Numerous DNA repair pathways are required for genomic stability and chromosome maintenance. XPF/ERCC1 protein complex is an evolutionally conserved structure-specific endonuclease involved in nucleotide excision repair (NER) and other DNA repair pathways, making it essential for viability. The pleiotropic phenotypes of XPF/ERCC1 deficiency in humans range from cancer-predisposition to accelerated aging and severe developmental abnormalities. The research outlined here combines biochemical and structural approaches to study XPF/ERCC1 incision 5′ to the lesion during NER. The proposal is guided by the premise that XPA and RPA proteins, two other NER core factors, stimulate XPF/ERCC1 activity. The primary aim is to structurally characterize the multiprotein-DNA assembly in order to elucidate the DNA binding and catalytic activity of XPF/ERCC1. To achieve this we will address how individual DNA binding domains of XPF/ERCC1 contribute to DNA cleavage, how XPA recruits XPF/ERCC1 to sites of NER, and how RPA in conjunction with XPA pre- organize the DNA structure at ds/ss junctions. The public health importance of this research comes from the increasing interest in the regulation of XPF/ERCC1 activity, due to its involvement in DNA repair pathways implicated in the resistance of tumors to chemotherapy. Cancer associated deaths are the number one cause of mortality worldwide and resistance to current chemotherapeutics remains a major clinical hurdle. Sensitizing cancer cells to treatment with DNA damaging chemotherapeutics holds great value. One way to achieve this result is the development of small drug-like molecules targeting DNA binding sites of NER proteins to disrupt the formation of a functional NER complex and decrease its capacity.