Excision repair of nitrogen mustard-DNA adducts in Saccharomyces cerevisiae

The bifunctional alkylating anticancer drug nitrogen mustard forms a variet y of DNA lesions, including monoadducts and intrastrand and interstrand cro sslinks. Although it is known that nucleotide excision repair (NER) is impo rtant in processing these adducts, the role of the other principal excision repair pathway, base excision repair (BER) is less well defined, Using iso genic Saccharomyces cerevisiae strains disrupted for a variety of NER and B ER genes we have examined the relative importance of the two pathways in th e repair of nitrogen mustard adducts, As expected, NER defective cells (rad 4 and rad14 strains) are extremely sensitive to the drug. One of the BER mu tants, a 3-methyladenine glycosylase defective (mag1) strain also shows sig nificant hypersensitivity, Using a rad4/mag1 double mutant it is shown that the two excision repair pathways are epistatic to each other for nitrogen mustard sensitivity, Furthermore, both rad14 and mag1 disruptants show elev ated levels of nitrogen mustard-induced forward mutation. Measurements of r epair rates of nitrogen mustard N-alkylpurine adducts in the highly transcr ibed RPB2 gene demonstrate defects in the processing of mono-adducts in rad 4, rad14 and mag1 strains. However, there are differences in the kinetics o f adduct removal in the NER mutants compared to the mag1 strain, In the mag 1 strain significant repair occurs within 1 h with evidence of enhanced rep air on the transcribed strand. Adducts however accumulate at later times in this strain. In contrast, in the NER mutants repair is only evident at tim es greater than 1 h. In a mag1/rad4 double mutant damage accumulates with n o evidence of repair. Comparison of the rates of repair in this gene with t hose in a different genomic region indicate that the contributions of NER a nd BER to the repair of nitrogen mustard adducts may not be the same genome wide.