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

用UVDDB-ubiquitin软管（E3）复合物在紫外线照射细胞中诱导XPC和DDB2泛素化，进行生化分析。在DDB2泛素化后，UVDDB失去了与紫外线损伤DNA的强结合活性，而泛素化后的XPC仍保留了其DNA结合活性。此外，只有当UV-DDB存在时，多泛素链的延伸似乎才需要体外核苷酸切除修复（NER）反应。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。使用稳定转化的表达gfp标记的XPC细胞系，通过FRAP（光漂后恢复流态）技术检测XPC的体内动力学。我们观察到细胞在紫外线照射后GFPXPC的固定化，这种固定化表现为双相剂量依赖性。SiRNA的敲低和DDB2的过表达表明，在相对低剂量（5-10 J/m^2）的紫外线照射下观察到的第一步固定依赖于UV- ddb。我们的研究结果表明，当只诱导少量紫外线光损伤时，UV- ddb介导的损伤识别途径可能占主导地位。为了研究紫外光诱导XPC可逆泛素化的生物学意义，我们筛选了XPC去泛素化相关酶的siRNA文库。已经获得了一种候选酶，目前正在研究中。此外，胸腺嘧啶DNA糖基化酶（TDG）和XPC都被SUMOylated。sumo化TDG的x射线晶体结构揭示了sumo化TDG功能调制的结构基础。我们还确定了XPC中的SUMOylation位点，这些位点的突变导致体内部分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.