The Molecular Mechanism Regulated By OGA In DNA Damage Repair

Posttranslational modifications play key roles in DNA damage repair,particularly in DNA double-strand break(DSB)repair.Following DSBs,a set of posttranslational modifications,such as phosphorylation,ubiquitination and ADP-ribosylation are induced,which regulate many biological events during DNA damage repair.Recently,it has been shown that other modifications,like protein O-GlcNAcylation can be induced by DNA damage.OGlcNAcylation is catalyzed by O-GlcNAc transferase(OGT).Using UDP-GlcNAc as a donor,OGT removes UDP from UDP-GlcNAc and covalently links GlcNAc residues to the side chains of serine or threonine residues in the target proteins via O-linked glycosidic bonds.In response to DNA damage,OGT relocates to DNA lesions and catalyzes O-GlcNAcylation at DNA lesions.Accumulated evidence shows that OGT-mediated O-GlcNAcylation plays an important role in response to DNA damage repair.In addition to OGT,the level of protein O-GlcNAcylation is also regulated by OGlcNAcase(OGA)that hydrolyzes the glycosidic bond and releases GlcNAc from respective proteins.However,it is unclear if the “eraser” O-GlcNAcase(OGA)participates in DNA damage repair.Human OGA(h OGA)is a 916-residue nucleocytoplasmic protein that consists of two unique functional domains including an N-terminal catalytic domain that is responsible for the removal of O-GlcNAc,and a C-terminal pseudo–histone acetyltransferase(HAT)domain(residues 707–916)that possesses sequence homology to HAT but lacks the key residues for the binding of acetyl-coenzyme A(acetyl-Co A).Thus,it may not have acetyltransferase activity.However,the pseudo HAT domain is evolutionarily conserved,indicating that this pseudo HAT domain may play an important role in the deglycosylationassociated functions.Here,we got the following achievement by examined the molecular mechanisms and biological functions of OGA in DNA damage repair.1.OGA mediates deglycosylation during DNA damage responseWe measured the kinetics of O-GlcNAcylation after DNA damage,and found that OGlcNAcylation reached the peak level following IR treatment and gradually reduced to the basal level.We treated cells with OGA inhibitor(Thiamet-G,TMG)to suppress the enzymatic activity of OGA or si RNA to knock down OGA,and found that the peak level of OGlcNAcylation was remarkably prolonged,suggesting that OGA mediates the removal of DNA damage-induced O-GlcNAcylation.Following laser treatment,OGA relocated to DNA lesions.When we pretreated cells with OSMI-1,an O-GlcNAcylation inhibitor,we found that the relocation of OGA was suppressed,suggesting that OGA was recruited by recognizing the substrates modified by O-GlcNAc at the DNA damage site,and participated in the DNA damage repair process by mediating the deglycosylation process of the substrate.2.The HAT domain of OGA regulates deglycosylationSince OGA has two prominent domains,we asked which domain mediated the relocation of OGA to DNA lesions.We generated two truncated OGA mutants by deleting either the Nterminal catalytic domain or the C-terminal HAT domain(C-OGA and N-OGA).Interestingly,only C-OGA but not N-OGA could relocate to DNA lesions,suggesting that the HAT domain of OGA mediates the recruitment.Moreover,when we treated cells with TMG or(Z)-PUGNAC,two different OGA inhibitors,to block the catalytic pocket of OGA,OGA was still able to be recruited to DNA lesions.Taken together,these results suggest that the HAT domain but not the catalytic domain is required for the recruitment.3.The HAT domain plays an important role in DNA damage repairSince OGA is the solo enzyme that hydrolases O-GlcNAcylation following DNA damage,OGA may play a critical role for DNA damage repair.To investigate the function of the catalytic domain and HAT domain of OGA in DNA damage repair,we used si RNA to knock down OGA and reconstituted the cells with full-length OGA,N-OGA or C-OGA.We treated cells with IR to induce DSBs.Then we measured repair kinetics of DSB using comet assays under neutral condition.Loss of OGA significantly suppressed DSB repair.In addition,loss of either the catalytic domain or the HAT domain of OGA,the cells were hypersensitive to IR treatment,suggesting that both the catalytic domain and the HAT domain of OGA played important roles in DNA damage repair.Moreover,we treated the cells with different doses of IR and performed colony formation assays to examine the cell viability.Consistently,cells lacking full-length OGA or the key functional domains were hypersensitive to DNA damaging.Collectively,these results demonstrate that OGA participates in DNA damage repair.In particular,the HAT domain is involved in DNA damage repair.4.The HAT domain of OGA recognizes substratesWe searched for the binding partner(s)of the HAT domain using unbiased affinity purification and mass spectrometry analysis.We found that the HAT domain associated with several NHEJ repair factors such as NONO and the Ku70/80 complex from the unbiased protein affinity purification.Both NONO and the Ku70/80 complex play important roles in the NHEJ pathway for the DSB repair.We examined the role of the interaction between the HAT domain and these NHEJ repair factors.We validated that the HAT domain of OGA(C-OGA)recognized these DNA damage repair factors but not N-OGA.Following IR treatment,we examined and found that the interactions between the HAT domain and these DNA damage repair factors were increased.5.Following DNA damage,OGA mediates the removal of O-GlcNAcylation of the substratesFollowing IR treatment,both NONO and the Ku70/80 complex were O-GlcNAcylated.Time course analyses show that these O-GlcNAcylation events started immediately following IR treatment,reached to the peak levels after DNA damage,and then gradually reduced.Moreover,with TMG treatment to suppress the enzymatic activity of OGA or si RNA treatment to knock down the expression of OGA,these O-GlcNAcylation events were remarkably prolonged,suggesting that OGA mediates the deglycosylation of NONO and the Ku70/80 complex during DNA damage repair.Moreover,the O-GlcNAcylation level was remarkably increased in the HAT domain-associated species,suggesting that the HAT domain recognizes O-GlcNAcylated NONO and the Ku70/80 complex following DNA damage.6.Deglycosylation of NONO regulates its chromatin association and the NHEJ repairFollowing laser microirradiation,both NONO and the Ku70/80 complex quickly relocated to DNA lesions.Using TMG or(Z)-PUGNAC to suppress deglycosylation,the retention of NONO at DNA lesions was remarkably prolonged.As an early DSB response factor,NONO was timely degraded in response to IR treatment,which was important to facilitate the subsequent loading of repair factors to repair DNA lesions in the next stage.With the treatment of TMG,we found that NONO degradation was suppressed,suggesting that blocking the removal of O-GlcNAcylation of NONO impaired the degradation of NONO.We performed the GFP reporter assays and found the suppression of OGA-mediated deglycosylation by either TMG or(Z)-PUGNAC impaired NHEJ.Our research shows that OGA relocates to the sites of DNA damage and the pseudo HAT domain of OGA plays a key role in the recruitment of OGA to DNA lesions and mediating the substrates recognition at DNA lesions.We identified DNA damage repair factors,the Ku70/80 complex and NONO as substrates of OGA.Suppression of the enzymatic activity of OGA prolongs O-GlcNAcylation at DNA lesions,which in turn delays NONO degradation at DNA damage sites and impairs NHEJ repair pathway.Taken together,these results suggest that OGAmediated deglycosylation promotes DNA damage repair.