Analysis of yeast DNA topoisomerase II mutants resistant to the antitumor drug amsacrine.

Site-directed mutagenesis of regions within a plasmid-borne yeast TOP2 gene encoding DNA topoisomerase II and hydroxylamine mutagenesis of the entire plasmid were carried out, and the mutagenized plasmid DNA pools were used separately to transform yeast with a temperature-sensitive top2-4 mutation in the chromosomal TOP2 locus. By selecting transformants that grow in the presence of the antitumor drug amsacrine at 35 degrees C, a nonpermissive temperature for the top2-4 allele, plasmid-borne top2 mutants expressing amsacrine-resistant and physiologically functional DNA topoisomerase II were readily obtained. The causality between amsacrine resistance and the presence of these mutations in yeast DNA topoisomerase II has been firmly established, and this causality in turn shows that, in yeast at least, DNA topoisomerase II is the only significant cellular target of amsacrine. Three classes of such mutants have been identified: one involves single or multiple changes in a sequence PLRGK-MLN located at positions 474-481 of yeast DNA topoisomerase II, a highly conserved motif in all type II DNA topoisomerases; a second involving a single mutation changing Ala642 to threonine or glycine; and a third involving deletions of portions of the carboxy-terminal domain of the yeast enzyme. The nature of drug resistance of these different classes of mutants is discussed. The approaches used in this work should be readily applicable to yeast cells expressing heterologous DNA topoisomerases such as human DNA topoisomerase II alpha. Other DNA topoisomerase II-targeting drugs can also be studied in such a system.