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首页> 外文期刊>Molecular biology of the cell >Essential Role of Phosphorylation of MCM2 by Cdc7/Dbf4 in the Initiation of DNA Replication in Mammalian Cells
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Essential Role of Phosphorylation of MCM2 by Cdc7/Dbf4 in the Initiation of DNA Replication in Mammalian Cells

机译:Cdc7 / Dbf4使MCM2磷酸化在哺乳动物细胞DNA复制的启动中的重要作用。

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摘要

We report the identification of Cdc7/Dbf4 phosphorylation sites in human MCM2 and the determination of the role of Cdc7/Dbf4 phosphorylation of MCM2 in the initiation of DNA replication. Using immunoblotting, immunofluorescence, and high-speed automated cell-imaging analyses with antibodies specific against MCM2 and Cdc7/Dbf4 phosphorylated MCM2, we show that the chromatin recruitment and phosphorylation of MCM2 are regulated during the cell cycle in HeLa cells. Chromatin-bound MCM2 is phosphorylated by Cdc7/Dbf4 during G1/S, which coincides with the initiation of DNA replication. Moreover, we show that baculovirus-expressed purified MCM2-7 complex and its phosphomimetic MCM2E-7 complex display higher ATPase activity when compared with the nonphosphorylatable MCM2A-7 complex in vitro. Furthermore, suppression of MCM2 expression in HeLa cells by siRNA results in the inhibition of DNA replication. The inhibition can be rescued by the coexpression of wild type MCM2 or MCM2E but not MCM2A. Taken together, these results indicate that Cdc7/Dbf4 phosphorylation of MCM2 is essential for the initiation of DNA replication in mammalian cells.
机译:我们报告了人类MCM2中的Cdc7 / Dbf4磷酸化位点的鉴定以及在DNA复制的启动中MCM2的Cdc7 / Dbf4磷酸化作用的确定。使用针对MCM2和Cdc7 / Dbf4磷酸化的MCM2特异的抗体的免疫印迹,免疫荧光和高速自动细胞成像分析,我们证明了MCM2的染色质募集和磷酸化在HeLa细胞的细胞周期中受到调节。染色质结合的MCM2在G1 / S期间被Cdc7 / Dbf4磷酸化,这与DNA复制的启动相吻合。而且,我们显示杆状病毒表达的纯化的MCM2-7复合物及其模拟磷酸MCM2E-7复合物与非磷酸化的MCM2A-7复合物相比在体外显示出更高的ATPase活性。此外,通过siRNA抑制HeLa细胞中MCM2表达可导致DNA复制受到抑制。可以通过野生型MCM2或MCM2E而不是MCM2A的共表达来挽救抑制作用。综上所述,这些结果表明,MCM2的Cdc7 / Dbf4磷酸化对于启动哺乳动物细胞中DNA复制至关重要。

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