Random mutagenesis of the B′A′ core domain of yeast DNA topoisomerase II and large-scale screens of mutants resistant to the anticancer drug etoposide

Mutagenic PCR method was applied to introduce point mutations to the B′A′ core domain of yeast DNA topoisomerase II. Screens for mutants resistant to the anticancer drug etoposide were carried out in a yeast ts system in the presence of high concentrations of the drug or in a drug-hypersensitive genetic background. 129 mutants were obtained from a total of 47,000 transformants. Nucleotide sequencing of 40 selected mutants showed that a large number of the mutations map to regions encoding the linker that joins the ATPase domain to the B′ module and the B′A′ linker. Significant reduction in catalytic activity was evident for a large fraction of mutant enzymes and all mutants were also resistant to amsacrine, another topoisomerase II drug with a different chemical structure, suggesting that few of the mutations reflect simple changes of specific amino acid side chains that are directly involved in enzyme-drug interactions.