【作者】 王超；

【导师】 洪泽辉；

【作者基本信息】 东南大学 ， 医学遗传学， 2022， 硕士

【摘要】 目的:p53蛋白作为重要的肿瘤抑制因子,在多种细胞应答中发挥着关键作用。细胞通过多种DNA修复途径修复由内源或外源性因素造成的DNA损伤,其中许多修复机制与p53相关。p53能够调控核苷酸切除修复中编码损伤识别蛋白DDB2、XPC的基因表达;在碱基切除修复中,p53能够调控DNA糖基化酶OGG1、MUTYH的表达;p53还能直接与Rad51、Rad54蛋白质相互作用参与同源重组修复。课题组前期观察到XRCC1在UV损伤位点的聚集受到PARP1和p53的共同调控,PARP1通过PARylation募集XRCC1至损伤位点,但p53调控XRCC1聚集的具体分子机制尚不明确。在本课题研究中,我们继续围绕p53如何调控XRCC1聚集至UV损伤位点来进一步挖掘p53在UV损伤应答中的分子机制。方法:1.构建稳定表达FLAG-XRCC1融合蛋白的U2OS细胞,进行免疫共沉淀实验,检测UV照射、PARP1抑制条件下细胞内p53与XRCC1蛋白质相互作用是否发生变化;2.对细胞进行亚细胞组分分级处理,考察不同条件下细胞内XRCC1在染色质处富集动态变化;3.蛋白质免疫印迹检测细胞中PARylation信号变化,研究p53对PARP1调控途径的影响。结果:1.p53与XRCC1存在蛋白质相互作用,UV照射后未发现相互作用改变;加入PARP1抑制剂ABT-888后发现p53与XRCC1相互作用增强;2.XRCC1在UV损伤位点富集随UV照射剂量和孵育时间增加而逐渐增强;3.加入PARP1抑制剂后,XRCC1更快地聚集到UV损伤位点;4.p53蛋白敲除后,XRCC1更快地聚集到UV损伤位点;5.PARP1和p53通路同时抑制后,XRCC1不能聚集到UV损伤位点;6.UV照射后,细胞内存在两次PARylation波段(0.5h和2h),且UV照射2h后,XRCC1的PARylation增加;7.p53敲除后,细胞内第二次PARylation波段消失,第一次波段增强。结论:1.p53通过与XRCC1蛋白相互作用募集XRCC1到UV损伤位点;UV照射不影响p53与XRCC1蛋白相互作用;但抑制PARP1活性后p53与XRCC1相互作用增强;2.XRCC1在UV损伤位点富集随UV辐照剂量和孵育时间增加而逐渐累积;3.PARP1与p53调控途径之间为相互制衡,相互竞争关系;4.UV损伤应答中第二次PAR信号的靶蛋白为XRCC1。更多还原

【Abstract】 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.更多还原