Inhibition of DNA binding of MCM2-7 complex by phosphorylation with cyclin-dependent kinases
Inhibition of DNA binding of MCM2-7 complex by phosphorylation with cyclin-dependent kinases
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DOI：
10.1093/jb/mvt062
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发表时间：
2013-10-01
影响因子：
2.7
通讯作者：
Ishimi, Yukio
中科院分区：
文献类型：
--
作者：
Moritani, Mariko;Ishimi, Yukio
细胞周期蛋白依赖性激酶 (CDK) 在防止 DNA 重新复制中发挥着核心作用，它会磷酸化多种复制蛋白以使其失活。 MCM2-7中的MCM4和RPA中的RPA2在体内被CDK磷酸化。这些蛋白质的磷酸化与其染色质结合之间存在反向相关性。在这里，我们利用 CDK2/cyclinE、CDK2/cyclinA、CDK1/cyclinB、CHK1、CHK2 和 CDC7/DBF4 激酶检测了 MCM2-7、RPA、TRESLIN、CDC45 和 RECQL4 人类复制蛋白的体外磷酸化。 MCM4、RPA2、TRESLIN 和 RECQL4 用 CDK 磷酸化。通过凝胶位移分析检查 CDK2/cyclinA 磷酸化对 MCM2-7 和 RPA 的 DNA 结合能力的影响。 RPA 的磷酸化不影响其 DNA 结合能力，但 MCM4 的磷酸化抑制 MCM2-7 的能力。 MCM4氨基末端区域中丝氨酸和苏氨酸的六个氨基酸改变为丙氨酸使得突变体MCM2-7对CDK的抑制不敏感。这些生化数据表明，CDK 在这些位点对 MCM4 进行磷酸化，在将 MCM2-7 从染色质上移出和/或防止复合物重新加载到染色质方面发挥着直接作用。
Cyclin-dependent kinase (CDK) that plays a central role in preventing re-replication of DNA phosphorylates several replication proteins to inactivate them. MCM4 in MCM2-7 and RPA2 in RPA are phosphorylated with CDK in vivo. There are inversed correlations between the phosphorylation of these proteins and their chromatin binding. Here, we examined in vitro phosphorylation of human replication proteins of MCM2-7, RPA, TRESLIN, CDC45 and RECQL4 with CDK2/cyclinE, CDK2/cyclinA, CDK1/cyclinB, CHK1, CHK2 and CDC7/DBF4 kinases. MCM4, RPA2, TRESLIN and RECQL4 were phosphorylated with CDKs. Effect of the phosphorylation by CDK2/cyclinA on DNA-binding abilities of MCM2-7 and RPA was examined by gel-shift analysis. The phosphorylation of RPA did not affect its DNA-binding ability but that of MCM4 inhibited the ability of MCM2-7. Change of six amino acids of serine and threonine to alanines in the amino-terminal region of MCM4 rendered the mutant MCM2-7 insensitive to the inhibition with CDK. These biochemical data suggest that phosphorylation of MCM4 at these sites by CDK plays a direct role in dislodging MCM2-7 from chromatin and/or preventing re-loading of the complex to chromatin.