Complementation of temperature-sensitive topoisomerase II mutations in Saccharomyces cerevisiae by a human TOP2β construct allows the study of topoisomerase IIβ inhibitors in yeast.

Abstract

We show herein that human DNA topoisomerase IIβ is functional in yeast. It can complement a yeast temperature-sensitive mutation in topoisomerase II. The effect on human topoisomerase IIβ of a number of topoisomerase II inhibitors was analysed in a yeast in vivo system and compared with that of human topoisomerase IIα and wild-type yeast topoisomerase II. A drug permeable yeast strain (JN394 top2 – 4) was used to analyse the in vivo effects of known anti-topoisomerase II agents on human topoisomerase IIβ transformants. A parallel analysis on human topoisomerase IIα transformants provides the first in vivo analysis of the responses of yeast bearing the individual isoforms to these drugs. The strain was analysed at 35°C, a non-permissive temperature at which only plasmid-borne topoisomerase II is active. A shuttle vector with either human topoisomerase IIβ, human topoisomerase IIα or yeast topoisomerase II under the control of a GAL1 promoter was used. The key findings were that amsacrine produced comparable levels of cell killing with both α and β, whilst etoposide, doxorubicin and mitoxantrone produced higher degrees of cell killing with α than with β or yeast topoisomerase II. Merbarone had the greatest effect on the yeast strain bearing plasmid-borne yeast topoisomerase II. Suramin, quercetin and genistein showed little cell killing in this system. This yeast in vivo system provides a powerful way to analyse the effects of anti-topoisomerase II agents on transformants bearing the individual human isoforms. This system also provides a means of analysing putative drug-resistance mutations in human topoisomerase IIβ or to select for drug-resistance mutations in human topoisomerase IIβ.