Topoisomerase II is an essential enzyme that modulates the topological state of DNA by passing an intact helix through a transient double-stranded break that it generates in a separate helix. The enzyme is required for proper chromosome structure and segregation and plays important roles in DNA replication and recombination. Beyond its critical physiological functions, topoisomerase II is the target for several of the most active anticancer agents currently used to treat human malignancies. These drugs elicit their cytotoxic effects by a mechanism that is markedly different than those of other enzyme-targeted agents. Rather than inhibiting the catalytic activity of the enzyme, anticancer drugs dramatically increase levels of covalent topoisomerase II-cleaved DNA complexes that are normal, but fleeting, intermediates ina the catalytic cycle of the enzyme. Thus, these agents poison topoisomerase II and convert it from an essential enzyme to a physiological toxin that generates DNA damage in treated cells. Despite the importance of topoisomerase II to the viability of eukaryotic cells and to the treatment of human cancers, interactions between the enzyme, its DNA and ATP substrates, and anticancer drugs have not been definitively characterized. Therefore, the ultimate goal of this proposal is to further delineate the mechanism by which topoisomerase II carries out its fundamental reactions and the mechanism by which anticancer drugs alter the catalytic function of the enzyme. More specifically, the aims of this proposal are 1) to further define the catalytic mechanism of topoisomerase II, 2) to further delineate the mechanism(s) by which anticancer drugs increase levels of topoismerase II-DNA cleavage complexes, and 3) to determine the mechanism(s) by which the enzyme becomes resistant or hypersensitive to anticancer drugs. The primary research models for this study will be Drosophila melanogaster and yeast (Saccharomyces cerevisiae). The Drosophila and yeast type II enzymes are the most well characterized of any eukaryotic topoisomerase (I or II) and yeast allows a degree of genetic manipulation an topoisomerase II overexpression that is unmatched by any other eukaryotic system. The proposed studies will take advantage of several recently developed assays. The catalytic mechanism of topoisomerase II will be characterized by analyzing interactions with its substrates and by determining how it cleaves DNA, catalyzes DNA strand passage, and selected sites of DNA cleavage. The mechanism of anticancer drug action will be addressed by determining how the ternary enzyme-drug-DNA complex is formed and how drugs interact with topoisomerase II and affect its catalytic activity. Finally, alterations in the sensitivity of topoisomerase II toward anticancer drugs will be assessed by generating and characterizing mutant enzymes that are either resistant or hypersensitive to these agents.
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