RECONSTITUTION OF HUMAN EXCISION NUCLEASE WITH RECOMBINANT XPF-ERCC1 COMPLEX

The human XPF-ERCC1 protein complex is one of several factors known to be required for general nucleotide excision repair, Genetic data indi cate that both proteins of this complex are necessary for the repair o f interstrand cross-links, perhaps via recombination, To determine whe ther XPF-ERCC1 completes a set of six proteins that are sufficient to carry out excision repair, the human XPF and ERCC1 cDNAs were coexpres sed in SF21 insect cells from a baculovirus vector, The purified compl ex contained the anticipated 5' junction-specific endonuclease activit y that is stimulated through a direct interaction between XPF and repl ication protein A (RPA), The recombinant complex also complemented ext racts of XP-F cells and Chinese hamster ovary mutants assigned to comp lementation groups 1, 4, and 11, Furthermore, reconstitution of the hu man excision nuclease was observed with a mixture of five repair facto rs (XPA, XPC, XPG, TFIIH, and RPA) and the recombinant XPF-ERCC1, thus verifying that no additional protein factors are needed for the speci fic dual incisions characteristic of human excision repair.