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.