TARGETED BASE SUBSTITUTIONS AND SMALL DELETIONS INDUCED BY NEOCARZINOSTATIN AT THE APRT LOCUS IN PLATEAU-PHASE CHO CELLS

Treatment of confluence-arrested CHO-D422 cells for 48 h with low conc entrations (0.5-3 nM) of the radiomimetic antibiotic neocarzinostatin resulted in an increase in up to Ii-fold in the frequency of mutations at the hemizygous APRT locus. Analysis by PCR and DNA sequencing reve aled that the mutations were a mixture of base substitutions, small de letions, and large-scale rearrangements. Base substitutions occurred p referentially at sequence positions where the drug is known to produce abasic sites with closely opposed strand breaks, e.g., AG (T) under b ar, TG (T) under bar and AG (C) under bar, where the abasic site occur s at the underlined base and the strand break occurs opposite the firs t base in each triplet. These results suggest that the substitutions w ere produced by replicative bypass of the abasic sites, perhaps during attempted repair of the accompanying strand break. Single-base deleti ons, which comprised nearly half of all deletions, were targeted to th ese same sequence positions, suggesting that they may have been genera ted either by replicative bypass of the abasic sites, or by end-joinin g repair of double-strand breaks, which are induced the same sites. Qu antitative analysis of neocarzinostatin-induced damage to APRT DNA in vitro confirmed the association between lesions involving concommitant damage to both DNA strands, and mutations. The results are consistent the hypothesis that agents which induce such bistranded DNA damage ca n produce biologically significant levels of mutagenesis even in nondi viding cells.