The roles of XPF/ERCC1 complex in DNA repair

描述(申请人提供)：由XPF和ERCC1蛋白组成的异二聚体蛋白复合体是一种进化上保守的DNA结构特异性内切酶。XPF/ERCC1复合体在核苷酸切除修复(NER)、DNA链间交联损伤(XL)修复和靶向同源重组中发挥作用。在这个提案中，我们将确定XPF/ERCC1复合体在哺乳动物细胞中NER和XL修复中的作用。 在NER过程中，DNA损伤处有两个DNA切口，分别为5‘和3’。NER因子XPA、RPA、XPC/HHR23B、TFIIH(包含XPB和XPD)和XPG可能为XPF/ERCC1复合体产生特定的解卷结构，以进行5‘切开。XPA和RPA被认为通过特定的蛋白质-蛋白质相互作用将XPF/ERCC1复合体带入病变。由于XPA和RPA是主要的DNA损伤识别因子，我们使用重组的NER系统测试了XPF/ERCC1复合体是否在NER的初始阶段发挥作用。有趣的是，XPA、RPA和XPF/ERCC1复合体与底物DNA预先孵育后，修复反应得到显著刺激。在这项提议中，我们将研究XPF/ERCC1复合体对NER的这种刺激作用的机制基础，特别是在DNA损伤识别步骤中。 ERCC1和ERCC4基因缺失的中国仓鼠卵巢细胞系(CHO)对DNA交联剂高度敏感，而ERCC-2(XP-B)等NER突变细胞对DNA交联剂仅轻度敏感。这些遗传数据表明，XPF/ERCC1复合体在XL修复中具有独特的功能，独立于其他NER因子。遗传数据还表明XRCC2和XRCC3参与了XL修复。XRCC2和XRCC3参与同源重组和DNA双链断裂修复。为了研究XL修复在人类细胞中的作用机制，我们建立了一种无细胞的XL修复系统，该系统特异性地依赖于XPF、ERCC1和XRCC3来去除XL。我们将用新开发的XL修复系统来确定XPF/ERCC1复合体和XRCC3的作用。这些研究将有助于理解哺乳动物细胞中XL修复的分子机制，也将为最终用纯化的蛋白质重建XL修复过程提供基础。

DESCRIPTION (provided by applicant): A heterodimer protein complex consisting of the XPF and ERCC1 protein is an evolutionally conserved DNA structure specific endonuclease. The XPF/ERCC1 complex plays roles in nucleotide excision repair (NER), DNA interstrand crosslink damage (XL) repair and targeted homologous recombination. In this proposal, we will define the roles of the XPF/ERCC1 complex in NER and XL repair in mammalian cells. Two DNA incisions, 5' and 3' to the DNA lesion, occur during NER. The NER factors, XPA, RPA, XPC/HHR23B, TFIIH (contains XPB and XPD) and XPG are supposed to generate a specific unwound structure for the XPF/ERCC1 complex to make the 5' incision. XPA and RPA are thought to bring the XPF/ERCC1 complex to the lesion by specific protein-protein interactions. Because XPA and RPA are primary DNA damage recognition factors, we have tested whether the XPF/ERCC1 complex functions in the initial stage of NER using a reconstituted NER system. Interestingly, the pre-incubation of the XPA, RPA and XPF/ERCC1 complex with the substrate DNA resulted in a marked stimulation of the repair reaction. We will study the mechanistic basis of this stimulatory effect of the XPF/ERCC1 complex on NER in this proposal, specifically, in DNA damage recognition step. Chinese hamster ovary cell lines (CHO) defective in ERCC1 and ERCC4 (XPF) are extremely sensitive to DNA crosslinking agents, while the other NER mutant cells such as ERCC-2 (XP-B) are only mildly sensitive to the same agents. These genetic data indicate that the XPF/ERCC1 complex has a distinct function in XL repair, independent of the other NER factors. The genetic data also indicate the involvement of XRCC2 and XRCC3 in XL repair. XRCC2 and XRCC3 are involved in homologous recombination and DNA double strand break (DSB) repair by recombination. To study the mechanism of XL repair in human cells, we have developed a cell free XL repair system that is specifically dependent on XPF, ERCC1 and XRCC3 for XL removal. We will define the role of XPF/ERCC1 complex and XRCC3 with the newly developed XL repair system. The proposed studies will yield some insights into the understanding of the molecular mechanism of XL repair in mammalian cells, and they will also provide fundamentals to eventually reconstitute the XL repair process with purified proteins.