Defective DMA Cross-Link Removal in Chinese Hamster Cell Mutants Hypersensitive to Bifunctional Alkylating Agents1

DNA repair-deficient mutants from five genetic complementa tion groups isolated previously from Chinese hamster cells were assayed for survival after exposure to the bifunctional alkylating agents mitomycin C or diepoxybutane. Groups 1, 3, and 5 exhibited 1.6to 3-fold hypersensitivity compared to the wildtype cells, whereas Groups 2 and 4 exhibited extraordinary hypersensitivity (30to 90-fold). Mutants from Groups 1 and 2 were exposed to 22 other bifunctional alkylating agents in a rapid assay that compared cytotoxicity of the mutants to the wild-type parental strain, AA8. With all but two of the compounds, the Group 2 mutant (UV4) was 15to 60-fold more sensitive than AA8 or the Group 1 mutant (UV5). UV4 showed only 6-fold hypersensitivity to quinacrine mustard. Alkaline elution measure ments showed that this compound produced few DNA interstrand cross-links but numerous strand breaks that were re vealed by proteinase treatment. Therefore, the extreme hyper sensitivity of mutants from Groups 2 and 4 appeared specific for compounds the main cytotoxic lesions of which were DNA cross links. Mutant UV5 was only 1to 4-fold hypersensitive to all the compounds. Repair kinetics of DNA interstrand cross-link pro duction and removal was measured by alkaline elution for AA8 and mutants UV4 and UV5 after exposure to diepoxybutane. Although the initial number of cross-links was similar for the three cell lines, during 24-h incubation, the efficiency of removal of cross-links was lowest in UV4 and intermediate in UV5. These results suggest that the different levels of sensitivity of the five complementation groups to bifunctional alkylation damage are specifically related to different efficiencies of DNA cross-link removal. The phenotype of hypersensitivity to both UV radiation and cross-link damage exhibited by the mutants in Groups 2 and 4 appears to differ from those of the known human DNA repair syndromes.