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.

迷你染色体维护（MCM）是真核生物期间DNA解开反应的中心成分。在DNA复制叉上需要DNA起始酶活性，​​以合成短RNA引物以在滞后链上的DNA链伸长。尽管在许多原核生物中已经知道了解旋酶和原始酶之间的直接物理和功能相互作用，但在真核生物中尚未探索解旋酶与原始酶之间的潜在相互作用。使用纯化的MCM和聚合酶α-原酶复合物，我们发现在甘油梯度离心机中，MCM4/6/7复合物与聚合酶α-蛋白酶复合物具有。在下拉测定中检测到MCM2〜7复合物和人DNA聚合酶α-原始酶亚基P48-P58异二聚体之间的直接物理相互作用。共存时，单链DNA结合活性都会增加。 MCM4/6/7和MCM2〜7复合物都刺激了体外原始酶的底漆RNA合成活性。但是，MCM4/6/7的解旋酶和ATP水解活性不受原始酶的影响。这些结果表明，原始酶和MCM之间的直接物理相互作用可能会激活前者蛋白质的启动反应。

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.

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.

ATP Binding Properties of Mouse CDT1 Protein

小鼠 CDT1 蛋白的 ATP 结合特性

• 发表时间: 2009
• 作者: You;Z.;Masai;H.
• 通讯作者: H.

Biochemical Characterization of MCM, the Essential DNA Helicase for Eukaryotic DNA Replication

MCM（真核 DNA 复制必需的 DNA 解旋酶）的生化表征

• 发表时间: 2012
• 作者: M. Yoshino;H. Takahashi;T. Takagi,T. Baba;T. Kanamori;M. Sonoyama;Zhiying You
• 通讯作者: Zhiying You