A Functional And Mechanistic Study For Bromodomain Of Protein Bdf1 In DNA Repair By Homologous Recombination

A variety of endogenous or exogenous factors such as chemicals,ultraviolet radiation,ionizing radiation,and reactive oxygen species can cause DNA damage in eukaryotes.DNA double-strand break(DSB)is one of the most toxic forms of DNA damage in which the phosphoric acid skeletons of two complementary DNA strands are broken at the same time.In order to maintain the stability of the genome,organisms have evolved a sophisticated DNA damage response system to detect and repair DSBs,including non-homologous end joining(NHEJ)and homologous recombination(HR).Homologous recombination,as a relatively accurate repair mechanism,is the predominant pathway for repairing DSBs in Saccharomyces cerevisiae.Bdf1,a member of the BET protein family,contains two tandem bromodomains and an extra C-terminus.The bromodomains of Bdf1 can recognize and bind acetylated histones H3 and H4.Bdf1 can recruit the TFIID complex to initiate transcription.In addition,Bdf1 is a subunit of the chromatin remodeling complex SWR and is involved in the regulation of chromatin structure.Tyrosine residues 187 and 354 are essential for the function of the bromodomains of Bdf1.Their mutation into a phenylalanine(bdf1-Y187FY354F)abolishes detectable binding of the bromodomains to the acetylated histone H4.Our previous study found that Bdf1 is involved in DNA damage repair,but its specific function and molecular mechanism are still unclear.In order to explore the role of Bdf1 in DSBs repair,we constructed a bdf1 mutant in which the two key residues were mutated(bdf1-2YF),and examined the role of the bromodomains of Bdf1 in DSBs repair.The following results were obtained:1)We tested the drug sensitivity for the bdf1-2YF,and found that the sensitivity of the bdf1-2YF to Camptothecin(CPT),Hydroxyurea(HU),Phleomycin and Methylmercuric sulfate(MMS)was significantly increased,indicating that the bromodomains of Bdf1 play an important role in DNA damage repair.Further,we explored the role of the bromodomains of Bdf1 on ectopic homologous recombination and chromatin based NHEJ.We found that the HR efficiency of the WT was 85%,while the efficiency of the bdf1-2YF reduced to 48%.Besides,the NHEJ efficiency of the bdf1-2YF was also reduced.These results suggest that the bromodomains of Bdf1 promote both NHEJ and HR repair.2)Chromatin remodeling complexes can facilitate DSB repair by relaxing chromatin structure,regulating DNA damage checkpoints,and promoting long-distance end resection.In order to explore the relationship between the bromodomain of Bdf1 and chromatin remodeling complexes,we tested the recruitment of chromatin remodeling complex at DSB end in the bdf1-2YF,and we found that compared to WT strains,the recruitment of Fun30,Ino80 and Rsc2 are decreased significantly,suggesting that the bromodomain of Bdf1 can recruit chromatin remodeling complexes to DSBs.Then we examined the chromatin structure by MNase digestion and by histone-loss experiment,and found that the bdf1-2YF does not affect the global or local chromatin structure.3)Our previous study found that the bromodomain of Bdf1 promote end resection.We tested the recruitment of Mre11,Dna2/Sgs1 and Exo1 by Ch IP-q PCR,and revealed that the recruitment of Dna2/Sgs1 and Exo1 at the DSB end in Y2 F cells are decreased significantly,suggesting that the bromodomain of Bdf1 may affect end resection.End resection defect can affect the activation of Mec1-dependent DNA damage checkpoint,so we examined the phosphorylation of Rad53 in the bdf1-2YF,and found that the phosphorylation of Rad53 in the bdf1-2YF was significantly delayed compared with the WT strains.We also counted the proportion of the cells in G2/M phase after induced DSBs for 4h,and found that the proportion of WT cells in G2/M phase was about 90%,while the proportion of the mutant cells in G2/M phase was 60%,which was significantly lower than that of WT cells.These results indicate that the bromodomain of Bdf1 can promote the activation of DNA damage checkpoint.Further,we detected the phosphorylation level of histone H2 A in the bdf1-2YF,which is upstream of the checkpoint,was also decreased,indicating that the bromodomain of Bdf1 may promote the activation of DNA damage checkpoint mediated by ?-H2 A.4)We tested the recruitment of RPA,Rad51 and y Ku70 at DSB ends.We found that the recruitment of these proteins in bdf1-2YF mutant was significantly reduced,indicating that the bromodomain of Bdf1 could promote the recruitment of these proteins.More importantly,overexpression of Rad51 can increase HR repair efficiency of the bdf1-2YF from 55% to 78%,completely rescue the HR repair defects,suggesting that the recruitment defect of Rad51 was the eventual reason for the defective HR repair in the mutant.Thus,we conclude that the bromodomain of Bdf1 can promote HR repair by regulating the recruitment of Rad51.