The Role Of Bdf1 Bromodomain In Yeast DNA Replication

Eukaryotes have evolved a highly sophisticated mechanism to ensure the fidelity of DNA replication.Histone acetylation plays critical roles in promoting DNA replication,but the underlying mechanism remains poorly unerstood.Acetyl-lysine is specifically recognized by a group of proteins harboring bromodomains(BRDs).Among BRD proteins,the BET(bromodomain and extraterminal domain)family members typically possess two tandem bromodomains and an extra terminal domain,and are associated with the formation and development of cancers.In this study,we investigated the function of Bdf1,a yeast BET family protein,in DNA replication.The BRDs of Bdf1 selectively recognize and bind acetylated histones H3 and H4,and the residues Y187 and Y354 are essential to maintain the BRD structure or to fulfil its physiological function.Here,we studied the role of BRD in DNA replication by using the point mutant in which both Y187 and Y354 were simultaneously mutated to phenylalanine(bdf1-Y2F).The following results were obtained:1)First,by using flow cytometry,we found that bdf1-Y2 F mutant displays significant delay in progressing through the S phase as compared to the wild-type(WT)cells.Next,we performed Brd U Ch IP and noted that Brd U incorporation in the genome is significantly reduced in bdf1-Y2 F mutant,indicating a defect in DNA synthesis.These results indicate that the BRD of Bdf1 plays an important role in promoting DNA replication.2)Previous studies showed that histone acetylation is increased at the replication origin.We examined the recruitment of Bdf1 at replication origins by Ch IP,and found that Bdf1 is highly enriched at active replication origins,but the enrichment for the mutant protein bdf1-Y2 F is dramatically decreased.Thus,Bdf1 is recruited to replication origins in a manner dependent on its BRD motifs.3)RPA is a single-stranded DNA specific binding protein complex that plays essential roles in the initiation and elongation of DNA replication.By using Coimmunoprecipitation assay,we found that Bdf1 physically interacts with RPA.Ch IPq PCR anaysis revealed that in the wild type cells,RPA is highly enriched at replication origions,while its enrichment is significantly impaired and delayed in bdf1-Y2 F mutant cells,indicating a role of Bdf1 BRD in facilitating RPA recruitment at replication origins.Indeed,over-expression of RPA partially rescue the defects of bdf1-Y2 F cells in progressing through the S phase.It suggests that the replication defect in bdf1-Y2 F cells is partly due to the impaired RPA recruitment.4)Next,we tested whether the recruitment of replication protein is affected in bdf1-Y2 F cells.The Ch IP results showed that recruitment of the replication origin recognition complex ORC is normal in bdf1-Y2 F mutant cells,while the recruitment of the MCM helicase complex and Pol 2 is significantly impaired in the mutant cells,indicating that the BRD of Bdf1 plays a role in promoting recruitment of the MCM complex during G1 phase.5)Finally,we assessed whether the BRD of Bdf1 is required for response to replication stresses.We noted that bdf1-Y2 F cells are highly sensitive to the DNA damaging agents inducing replication stress.In addition,the mutant also showed delayed cell cycle progression and checkpoint activation upon replication stress,indicating that the BRD of Bdf1 is important for response to replication stresses.Our results establish a critical role of BRD motifs in DNA replication,and provide insights into how histone acetylation regulates DNA replication.