Han Xiangzi, Mayca Pozo Franklin, Wisotsky Jacob N, Wang Benlian, Jacobberger James W, Zhang Youwei
From the Departments of Pharmacology and.
Genetics and Genome Science.
J Biol Chem. 2015 May 8;290(19):12370-8. doi: 10.1074/jbc.M114.621532. Epub 2015 Mar 25.
Mechanisms controlling DNA replication and replication checkpoint are critical for the maintenance of genome stability and the prevention or treatment of human cancers. Checkpoint kinase 1 (Chk1) is a key effector protein kinase that regulates the DNA damage response and replication checkpoint. The heterohexameric minichromosome maintenance (MCM) complex is the core component of mammalian DNA helicase and has been implicated in replication checkpoint activation. Here we report that Chk1 phosphorylates the MCM3 subunit of the MCM complex at Ser-205 under normal growth conditions. Mutating the Ser-205 of MCM3 to Ala increased the length of DNA replication track and shortened the S phase duration, indicating that Ser-205 phosphorylation negatively controls normal DNA replication. Upon replicative stress treatment, the inhibitory phosphorylation of MCM3 at Ser-205 was reduced, and this reduction was accompanied with the generation of single strand DNA, the key platform for ataxia telangiectasia mutated and Rad3-related (ATR) activation. As a result, the replication checkpoint is activated. Together, these data provide significant insights into the regulation of both normal DNA replication and replication checkpoint activation through the novel phosphorylation of MCM3 by Chk1.
控制DNA复制和复制检查点的机制对于维持基因组稳定性以及预防或治疗人类癌症至关重要。检查点激酶1（Chk1）是一种关键的效应蛋白激酶，可调节DNA损伤反应和复制检查点。异源六聚体微小染色体维持（MCM）复合物是哺乳动物DNA解旋酶的核心成分，并与复制检查点激活有关。在此，我们报告在正常生长条件下，Chk1在Ser-205位点磷酸化MCM复合物的MCM3亚基。将MCM3的Ser-205突变为丙氨酸会增加DNA复制轨道的长度并缩短S期持续时间，表明Ser-205磷酸化对正常DNA复制起负调控作用。在复制应激处理后，MCM3在Ser-205位点的抑制性磷酸化减少，并且这种减少伴随着单链DNA的产生，单链DNA是共济失调毛细血管扩张症突变和Rad3相关蛋白（ATR）激活的关键平台。结果，复制检查点被激活。总之，这些数据通过Chk1对MCM3的新型磷酸化作用，为正常DNA复制和复制检查点激活的调控提供了重要见解。
