The top2-5 mutant of yeast topoisomerase II encodes an enzyme resistant to etoposide and amsacrine

Abstract

We have characterized a temperature-sensitive mutant in the yeast TOP2 gene that shows resistance to the anti-topoisomerase II agents amsacrine and etoposide. Cells carrying the top2-5 mutant have a minimum lethal concentration of amsacrine of greater than 100 micrograms/ml, compared to 10 micrograms/ml in isogenic wild-type cells, and a minimum lethal concentration of greater than 100 micrograms/ml etoposide, compared with 50 micrograms/ml for cells carrying wild-type topoisomerase II. We have cloned the top2-5 allele into a yeast vector that allows high level overexpression of the protein. As expected, the purified top2-5 activity is temperature-sensitive. The protein shows a 3-fold reduction of amsacrine-stabilized cleavage at the permissive temperature, confirming that the top2-5 protein is resistant to amsacrine in vitro. The protein also exhibits reduced cleavage in the presence of etoposide, with the largest effect at low concentrations of the drug. These results suggest that the top2-5 protein is altered in its sensitivity to anti-topoisomerase II agents. The relevant portion of the mutant allele has been sequenced, and several tightly clustered mutations have been identified. The location of the mutations identify a domain of the topoisomerase II protein that may be important in the interaction of the protein with anti-topoisomerase II anti-cancer drugs.