Use of Yeast in the Study of Anticancer Drugs Targeting DNA Topoisomerases: Expression of a Functional Recombinant Human DNA Topoisomerase IIα in Yeast

Abstract A plasmid was constructed for the expression of human DNA topoisomerase IIα in yeast from a galactose-inducible promoter of the yeast GAL1 gene. Expression of a recombinant human enzyme, in which the first 28 of the 1531 codons of human DNA topoisomerase IIα were replaced by the first five codons of yeast DNA topoisomerase II, was shown to rescue the lethal phenotype of thermal sensitive yeast DNA topoisomerase II mutants at 35°C. Purification of the human enzyme overexpressed in yeast yielded a single polypeptide with an apparent mass of 170 kDa, and the properties of the purified recombinant enzyme were found to be the same as those reported for human DNA topoisomerase IIα purified from HeLa cells. Studies with the anticancer drug amsacrine indicated that the human enzyme, either inside yeast cells or in its purified form, is a target of the drug; inhibition of the purified enzyme by teniposide (VM-26) and merbarone was also demonstrated. These studies demonstrate that yeast strains expressing human DNA topoisomerase IIα provide a convenient system for studying drugs targeting the enzyme; unlike mammalian systems, potential complications due to the presence of human DNA topoisomerase IIβ can be eliminated in this system. Overexpression of human DNA topoisomerase IIα in yeast also provides a convenient source of the enzyme for in vitro studies.