The Mechanism By Which Replication Stress Inhibits Homologous Recombination

The genomic DNA is continuously challenged by both endogenous and exogenus DNA damage-inducing agents,which lead to many types of lesions in the DNA.DNA double-strand break(DSB)is one of the most cytotoxic DNA damages.If DSB cannot be correctly and effectively repaired,it will lead to genome instability.Replication stress may potentially lead to DSBs.Homologous recombination is one of two major pathways for repair of DSBs.A key question remains to be addressed is that how cells coordinate the response to replication stress and the repair by homologous recombination when DSB occurs under replication stress.In this study,we investigated the role of Mrc1,a mediator protein of the DNA replication checkpoint,in coordinating the response to replication stress and the repair by homologous recombination.We obtained the following results:Homologous recombination is one of two major pathways for repair of DSBs.A key question remains to be addressed is that how cells coordinate the response to replication stress and the repair by homologous recombination when DSB occurs under replication stress.In this study,we investigated the role of Mrc1,a mediator protein of the DNA replication checkpoint,in coordinating the response to replication stress and the repair by homologous recombination.We obtained the following results:1)Replication stress inhibits DSB end resection.We found that the hydroxyurea(HU)treatment severely impaired the speeds of DSB end resection.Both initial resection and the Exo1-or Sgs1/Dna2-mediated long-range resection were suppressed.2)The inhibition of end resection by replication stress is mediated by Mrc1.We found that deletion of MRC1 can greatly diminish the inhibition of end-resection by replication stress.This indicates that Mrc1 plays a key role in mediating replication stress-induced inhibition of DSB end resection.3)Rad9 binds chromatin in the vicinity of DSB and forms an obstacle that can inhibit long-range resection.Depletion of Rad9 or Dotl,a methyltransferase required for Rad9 recruitment,did not restore the rate of DSB end resection under replication stress,suggesting that replication stress may affect steps uspstream of Rad9 recruitment,such as,histone modification and chromatin remodeling.4)The S-phase checkpoint pathways have both positive and negative effects on end resection.We showed that HU-induced suppression of resection was relieved shortly after removal of HU,and this correlated with the activation and inactivation of replication checkpoint,respectively.It suggests that the maintenance of replication checkpoint is essential to sustain the inhibition of resection.Importantly,we found that resection was nearly abolished in the absence of the kinase Mec1 or Rad53 upon replication stress,suggesting that the two kinases also play a role in promote resection under replication stress.5)Mrcl is critical for DSB repair by homologous recombination under replication stress.There was no significant difference in the repair kinetic between WT and mrc1?cells in the absence of replication stress.While under HU treatment,WT cells successfully repaired DSBs by homologous recombination although with a significant delay,while the mrc1? cells failed to repair DSBs.Thus,Mrc1 plays a key role in promoting DSB repair by homologous recombination under replication stress although it also acts to inhibit resection.These results indicate that Mrc1 plays important functions in coordinating replication stress and repair by homologous recombination.