The Bromodomain Proteins Bdf1 Promotes End Resection And Checkpoint Activation In Response To DNA Double-strand Break

The genome of cells suffers from endogenous and extraneous genotoxic insults.One of the most dangerous DNA lesions is the DNA double-strand breaks(DSBs).In order to maintain genomic integrity and stability,cells have employed an elaborate DNA damage response system to repair DSBs.DSBs-induced DNA damage response system includes the DNA damage checkpoint signaling pathway and DSB repair pathway.DNA damage checkpoint can sense and amplify the DNA damage signal so as to regulate the cell cycle to cooperate with repair.The DSB repair pathway includes non-homologous end joining(NHEJ)and homologous recombination(HR).HR is regulated by the cell cycle and the resection of DNA ends.HR is a high-fidelity repair mechanism that requires the processing of DSB ends to yield 3'-single strand DNA(ssDNA)and the invasion of ssDNA into homologous template to direct the DNA repair.Bdfl is one of the bromodomain and extraterminal protein(BET)family members.It contains two tandem bromodomains and an additional C-terminus.The two bromodomains can specifically recognize and preferentially bind to acetylated N-terminal tail of the histone H3 and H4.Bdfl is involved in the transcription initiation mediated by TFIID complex and the formation of chromatin remodeling complex SWR1.Bdfl has been reported to participate in the maintenance of genome stability,but the detailed mechanism remains to be explored.To investigate the function of Bdfl in DNAdamage response upon DSBs,we examined the effect of Bdfl on DSB end resection and the activation of the DNA damage checkpoint.The results are presented in the following:1)By using Southern Blot assay,we demonstrated that Bdfl promotes the DSB end resection.Further analysis revealed that Bdfl promotes both Exo1 and Sgs1-Dna2 resection pathways,and the Exol pathway appears to be more dependent on Bdf1.2)Bdf1 also promotes the DNA damage checkpoint activation.By using Western Blot,we monitored the effect of Bdf1 on Rad53 phosphorylation following DSB induction.The results revealed that Bdf1 is essential for DSB-induced Rad53 phosphorylation.Further studies revealed that Bdf1 is critical for maintaining the protein level of Rad9.Moreover,Bdfl is important for the expansion of ?-H2A along chromatin after DSBs although H2A can be efficiently produced in the absence of Bdfl.These results suggest that Bdfl promote the amplification and transduction of checkpoint signal.3)Since Bdf1 plays a role in both DSB ends resection and checkpoint activation,we reasoned that Bdf1 may affect the loading or expression of Mrell-Rad50-Xrs2(MRX complex),an initial protein complex recognizing DSBs.Indeed,our analysis showed that Bdfl is essential for the maintenance of MRX protein level,and overexpression of MRX complex in bdf1 mutant partially restored end resection and DNA damage checkpoint activation.4)Further,we analyzed the role of different domains of Bdf1 protein in resection and checkpoint activation.The results indicated that the bromodomains play an importantrole in promoting DNA end resection,while the C-terminus is not required for resection.However,both the bromodomains and C-terminus are needed to promote DNA damage checkpoint activation.5)By mass spectrometry analysis,we identified the proteins Arp4,Ino80,Swc4 and snf5 as Bdf1 interactors.Furthermore,the interaction between Bdfl and Arp4 or Rdh54 was confirmed by Co-immunoprecipitation assay.However,Bdf1 does not interact with RPA and Rad52.6)Overexpressing of BDF2 or ESA1,which encodes the catalytic subunit of NuA4 complex in bdfl mutant significantly improved cell resistance to the DNA-damaging agents,but failed to restore checkpoint activation.These results suggest Bdf1 is partially redundant with Bdf2 in DNA damage response,and that Bdf1 may function through acetylation of histone H4.Altogether,these results demonstrated that Bdf1 affects DSB ends resection and checkpoint activation partially through influencing MRX protein level.Both the bromodomains and the C-terminus of Bdf1 are important for promoting checkpoint activation.However,only the bromodomains are needed for DSB end resection.Bdf1 appears to promote resection in a manner independent of its roles in transcription initiation.Given that Bdf1 can recognize acetylated histone H3 and H4,and that overexpression of ESA1 can improve the drug resistance of bdf1 mutant,it is likely that the functions of Bdf1 are at least partially related to histone acetylation.