Enhanced amsacrine-induced mutagenesis in plateau-phase Chinese hamster ovary cells, with targeting of+1 frameshifts to free 3 ' ends of topoisomerase II cleavable complexes

Previous work showed that the DNA double-strand cleaving agents bleomycin a nd neocarzinostatin were more mutagenic in plateau-phase than in log-phase cells. To determine whether topoisomerase Il poisons that produce double-st rand breaks by trapping of cleavable complexes would, likewise, induce muta tions specific to plateau-phase cells, aprt mutations induced by amsacrine in both lag-phase and plateau-phase CHO cells were analyzed. The maximum ap rt mutant frequencies obtained were 7 x 10(-6) after treatment with 0.02 mu M amsacrine in log phase and 27 x 10(-6) after treatment with 1 mu M amsac rine in plateau phase, compared with a spontaneous frequency of <1 x 10(-6) , Base substitutions dominated the spectrum of mutations in log-phase cells , but were much less prevalent in plateau-phase cells. Both spectra also in cluded small deletions, insertions and duplications, as well as few large-s cale deletions or rearrangements. About 5% of the log-phase mutants and 16% of the plateau-phase mutants were +1 frameshifts, and all but one of these were targeted to potential free 3' termini of cleavable complexes, as dete rmined by mapping of cleavage sites in DNA treated with topoisomerase II pl us amsacrine in vitro. Thus, these insertions may arise from templated exte nsion of the exposed 3' terminus by a DNA polymerase, followed by resealing of the strand, as shown previously for acridine-induced frameshifts in T4 phage.