Characterization of the XPF endonuclease complex

The XPF endonuclease complex is an essential component of the Nucleotide Excision Repair pathway, functioning in the removal of damaged DNA. XPF has sequence homology to yeast and drosophila proteins that function in both excision repair and genetic recombination. The complex formed by XPF and ERCC1 makes the incision on the 5' side of the DNA damage; however, the precise contribution of the individual XPF and ERCC1 proteins to this activity is unclear. To address this and related questions concerning recombination. we expressed XPF in E. coil as a fusion protein (trxXPF). Both trxXPF and ERCC1 were overexpressed from the same plasmid in order to isolate the XPFERCC1 complex. In addition, several antibodies targeted to different regions of XPF were produced. When tested against supercoiled DNA, both XPF and the XPF-ERCC1 complex exhibited endonnclease activity that was stimulated by addition of anti-XPF antibodies. Thus, the XPF protein appears to be the catalytic component of the endonuclease complex, and the antibody effect is presumably due to stabilization of the active form of XPF. To study the interaction of the XPF endonuclease complex with other repair proteins, XPF and ERCC1 were co-expressed using a baculovirus vector. This complex displayed 5'-junction specific endonuclease activity which was stimulated in the presence of RPA. Furthermore, excision nuclease activity was reconstituted in a mixture of 5 other repair factors (XPA, XPC, XPG, TFIIH, RPA) that was completed by addition of the XPF-ERCC1 complex.