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

为了研究在DNA损伤时特异性诱导的XPC蛋白泛素化的可能性，在XP4PASV细胞中同时过表达FLAG标记的XPC(FLAG-XPC)和HA标记的泛素，XP4PASV细胞来自XP C组患者，不表达内源性XPC蛋白。通过从细胞提取液中免疫沉淀FLAG-XPC，证实了XPC在体内确实可以泛素化。从使用不同截短的XPC蛋白的类似实验中，我们发现了XPC中的一个结构域，它相对更容易泛素化。为了研究XPC泛素化的可能作用，我们建立了一个稳定表达生理性数量的FLAG-XPC的细胞系。当用紫外线和4-NQO等DNA损伤剂处理细胞时，一小部分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。此外，我们发现XPC泛素化发生在细胞周期无关的方式，至少从Gl/S边界到M期。为了探索与XPC相互作用的新因素，我们进行了酵母双杂交筛选，得到了泛素样蛋白SUM0-1和SUMO-1结合酶Ubc9。在泛素化方面，我们证明了XPC在细胞中也可以通过同时过表达FLAG-XPC和HA-SUMO-1来实现。结果表明，HR23B的N末端泛素样结构域既不是XPC细胞内稳定所必需的，也不是从全球基因组中去除紫外线损伤所必需的。

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.