ISOLATION OF MAMMALIAN-CELL MUTANTS THAT ARE X-RAY SENSITIVE, IMPAIRED IN DNA DOUBLE-STRAND BREAK REPAIR AND DEFECTIVE FOR V(D)J RECOMBINATION

The Chinese hamster lung V79-4 cell line was infected with a Moloney m urine leukemia retrovirus and the infected cells were subsequently scr eened for mutants that were sensitive to X-rays using a toothpicking/9 6-well replica plating technique. Four independent mutants that were s ensitive to X-irradiation (sxi-1 to sxi-4) were isolated from 9000 ret rovirally infected colonies. A pulse-field gel electrophoresis (PFGE) assay demonstrated that all of the sxi mutants were impaired in DNA do uble-strand break (DSB) repair, thus providing a molecular explanation for the observed X-ray sensitivity. Interestingly, additional PFGE ex periments demonstrated that for any given X-ray dose all of the mutant s incurred more DNA DSBs than the parental V79-4 cell line indicating there may be some inherent fragility to sxi chromosomes. Cross-sensiti vity to other DNA-damaging agents including bleomycin, mitomycin C and methyl methanesulfonate indicated that sxi-2 sxi-3 and sxi-4 appear t o be specifically hypersensitive to genotoxic agents that cause DNA DS Bs, whereas sxi-1 appeared to be hypersensitive to multiple types of D NA lesions. Lastly, in preliminary experiments all of the sxi mutants demonstrated an inability to carry out V(D)J recombination, a somatic DNA rearrangement process required for the assembly of lymphoid antige n receptor genes. Thus, the sxi cell lines have interesting phenotypes which should make them valuable tools for unraveling the mechanism(s) of DNA DSB repair and recombination in mammalian cells.