The Molecular Mechanism Of P53-mediated XRCC1 Accumulation At UV-induced DNA Damage Sites

Objective: p53 is an important member of tumor suppressor genes,known as ‘genome keeper',acting as a regulatory factor among several cell life activities,such as cell cycle regulation,DNA repair,cell differentiation,vital in maintaining genome stability and normal physiological function of cells.Studies have shown that p53 protein is involved in the regulation of multiple DNA damage repair pathways in maintaining genomic stability.In BER and NHEJ pathways,p53 can affect the expression of DNA pol ? and ku70,ku80 respectively through its transactivition function;in NER,p53 regulates the expression of damage recognition factors,XPC and DDB2;p53 protein can also act in HR pathway via its interaction with Rad51,Rad54.Previous study in the laboratory have shown that the recruitment of XRCC1 at UV-induced DNA damage sites is regulated by PARP1 and p53,however,the specific regulatory mechanism is not clear.Hence the molecular mechanism of p53-mediated accumulation of XRCC1 at damage sites is under research,hoping to better understand the mechanism p53 participates in response to UV-induced DNA damage.Methods: 1.Duplicate test of the influence of PARP1 and p53 protein on XRCC1 recruited to UV-induced damage sites in U2 OS and HCT116 cells;2.Construction of U2 OS cell line stably expressing FLAG-XRCC1(311-537)fusion protein,in which Co IP assay is carried out to elucidate the mechanism of PARP1-regulated XRCC1 recruitment;3.WB is used to detect the effect of p53 on the expression of XRCC1 protein;4.Immunofluorescence staining is used to study the aggregation of p53 after UV injury;5.Research on the molecular mechanism of p53-mediated XRCC1 accumulation via domain analysis and Co IP assay;6.Study on upstream transduction pathway of p53-mediated XRCC1 accumlation through inhibition of upstream signals and immunofluorescence technic;7.Analysis of the relationship between the two regulation approches by examining ?H2AX level after UV radiation via western blotting.Results: 1.U2 OS,P53-KD cells and HC116,HCT116 p53-/-cells are exposed to UV light after treatment of PARP inhibitors,3-AB and ABT-888,after which XRCC1 accumulation at UV-induced DNA damage sites are observed.In U2 OS and HCT116 cells,the accumulation cannot be affected by PARP inhibitors,while in P53-KD and p53-/-cells,the accumulation is inhibited after treatment of PARP inhibitors;2.The interaction between XRCC1(311-537)fragment and PARP1 is proved in U2 OS cells stably expressing FLAG-XRCC1(311-537),with which can be PARylated after UV radiation,and the PARylation can be enhanced by UV;3.p53 in U2 OS and HCT116 cells had no significant effect on the expression of XRCC1 protein after UV injury.The expression of XRCC1 protein is not significantly different in U2 OS,P53 KD,HCT116,p53-/-control group and UV treatment group.4.UV irradiation on U2 OS cells,HCT116 cells shows that p53 protein could aggregate to the damage site in some way.5.Co IP assay is carried out on U2 OS stable cell lines expressing FLAG-XRCC1(1-310)and FLAG-XRCC1(311-537)fusion proteins.With PCNA and PARP1 as positive controls,no direct interaction between p53 and XRCC1 protein fragments 1-310 and 311-537 is found;6.Inhibitors,like BEZ235,Ku-55933,of p53 upstream signals such as ATM,ATR,DNA-PK are used to treat U2 OS cells together with 3-AB,the aggregation of XRCC1 to damage sites has no significantly change;7.WB is used to detect the level of ?H2AX in U2 OS and HCT116 cells at 0,1,4 and 8 hours after UV irradiation,when PARP1 and p53 regulatory pathways are inactivated.Compared with U2 OS group,the amount of ?H2AX in U2OS+3AB,P53-KD and P53-KD+3AB groups increase significantly at 1h,4h and 8h after UV irradiation,but there is no significant difference among the latter three groups.Conclusion: 1.XRCC1 participates in UV-induced DNA damage repair is regulated by PARP1 and p53 protein;2.After UV radiation,the accumulation of XRCC1 to damage sites is regulated by PARP1-mediated PARylation;3.p53 protein can aggregate to damage sites after UV irradiation and may regulate the accumulation of XRCC1 through protein interaction.