Abstract
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.
Highlights
Checkpoint kinase 1 (Chk1) and the minichromosome maintenance (MCM) complex play important roles in DNA replication and replication checkpoint
Ser-205 Phosphorylation Is Involved in Replication Checkpoint Activation—We recently reported that the MCM complex plays an important role in recruiting Chk1 onto chromatin, a critical step for DNA damage-induced Chk1 phosphorylation [25]
We have illustrated a novel mechanism by which Chk1 regulates DNA replication and replication checkpoint through inducing an inhibitory phosphorylation of MCM3
Summary
Chk and the MCM complex play important roles in DNA replication and replication checkpoint. Conclusion: Phosphorylation of MCM3 by Chk regulates normal DNA replication and checkpoint. 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 Rad3related (ATR) activation. In addition to its roles in the DNA damage response and checkpoint activation, Chk regulates DNA replication under normal growth conditions. The reduction in Ser205 phosphorylation of MCM3 is accompanied with the generation of long stretches of single strand DNA (ssDNA), the critical platform for maximal activation of ATR. This seems to be important for subsequent Chk phosphorylation and likely the amplification and maintenance of replication checkpoint
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