Repair mechanism of UV-induced DNA damage and roles of ubiquitination

用UVDDB-泛素软管(E3)络合物在紫外光照射的细胞中诱导XPC和DDB2泛素化。当DDB2泛素化后，UVDDB失去了与紫外线损伤的DNA的强结合活性，而泛素化的XPC仍然保持了它的DNA结合活性。此外，只有在UV-DDB存在的情况下，体外核苷酸切除修复(NER)反应似乎才需要延长多泛素链。基于这些结果，我们提出了一个关于UVDDB和泛素化在NER损伤识别过程中的可能作用的模型。利用稳定转化的表达GFP标记的XPC的细胞系，采用FRAP(Fumesmnce Recovly After Photobleding)技术研究了XPC的体内动力学。紫外光照射细胞后，我们观察到GFPXPC的固定化，表现出双相紫外光剂量依赖关系。SiRNA的敲除和DDB2DNA的过表达表明，在较低剂量(5-10J/m^2)的UV照射下观察到的第一步固定化依赖于UV-DDB。我们的结果表明，当只诱导少量UV光刻时，UV-DDB介导的损伤识别途径可能占主导地位。为了研究紫外线诱导的可逆泛素化XPC的生物学意义，我们筛选了参与XPC去泛素化的酶的siRNA文库。已经获得了一种候选酶，目前正在进行研究。此外，胸腺嘧啶DNA糖基酶(TDG)和XPC都是SUMO化的。糖基化TDG的X-射线晶体结构揭示了糖基化对TDG功能调节的结构基础。我们还确定了XPC上的SUMO化位点，这些位点的突变在体内导致了部分NER缺陷。

Biochemical analyses were carried out on ubiquitylation of XPC and DDB2 by the UVDDB-ubiquitin hose (E3) complex induced in UV-irradiated cells. Upon ubiquitylation of DDB2, UVDDB lost its strong binding activity to UV-damaged DNA, whereas ubiquitylated XPC still retained its DNA binding activity. Furthermore, elongation of polyubiquitin chains seemed to be required for in vitro nucleotide excision repair (NER) reactions only when UV-DDB was present. Based on these results, we proposed a model concerning possible roles of UVDDB and ubiquitylation in the damage recognition process in NER.In vivo dynamics of XPC were examined by the FRAP (flumesmnce recovely after photobleaching) technique using a stably transformed cell line expressing GFP-tagged XPC. We observed immobilization of GFPXPC after UV-irradiation of cells, which showed a biphasic UV-dose dependency. SiRNA knockdown and overexpression of DDB2 indicated that the first step immobilization observed after relatively low doses (5-10 J/m^2) of UV depended on UV-DDB. Our results suggest that the UV-DDB-mediated damage recognition pathway may be predominant when only a small number of UV photolesions are induced.To investigate biological meanings of the reversible ubiquitylation XPC induced by UV-irradiation, we screened siRNA libraries for enzymes involved in deubiquitylation of XPC. A candidate enzyme has been obtained, which is now under investigation. In addition, both thymine DNA glycosylase (TDG) and XPC have been known to be SUMOylated. Structural basis of the functional modulation of TDG by SUMOylation could be revealed by the X-ray crystal structures of SUMOylated TDG. We also determined SUMOylation sites in XPC and mutations at these sites caused partial NER defects in vivo.