HUMAN ERCC5 CDNA-COSMID COMPLEMENTATION FOR EXCISION-REPAIR AND BIPARTITE AMINO-ACID DOMAINS CONSERVED WITH RAD PROTEINS OF SACCHAROMYCES-CEREVISIAE AND SCHIZOSACCHAROMYCES-POMBE

Several human genes related to DNA excision repair (ER) have been isol ated via ER cross-species complementation (ERCC) of UV-sensitive CHO c ells. We have now isolated and characterized cDNAs for the human ERCC5 gene that complement CHO UV135 cells. The ERCC5 mRNA size is about 4. 6 kb. Our available cDNA clones are partial length, and no single clon e was active for UV135 complementation. When cDNAs were mixed pairwise with a cosmid clone containing an overlapping 5'-end segment of the E RCC5 gene, DNA transfer produced UV-resistant colonies with 60 to 95% correction of UV resistance relative to either a genomic ERCC5 DNA tra nsformant or the CHO AA8 progenitor cells. cDNA-cosmid transformants r egained intermediate levels (20 to 45%) of ER-dependent reactivation o f a UV-damaged pSVCATgpt reporter plasmid. Our evidence strongly impli cates an in situ recombination mechanism in cDNA-cosmid complementatio n for ER. The complete deduced amino acid sequence of ERCC5 was recons tructed from several cDNA clones encoding a predicted protein of 1,186 amino acids. The ERCC5 protein has extensive sequence similarities, i n bipartite domains A and B, to products of RAD repair genes of two ye asts, Saccharomyces cerevisiae RAD2 and Schizosaccharomyces pombe rad1 3. Sequence, structural, and functional data taken together indicate t hat ERCC5 and its relatives are probable functional homologs. A second locus represented by S. cerevisiae YKL510 and S. pombe rad2 genes is structurally distinct from the ERCC5 locus but retains vestigial A and B domain similarities. Our analyses suggest that ERCC5 is a nuclear-l ocalized protein with one or more highly conserved helix-loop-helix se gments within domains A and B.