The Molecular Mechanism Of P53 Regulating XRCC1 In UV Damage Response

Objective: As an important tumor suppressor,p53 plays a key role in many cellular responses.Cells repair DNA damage caused by endogenous or exogenous factors through varieties of DNA repair ways,many of which are correlated to p53.p53 can regulate the gene expression of damage recognition proteins DDB2 and XPC in nucleotide excision repair;In base excision repair,p53 can regulate the expression of DNA glycosylase OGG1 and MUTYH;p53 can also directly interact with Rad51 and Rad54 proteins to participate in the homologous recombination pathway.In the previous work,our research group observed that XRCC1 recruitment at UV damage sites was jointly regulated by PARP1 and p53.PARP1 recruited XRCC1 to the damage sites through PAR modification,but the specific molecular mechanism of p53 regulating XRCC1 recruitment is still unclear.In this research,how p53 regulates the recruitment of XRCC1 to UV damage sites is explored.The results of this paper has provided new clues for the molecular mechanism of p53 in UV damage response.Method: 1.U2OS cells stably expressing FLAG-XRCC1 fusion protein were constructed,and the immunoprecipitation experiment was carried out to detect whether the protein interaction between p53 and XRCC1 changed under UV irradiation and PARP1 inhibition.2.The subcellular classification of cells was carried out to study the dynamic changes of XRCC1 enrichment in chromatin under different conditions;3.Western blot is used to detect the changes of PARylation signal in cells to study the effect of p53 on PARP1 regulatory pathway.Results: 1.p53 protein recruit XRCC1 to UV damage site through protein interaction with XRCC1.The interaction cannot be affected by UV irradiation,but is enhanced when PARP1 activity was inhibited after UV irradiation;2.The enrichment of XRCC1 in UV damaged sites gradually increased with the increase of UV irradiation dose and incubation time.3.XRCC1 gathered to chromatin in advance after UV irradiation after adding PARP1 inhibitor.4.XRCC1 gathered to chromatin in advance after UV irradiation after p53 knock-out.5.XRCC1 was not gathered in chromatin after both PARP1 and p53 pathways were inhibited.6.Two distinct waves of PARylation(0.5h and 2h)after UV irradiation,and the PARylation of XRCC1 increased after UV irradiation.7.The second distinct waves of PARylation disappeared and the first band increased after p53 knockout.Conclusion: 1.The protein interaction exists with p53 and XRCC1,and the interaction remains unchanged after UV irradiation,but enhanced after inhibiting the activity of PARP1.2.The enrichment of XRCC1 in UV damaged sites gradually accumulated with the increase of UV irradiation dose and incubation time.3.The regulatory pathways of PARP1 and p53 are mutually balanced and competitive;4.The target protein of the second PAR signal in response to UV damage is XRCC1.