MOLECULAR-CLONING OF THE HUMAN NUCLEOTIDE-EXCISION-REPAIR GENE ERCC4

ERCC4 was previously identified in somatic cell hybrids as a human gen e that corrects the nucleotide-excision-repair deficiency in mutant ha mster cells. The cloning strategy for ERCC4 involved transfection of t he repair-deficient hamster cell line UV41 with a human sCos-1 cosmid library derived from chromosome 16. Enhanced UV resistance was seen wi th one cosmid-library transformant and two secondary transformants of UV41. Cosmid clones carrying a functional ERCC4 gene were isolated fro m a library of a secondary transformant by selecting in Escherichia co li for expression of a linked neomycin-resistance gene that was presen t in the sCos-1 vector. The cosmids mapped to 16p13.13-p13.2, the loca tion assigned to ERCC4 by using somatic cell hybrids. Upon transfectio n into UV41, six cosmid clones gave partial correction ranging from 30 % to 64%, although all appeared to contain the complete gene. The capa city for in vitro excision of thymine diners from a plasmid by transfo rmant cell extracts correlated qualitatively with enhanced UV resistan ce.