Study For The Role Of Bre1-dependent H2B Ubiquition In DNA Damage Response And Repair

Cellular genomic DNA is susceptible to different types of DNA damages caused by internal or external factors.To cope with DNA damages,cells have evolved complex and accurate DNA damage response systems(DDR),including DNA damage checkpoints and DNA repair systems.DNA double strand break(DSB)is a type of DNA lesion highly toxic to cells.It is mainly repaired by two pathways,homologous recombination(HR)and non homologous end joining(NHEJ).DSB repair occurs at chromatin level and is tightly regulated by histone modifications in cells.Histone H2 B in yeast cells can be mono-ubiquited(H2Bub)by the concerted action of ubiquitin-conjugating enzyme Rad6(E2)and ligase Bre1(E3).H2 Bub participates in multiple essential cellular processes,such as transcription,replication,DNA repair,telomere maintenance and so on.However,the role and mechanism of H2 Bub in repair of DSBs are still not fully understood.In this study,we explored the function and molecular mechanism of H2 Bub in DSB repair and damage response using Saccharomyces cerevisiae as a model.First,we have revealed that Bre1 deficiency leads to the cell sensitivity to a variety of DNA damaging agents,which can be rescued by introducing of an exogenous BRE1 gene.Meanwhile,Bre1 deficient cells accumulated a large amount of ?-H2 A upon DSB induction.Further,we demonstrated that the role of Bre1 in DNA damage response is acting through H2B-K123 ubiquitination.Second,we examined the role of Bre1 in homologous recombination and non homologous end joining repair.The fluorescence microscope analysis showed that a large number of Rad52-YFP foci were accumulated in the bre1? mutant cells during the recovery from phleomycin treatment as compared to the wild type cells,and there was no significant decrease with the prolongation of the recovery time.Further,we measured the efficiency of DSB repair by ectopic recombination and found that the efficiency was significantly reduced in the mutant.Accordingly,the bre1?yku70? double mutant strain was more sensitive to the DNA damages than each single mutant.In addition,we found that Bre1 promotes DSB repair by NHEJ at both chromatin level and plasmid-based assay.Thus,our results indicate that Bre1 promotes DSB repair by both HR and NHEJ through H2B-K123 ubiquitination.Finally,we found that Bre1 is essential for checkpoint activation upon DSBs,and the RING and RBD domains of Bre1 are also crucial for the checkpoint activation.We showed that Bre1-H2 Bub deficiency impaired the formation of ?H2A and H3K79 methylation on chromatin,leading to defective Rad9 recruitment and Rad53 phosphorylation.