的注射焦点，这些焦点主要与ORC1P重叠，ORC1P是ORC.Furthermore的一个组件，我们发现MCM10P与ORC与ORC物理相关的MCMMCIPITITION。在染色体DNA复制的开始期间，染色质预复原蛋白并与兽人一起起作用；此外，MCM10P在DNA复制的伸长过程中起着精致的作用。

项目成果

Lisa Homesley: "Mcm10 and the MCM2-7 complex interact to initiate DNA synthesis and to release replication factors from origins."Genes and Development. 14・8. 913-926 (2000)

Lisa Homesley：“Mcm10 和 MCM2-7 复合物相互作用，启动 DNA 合成并从起点释放复制因子。”14・8 (2000)。

Makoto Kihara: "Characterization of the yeast Cdc7p/Dbf4p complex purified from insect cells : its protein kinase activity is regulated by Rad53p."Journal of Biological Chemistry. 275・45. 35051-35062 (2000)

Makoto Kihara：“从昆虫细胞中纯化的酵母 Cdc7p/Dbf4p 复合物的表征：其蛋白激酶活性受 Rad53p 调节。”生物化学杂志 275・45（2000）。

Tomoko Ohya et al.: "Structure and function of the fourth subunit (Dpb4p) of DNA polymerase εin Saccharomyces cerevisiae."Nucleic Acids Research. 28-20. 3846-3852 (2000)

Tomoko Ohya 等人：“酿酒酵母 DNA 聚合酶 ε 的第四亚基 (Dpb4p) 的结构和功能。核酸研究”28-20 (2000)。

Yasuo Kawasaki: "Interactions between Mcm10p and other replication factors are required for proper initiation and elogation of chromosomal DNA replication in Saccharomyces cerevisiae."Genes to Cells. 5・12. 975-989 (2000)

Yasuo Kawasaki：“Mcm10p 和其他复制因子之间的相互作用对于酿酒酵母中染色体 DNA 复制的正确启动和延伸是必需的。”Genes to Cells 5·12 (2000)。

Masako Izumi: "The human homolog of Saccharomyces cerevisiae Mcm10 interacts with replication factors and dissociates from nuclear structure in G2 phase."Nucleic Acids Research. 28・23. 4769-4777 (2000)

Masako Izumi：“酿酒酵母 Mcm10 的人类同源物与复制因子相互作用并在 G2 期从核结构中解离。”核酸研究 28・23 4769-4777（2000）。