Study Of Artemis In Regulation Cell Cycle Recovery From Replication Checkpoint

To ensure the fidelity of DNA replication,cells activate a stress-response pathway when DNA replication is perturbed.Replication stress induced phosphorylation enables the rapid activation of numerous proteins involved in DNA replication,DNA repair and cell cycle checkpoints.Mounting evidence has suggested that this pathway is important for the maintenance of genomic integrity.Artemis is a known phosphorylation substrate of DNA PK,ATM and ATR,and it was shown to have a role in the cell cycle recovery from G2/M checkpoint.Here,we studied how Artemis is involved in the replication stress response pathway and how it regulates the cell cycle machinery to recover from the stress.We have previous identified four serine residues (S516,S534,S538 and S645) that are subject to phosphorylation in response to IR (ionizing radiation) and the major kinase is ATM.In this study,we show that the above sites in Artemis are also phosphorylated by ATR in response to replication stress. We show here that mutation of S516 and S645 on Artemis exhibits a prolonged S phase arrest after treated with DNA replication inhibiting reagents.The prolonged arrest is not due to enhanced checkpoint pathway or repair failure.The evidence provided by this study indicates that phosphorylation of Artemis at serines 516 and 645 by ATR under replication stress plays a role in the recovery of cell cycle from replication checkpoint.We further showed that the delayed recovery process in HEK293 cells expressing S516/645A Artemis mutant coincides with a prolonged high expression level of cyclin E protein which attenuates cyclin A-CDK2 complex formation and kinase activity.We found that depletion of Artemis from cultured cells also induces a high expression of cyclin E.The striking discovery of this study is that Artemis physically interacts with Fbw7 complex proteins which regulates cyclin E by ubiquitination.The phosphorylation of Artemis at serines 516 and 645 by ATR after replication stress promotes the association between Artemis and Fbw7 complex proteins.The data of this study suggested a new model that cyclin E as the regulation center involved in recovery of cell cycle from S phase checkpoint after replication stress,which is regulated by the phosphorylation of Artemis.