Artemis蛋白磷酸化对紫外线C引发的复制检测点的调控

Objective To investigate Artemis phosphorylation on S516 and S645 in response to stalled replication forks and its role in regulation of cell cycle replication checkpoint.Methods Western-blotting was used to measure the expression of phosphorylation of Artemis on S516 and S645 after UVC irradiation.The nonphosphorylatable double mutant (S516-645A) and the mimicking phosphorylation mutant (S516-645D) plasmids were constructed.HEK 293 cells with stable expression of wild type Artemis and the corresponding mutants were established by transfection.Cell cycles of the cells treated with UVC irradiation were analyzed by flow cytometry,Western-blotting was used to measure the expression of Chk1,γ-H2AX and Cdk2.IP-kinase assay was used to measure the kinase activity of Cdk2 2,6 and 12 h after UVC irradiation.Results Artemis got rapid and prolonged pbosphorylation on S516 and S645 after treatment with UVC irradiation and the major responsible kinase was ATR.The S516-645A mutant caused prolonged arrest in replication checkpoint in S phase.The Cdk2 IP kinase activity was inhibited in S516-645A mutant ceils,but the expression levels of Chk1,Cdk2 and γ-H2AX were not affected.Conclusion The ATR phosphorylation on S516 and S645 of Artemis promotes cell cycle recovery from UVC induced replication checkpoint.

Regulation of artemis phosphorylation on replication checkpoint induced by UVC irradiation