Reconstitution of human excision nuclease with recombinant XPF-ERCC1 complex

Abstract

The human XPF-ERCC1 protein complex is one of several factors known to be required for general nucleotide excision repair. Genetic data indicate that both proteins of this complex are necessary for the repair of interstrand cross-links, perhaps via recombination. To determine whether XPF-ERCC1 completes a set of six proteins that are sufficient to carry out excision repair, the human XPF and ERCC1 cDNAs were coexpressed in Sf21 insect cells from a baculovirus vector. The purified complex contained the anticipated 5' junction-specific endonuclease activity that is stimulated through a direct interaction between XPF and replication protein A (RPA). The recombinant complex also complemented extracts of XP-F cells and Chinese hamster ovary mutants assigned to complementation groups 1, 4, and 11. Furthermore, reconstitution of the human excision nuclease was observed with a mixture of five repair factors (XPA, XPC, XPG, TFIIH, and RPA) and the recombinant XPF-ERCC1, thus verifying that no additional protein factors are needed for the specific dual incisions characteristic of human excision repair.