Defining the roles of nucleotide excision repair and recombination in the repair of DNA interstrand cross-links in mammalian cells

The mechanisms by which DNA interstrand cross-links (ICLs) are repaired in mammalian cells are unclear. Studies in bacteria and yeasts indicate that b oth nucleotide excision repair (NER) and recombination are required for the ir removal and that double-strand breaks are produced as repair intermediat es in yeast cells. The role of NER and recombination in the repair of ICLs induced by nitrogen mustard (HN2) was investigated using Chinese hamster ov ary mutant cell lines. XPF and ERCC1 mutants (defective in genes required f or NER and some types of recombination) and;XRCC2 and XRCC3 mutants (defect ive in RAD51-related homologous recombination genes) were highly sensitive to HN2. Cell lines defective in other genes involved in NER (XPB, XPD, and XPG), together with a mutant defective in nonhomologous end joining (XRCC5) , showed only mild sensitivity. In agreement with their extreme sensitivity , the XPF and ERCC1 mutants were defective in the incision or "unhooking" s tep of ICL repair. In contrast, the other mutants defective in NER activiti es, the XRCC2 and XRCC3 mutants, and the XRCC5 mutant all showed normal unh ooking kinetics. Using pulsed-field gel electrophoresis, DNA double-strand breaks (DSBs) were found to be induced following nitrogen mustard treatment . DSB induction and repair were normal in all the NER mutants, including XP F and ERCC1. The XRCC2, XRCC3, and XRCC5 mutants also shelved normal induct ion kinetics. The XRCC2 and XRCC3 homologous recombination mutants were, ho wever, severely impaired in the repair of DSBs. These results define a role for,XPF and ERCC1 in the excision of ICLs, but not in the recombinational components of cross-link repair. In addition, homologous recombination but not nonhomologous end joining appears to play an important role in the repa ir of DSBs resulting from nitrogen mustard treatment.