Overexpression and Purification of DNA Topoisomerase I from Yeast

The mechanism of action of DNA topoisomerase I in catalyzing the relaxation of supercoiled DNA and how this reaction is perturbed by the antitumor drug camptothecin have been the subject of intense investigation (reviewedin (1 -3 ). Much of this effort has focused on structure/function studies of wild-type and mutant forms of the enzyme, derived from a variety of sources (1 ,4 -20 ). The budding yeast Saccharomyces cerevisiae has proven particularly amenable to the genetic manipulations required to overexpress and purify wild-type and mutant forms of eukaryotic DNA topoisomerase I (3 ,4 ,12 ,13 ).