Molecular analysis and regulation of replication fork complex, especially focus on MCM helicase

复制叉复合物的分子分析和调控，特别关注MCM解旋酶

• 批准号: 21570156
• 负责人: ZHIYING You
• 金额: $ 3.08万
• 依托单位: 財団法人東京都医学総合研究所 (2011)Tokyo Metropolitan Organization for Medical Research (2009-2010)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2009
• 资助国家: 日本
• 起止时间: 2009 至 2011
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-21570156/
• 关键词: 酵素の作用機作と調節; DNA複製; MCMヘリカーゼ; DNA結合; 複製フォーク; ATP結合活; プライマーゼ; Cdt1; ATP結合活性

Mini-chromosome maintenance(Mcm) is a central component for DNA unwinding reaction during eukaryotic DNA replication. DNA primase activity is required at the DNA replication fork to synthesize short RNA primers for DNA chain elongation on the lagging strand. Although direct physical and functional interactions between helicase and primase have been known in many prokaryotic and viral systems, potential interactions between helicase and primase have not been explored in eukaryotes. Using purified Mcm and polymeraseα-primase complexes, we find that the Mcm4/6/7 complex co-sediments with the polymeraseα-primase complex in the glycerol gradient centrifugation. A direct physical interaction between the Mcm2～ 7 complex and human DNA polymeraseα-primase subunits p48-p58 hetero-dimer is detected in pull-down assay. Single-stranded DNA binding activities of both primase and Mcm2～ 7 increase when they coexist. Both the Mcm4/6/7 and Mcm2～ 7 complexes stimulate primer RNA synthesis activity of primase in vitro. However, the helicase and ATP hydrolysis activities of Mcm4/6/7 are not affected by primase. These results indicate a possibility that a direct physical interaction between primase and Mcm may activate priming reaction by the former protein.

微染色体维持（Mcm）是真核生物DNA复制过程中DNA解绕反应的核心组成部分。DNA引物酶的活性需要在DNA复制叉合成短RNA引物，以延长DNA链上的滞后链。虽然在许多原核生物和病毒系统中，解旋酶和引物酶之间的直接物理和功能相互作用是已知的，但在真核生物中尚未探索解旋酶和引物酶之间的潜在相互作用。使用纯化的Mcm和聚合酶α-引物酶复合物，我们发现Mcm4/6/7复合物在甘油梯度离心中与聚合酶α-引物酶复合物共沉淀。Mcm2~ 7复合物与人DNA聚合酶α-引物酶亚基p48-p58异二聚体之间存在直接的物理相互作用。引物酶和Mcm2~ 7共存时单链DNA结合活性增加。Mcm2 /6/7和Mcm2~ 7复合物均能刺激引物酶的体外引物RNA合成活性。而Mcm4/6/7的解旋酶和ATP水解活性不受引物酶的影响。这些结果表明，引物酶和Mcm之间的直接物理相互作用可能会激活前者蛋白的引物反应。

项目成果

Actions of Mcm helicase and fork stabilizing factors at replication forks

Mcm 解旋酶和复制叉稳定因子的作用

• 发表时间: 2009
• 作者: Masai;H.;You;Z.;Tanaka;T.;Yokoyama;M.
• 通讯作者: M.

Fission Yeast Swi1-Swi3 Complex Facilitates DNA Binding of Mrc1*

• DOI: 10.1074/jbc.m110.173344
• 发表时间: 2010-10
• 期刊: The Journal of Biological Chemistry
• 作者: Taku Tanaka;M. Yokoyama;S. Matsumoto;Rino Fukatsu;Z. You;H. Masai

MCM複合体によるDNAプライマーゼ活性の促進

MCM 复合物促进 DNA 引物酶活性

• 发表时间: 2011
• 作者: Zhiying You;Sara De Falco;Francesca M. Pisani,正井久雄
• 通讯作者: Francesca M. Pisani,正井久雄

Purification and Biochemical analysis of replication factor Claspin

复制因子Claspin的纯化及生化分析

• 发表时间: 2009
• 作者: Uno;S.;You;Z.;Kakusho;N.;Masai;H.
• 通讯作者: H.

EBNA1-dependentassociation of mammalian pre-RC components with oriP of Epstein-Barr Virus in vitro

哺乳动物前 RC 成分与 Epstein-Barr 病毒 oriP 体外的 EBNA1 依赖性关联

• 发表时间: 2009
• 作者: 森山賢治;高井裕子;正井久雄
• 通讯作者: 正井久雄