Studies on novel molecular mechanisms that regulate repair of genomic DNA damage caused by environmental stresses

本研究旨在阐明哺乳动物核苷酸切除修复（NER）时空调控的新分子机制，重点研究损伤识别蛋白XPC和DDB2的翻译后修饰（泛素化和sumo化）。对于紫外光诱导的XPC可逆泛素化，研究发现，敲低鉴定的去泛素化因子会导致泛素化XPC的积累，从而显著影响XPC对受损DNA位点的招募。我们还成功地重建了XPC的体外summoylation并鉴定了修饰位点。稳定表达非SUMO化突变体XPC的细胞在紫外线照射后表现出NER阻滞和XPC泛素化诱导受损，这可能是因为XPC和DDB2之间的功能相互作用可能是由SUMO介导的。最后，我们确定了通过CRL4^<DDB2>泛素连接酶实现DDB2自身泛素化的靶位点，这表明DDB2泛素化可能具有不同的作用，取决于修饰位点的不同，包括蛋白质降解和受损DNA的解离。

This study has been aimed to elucidate novel molecular mechanisms involved in spatio-temporal regulation of mammalian nucleotide excision repair(NER), focusing on post-translational modifications(ubiquitylation and sumoylation) of the damage recognition proteins, XPC and DDB2. Concerning the UV-induced reversible ubiquitylation of XPC, knockdown of the identified deubiquitylation factor was found to result in accumulation of ubiquitylated XPC, which significantly compromised recruitment of XPC to damaged DNA sites. We also succeeded in reconstitution of in vitro sumoylation of XPC and identification of the modification sites. The cells stably expressing the non-sumoylated mutant XPC exhibited retardation of NER and impaired induction of XPC ubiquitylation after UV irradiation, possibly because the functional interaction between XPC and DDB2 may be mediated by SUMO. Finally, target sites for DDB2 self-ubiquitylation by the CRL4^<DDB2> ubiquitin ligase were identified, suggesting the possibility that DDB2 ubiquitylation may have distinct roles, protein degradation and dissociation from damaged DNA, depending on the modification sites.