SUMO2/3 Modification Of TIP60 Mediates DNA Damage Repair Through Regulating DNA-PKcs Activity

Background: DNA damage can be induced by radiation therapy and many chemotherapy drugs,which have been widely used in the treatment of tumors.Disruptions in DNA repair pathways,which is a common method for clinical treatment of tumors,predispose tumor cells to accumulating DNA damage after radiotherapy and chemotherapy,thereby induceing programmed cell death of tumor cells.Depth research on the mechanism of DNA damage repair has important implications for guiding us in the clinical practice of individualized tumor treatment.In case of DSBs,Ku quickly senses and binds to the DSB,then recruits DNA-PKcs to the site of the DNA break and activites DNA-PKcs.Activation of DNA-PKcs is critical for effective NHEJ and DSBs repair.As a highly conserved acetyltransferase in eukaryotes,TIP60 promotes activativity of DNA-PKcs through its acetyltransferase activity in DDR.But the mechanism of TIP60 regulating DNA-PKcs activity is far from clear.In this study,we are aimed to explore the mechanisms by which the SUMO2/3modification of TIP60 mediates DNA damage response through regulating DNA-PKcs activity,thereby deepening our understanding of the function of TIP60 and DNA-PKcs in DNA damage response.Through this study,We also expect to provide guideline and find a new target for cancer thearpy.Methods: 1.Myc-TIP60 plasmid was co-transfected with His-SUMO2/3,His-vector into 293 T cells.Cells were lysed 36 hours after transfection,and then N2+binding assay was performed.The samples were detected through Westernbolting by running SDS-page,myc-antibody was used to investigate Myc-TIP60 sumoylation;Co-IP assay was performed to detect the interaction between SAE1/2 and TIP60 by overexpression HA-SAE1/2 and Myc-TIP60 or HA-TIP60 and Myc-SAE1/2 in 293 T cells;Overexpression GFP-TIP60 and RFP-SAE1/2 or RFP-UBC9,CFP-SUMO2/3 in Hela cells,treated cells with 4Gy IR 36 hours after transfection,fluorescence microscope was used to observe the localization of the proteins;2.To confirm the E3 and desumoylation enzyme of TIP60 sumo2/3 modification.We overexpression differnet sumo E3 and desumoylation enzyme along with HA-SUMO2/3 and Myc-TIP60,then N2+ binding assay was performed.The samples were detected through Westernbolting by running SDS-page;The SUMO2/3modification sites of TIP60 were predicted by bioinformatics technology,the different sites mutant myc-TIP60 plasmids were constructed,then detected the sumoylation change of the mutant TIP60 by N2+ binding assay;3.To study the proteins effects TIP60 SUMO2/3 modification,we transfected theip60-interaction protein overexpreesion plasmid along with Myc-TIP60 and His-SUMO2.3 respectively,then N2+ binding assay was performed 36 hours after transfection;Thy and Noc were used to block Hela cells to S phase and G2/M phase to explore whether cell cycle effects TIP60 SUMO2/3 modification;4.Study the interaction domain between TIP60 and DNA-PKcs.According to the NCBI database and published paper,wo designed different TIP60 and DNA-PKcs mutant constructions,then transfected 293 T cells.Co-IP assay and Westernblotiing were conducted to verify their interaction domain.GST-pull down assay was performed to verify their interaction;5.To study whether SUMO2/3 modification of TIP60 regulates the interaction between TIP60 and DNA-PKcs in a cell-cycle dependent way.Cells were collected before and after irradiation,followed by IP TIP60,DNA-PKcs,then ran SDS-page.GST-TIP60 and GST-DNA-PKcs H domain were purified by prokaryotic expression,then GST-pull down assay was performed.TIP60 WT and TIP60 K430 R de-SUMO mutant in-vitro potein were overexpressed in 293 T cells,then Co-IP and Western blotting were conducted to detect their interactions with DNA-PKcs before and after irradiation;6.Hela cells were blocked to different cell cycle phase,then Co-IP assay was conducted to investigate the interaction between DNA-PKcs and TIP60.Subsequently,GST-pull down assay was performed to verify the resule;The effects of TIP60SUMO2/3 modification on the interaction between TIP60 and DNA-PKcs were studied by knockdown and complement of PIAS4 and SENP3,respectively.7.TIP60 WT and TIP60 K430 R Hela cells were treated with IR,the cells were collected at 0 h,1 h,2 h,4 h,8 h,12 h,then IF assay was performed to observe 53BP1 foci,and Western blotting to detect the activity of DNA-PKcs p S2056.HR and NHEJ assay were conducted to study whether TIP60 K430 R effects the DSBs repair pathway choices;8.Colony formation and MTT assay were conducted to detect whether TIP60K430 R mutant effects the sensitivity of Hela cells to irradiation and DNA damage chemotherapy drugs;Flow cytometry was used to detect percentage of apoptosis cells.Results:1.PIAS4 sumoylates TIP60 by using SUMO2/3 at K430 site,and SNEP3 is the desumoylation enzyme of TIP60 SUMO2/3 modification;2.SUMO2/3 modification of TIP60 is mediated by cell cycle and DNA damage.After irradiation,TIP60 is de-sumolated quickly.In addition,PLK1 promotes TIP60 sumoylation in S phase;3.The SUMO2/3 modification of TIP60 blocks its interaction with DNA-PKcs,andregulates DNA-PKcs activity in a cell cycle and DNA damage dependent way;4.TIP60 K430 R de-SUMO mutation induces DNA-PKcs p S2056 increased aberrantly in S phase,thereby promotes NHEJ and inhibits HR pawathway,impairs DNA damage repair(p<0.05).5.TIP60 K430 R mutatation increases cell apoptosis induced by radiation,consequently results in hypersensitivity to irradiation and Olarparib.In addition,mutations at this site increases the sensitivity of tumor cells to DNA-damage chemotherapy drugs(p<0.05).Significance:In this study,we find the new mechanism in which the SUMO2/3modification of TIP60 regulates HR,NHEJ pathway choices and DDR through mediating DNA-PKcs activity.In addition,TIP60 K430 R mutation results in tumor cells hypersensitivity to DNA damage and PARPi,which would be a useful guideline for the precise treatment of clinical tumors and might be used as a new target of PARPi in cancer therapy.