Studies on functions of mammalian RAD23 homologs that link DNA repair and protein degradation systems

为了检查DNA损伤后特异性诱导的XPC蛋白泛素化的可能性，在XP 4PASV细胞中同时过表达FLAG标记的XPC（FLAG-XPC）和HA标记的泛素，所述XP 4PASV细胞来源于XP C组患者并且不表达内源性XPC蛋白。通过从细胞提取物中免疫沉淀FLAG-XPC，证明XPC确实可以在体内被泛素化。从使用各种截短的XPC蛋白的类似实验中，发现XPC中的结构域相对更容易被泛素化。为了研究XPC泛素化的可能作用，我们建立了稳定表达生理量的FLAG-XPC的细胞系。当细胞用DNA损伤剂如UV和4-NQO处理时，一部分XPC与染色质结合得更紧密，而大多数泛素化的XPC在相同条件下可以溶解。为了探索与XPC相互作用的新因子，我们进行了酵母双杂交筛选，得到了泛素样蛋白SUMO-1和SUMO-1结合酶Ubc 9。我们证明，通过同时过表达FLAG-XPC和HA-SUMO-1，XPC也可以在细胞中被SUMO化。使用同时缺乏HR 23 A和HR 23 B的小鼠胚胎成纤维细胞，我们建立了稳定表达各种突变HR 23 B蛋白的细胞系。结果表明，HR 23 B的N-末端泛素样结构域对于XPC的细胞内稳定性和从整体基因组中去除UV损伤都是必不可少的。

To examine the possibility of ubiquitylation of the XPC protein that is specifically induced upon DNA damage, FLAG-tagged XPC (FLAG-XPC) and HA-tagged ubiquitin were simultaneously overexpressed in XP4PASV cells, which are derived from a XP group C patient and do not express endogenous XPC protein. By immunoprecipitation of FLAG-XPC from the cell extract, it was demonstrated that XPC can be indeed ubiquitylated in vivo. From similar experiments using various truncated XPC proteins, a domain in XPC was found, which is relatively more susceptible to the ubiquitylation. To investigate possible roles of the XPC ubiquitylation, we established a cell line that stably expresses a physiological amount of FLAG-XPC. When the cells were treated with DNA damaging agents such as UV and 4-NQO, a fraction of XPC became more tightly bound to chromatin, while the majority of ubiquitylated XPC could be solubilized under the same conditions. Moreover, we found that the XPC ubiquitylation occurred in a cell-cycle-independent manner, at least from the Gl/S boundary to the M phase.To explore novel factors that interact with XPC, the yeast two-hybrid screening was carried out, which resulted in the ubiquitin-like protein SUM0-1 as well as the SUMO-1 conjugating enzyme Ubc9.As done for ubiquitylation, we demonstrated that XPC can be also sumoylated in cells by simultaneous overexpression of FLAG-XPC and HA-SUMO-1.Using mouse embryonic fibroblast cells that lack both HR23A and HR23B, we established cell lines that stably express various mutant HR23B proteins. It was shown that the N-terminal ubiquitin-like domain of HR23B is essential neither for intracellular stabilization of XPC nor removal of UV-damage from the global genome.