Functions Of Histone Deacetylases Rpd3 And Hda1 In DNA Repair

DNA double strand Break(DSB)threatens genome stability.Cells repair DSBs by two major pathways: homologous recombination(HR)and non-homologous end joining(NHEJ).Dynamic histone acetylation and deacetylation is critical for proper DSB repair,while its dysfunction is associated with multiple human diseases including cancer.However,how this dynamics affects DSB repair remains partially understood.In this study,we explored the roles of Rpd3 and Hda1,two yeast histone deacetylases,in DNA damage response and HR repair.We found that:1.Compare with the wild type(WT)cells,hda1? single mutant only displaysslight sensitivity to MMS,but rpd3? mutant exhibits significant sensitivity to CPT,MMS and phleomycin,indicating that Rpd3 is important for DNA damage response and repair.It is noteworthy that cells lacking of both Rpd3 and Hda1 show more severe defect in resistance to DNA damage agents.Similarly,the double mutant rpd3?hda1? exhibits a reduced survival rate upon acute MMS treatment ascompared with each single mutant.These results suggest that Rpd3 and Hda1 play redundant functions in DNA damage response and repair.2.Next,we evaluated the roles of Hda1 and Rpd3 in DNA repair by monitoring Rad52 foci along the recovery following phleomycin treatment.We noted that the rpd3? hda1? double mutant possesses a higher level of spontaneous DNA damagethan the WT cells or rpd3? and hda1? single mutants.However,phleomycintreatment induces a great increase of Rad52 foci number in all of these strains.Along the recovery,Rad52 foci number decreases quickly to the normal level in the WT and hda1? mutant cells.However,it descends slower in rpd3? single mutant or rpd3?hda1? double mutant.These results indicate that Rpd3 plays an important role inDNA repair and it acts redundantly with Hda1.To verify this result,we tested HR efficiency for these strains by gene targeting.The results show that deletion of RPD3 leads to partial defects in HR,while deletion of both RPD3 and HDA1 results in a much severe defect in HR.3.To explore how Rpd3 and Hda1 may affect HR,we examined the rate of DSB end resection for these strains by Southern blot.We found that resection proceeds normally in hda1? single mutant,while it is significantly impaired in rpd3? singlemutant.Notably,the defect became more severe in the rpd3? hda1? double mutant.To confirm this result,we tested the recruitment of RPA and Rad51 at DSB by using chromatin immunoprecipitation.As expected,the recruitment of both proteins remains unaffected in hda1? mutant,yet it is slightly reduced in the rpd3? single mutant.Strikingly,the recruitment of RPA and Rad51 is greatly impaired in rpd3? hda1?double mutant.4.Finally,we assessed the function of Rpd3 and Hda1 in checkpoint activation.By following cell cycle arrest or by Western blot,we found that both WT and hda1?mutant activate checkpoint normally in response to MMS treatment or DSBs,while rpd3? mutant exhibits a delayed checkpoint activation.Notably,deletion of RPD3 and HDA1 simultaneously results in a more severe defect,indicating a redundant function of Rpd3 and Hda1 in DNA damage response.In summary,our results reveal that Rpd3 plays important roles in DNA damage response and HR repair,and it shares redundant roles with Hda1 during these processes.Our study lays the foundation for further exploration of role and mechanism of HDAC in DNA damage response and maintenance of genome stability.