PHENOTYPIC HETEROGENEITY IN NUCLEOTIDE EXCISION-REPAIR MUTANTS OF RODENT COMPLEMENTATION GROUP-1 AND GROUP-4

Rodent ultraviolet light (UV)-sensitive mutant cells in complementatio n groups (CGs) 1 and 4 normally are known for their extraordinary (sim ilar to 80-100 x) sensitivity to mitomycin C (MMC), although some CG1 mutants with reduced MMC sensitivity were previously reported (Stefani ni et al. (1987) Cytotechnology 1, 91). We report here new CG1 and CG4 mutants with only 1.6-10 x wild-type MMC sensitivity despite low unsc heduled DNA synthesis (UDS) levels, Mutant UV140, in UV CG4, has simil ar to 3.8 x the UV sensitivity of parental line AA8, similar to 1.6 x wild-type MMC sensitivity, wild-type X-ray and ethyl methanesulfonate (EMS) sensitivity, and is only slightly (similar to 1.4 x)hypermutable to 8-azaadenine resistance by UV light. It has moderately decreased i ncision of UV-damaged DNA, has moderately decreased removal of (6-4) p hotoproducts, and is profoundly deficient in UDS after UV. After UV, i t shows abnormally decreased DNA synthesis and persistently decreased RNA synthesis. In addition a cell-free extract of this mutant displays strongly reduced nucleotide excision repair synthesis using DNA treat ed with N-acetoxy-acetyl-amino-fluorene (AAF). The extract selectively fails to complement extracts of group 1 and 4 mutants consistent with the notion that the affected proteins, ERCC1 and ERCC4, are part of t he same complex and that mutations in one subunit also affect the othe r component. Mutant UV212 is a CG1 mutant with similar to 3.3 x wild-t ype UV and similar to 5-10 x wild-type MMC sensitivity, with profoundl y deficient UDS and hypermutability (similar to 5.8 x) by UV. Mutant U V201, probably in CG1, is only slightly (similar to 1.5 x) UV-sensitiv e and has near wild-type (1.02 x) UV mutability. These unusual group 1 and 4 mutants demonstrate that the unique UV and MMC sensitivity phen otypes displayed by these groups can be separated and support the idea that they are the result of distinct repair functions of the correspo nding ERCC1 and ERCC4 genes: nucleotide excision repair for UV lesions and a separate repair pathway for removal of interstrand crosslinks.