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 concentrations (0.5-3 nM) of the radiomimetic antibiotic neocarzinostatin resulted in an increase in up to 11-fold in the frequency of mutations at the hemizygous APRT locus. Analysis by PCR and DNA sequencing revealed that the mutations were a mixture of base substitutions, small deletions, and large-scale rearrangements. base substitutions occurred preferentially at sequence positions where the drug is known to produce abasic sites with closely opposed strand breaks, e.g., AGT, TGT and AGC, where the abasic site occurs at the underlined base and the strand break occurs opposite the first base in each triplet. These results suggest that the substitutions were produced by replicative bypass of the abasic sites, perhaps during attempted repair of the accompanying strand break. Single-base deletions, which comprised nearly half of all deletions, were targeted to these same sequence positions, suggesting that they may have been generated either by replicative bypass of the abasic sites, or by end-joining repair of double-strand breaks, which are induced the same sites. Quantitative 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 can produce biologically significant levels of mutagenesis even in nondividing cells.
Wang
P
P
Department of Pharmacology and Toxicology, Medical College of Virginia, Richmond, USA.
Povirk
L F
LF
D000228:Adenine Phosphoribosyltransferase / Q000235:genetics*; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D001483:Base Sequence; D016466:CHO Cells; D002478:Cells, Cultured; D002874:Chromosome Mapping; D006224:Cricetinae; D004247:DNA / Q000187:drug effects; D004249:DNA Damage; D053938:DNA Fragmentation / Q000187:drug effects; D004260:DNA Repair; D008954:Models, Biological; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000187:drug effects*; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion / Q000187:drug effects*; D009353:Zinostatin / Q000494:pharmacology*
D000903:Antibiotics, Antineoplastic; D004247:DNA; D009353:Zinostatin; D000228:Adenine Phosphoribosyltransferase