DNA LESIONS AS ENDOGENOUS TOPOISOMERASE POISONS

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Topoisomerase II is the cellular target for several of the most active antineoplastic agents currently used for the treatment of human cancers. These drugs elicit their cytotoxic effects by a unique mechanism. Rather than acting by 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 in the catalytic cycle of the enzyme. When the resulting enzyme-associated double-stranded DNA breaks are present in the genome in high concentrations, they generate mutations, chromosomal aberrations, and under extreme conditions, cell death. Thus, anticancer drugs poison topoisomerase II and convert it from an essential enzyme into a physiological toxin. The unusual mechanism of action of topoisomerase II poisons raises the possibility that these drugs represent exogenous counterparts of cellular components that induce DNA recombination, mutagenesis, or cell death pathways. Previous results from this laboratory indicate that abasic sites, which are the most commonly formed lesion in DNA and are generated by a myriad of DNA damaging events, stimulate topoisomerase II-mediated double-stranded DNA cleavage. The efficacy of this cleavage stimulation is similar to that of etoposide (which is the most widely prescribed anticancer agent in clinical use). However, the potency of abasic sites is about 2,000-fold greater than that of the drug. Therefore, the ultimate goals of this proposal are to define interactions between abasic sites and the type II enzyme and to determine whether abasic sites function as endogenous topoisomerase II poisons. More specifically, the aims of this proposal are 1) to determine the mechanism by which abasic sites enhance topoisomerase II-mediated cleavage, 2) to delineate the mechanism by which the enzyme recognizes abasic sites, 3) to define relationships between the mechanism of action of abasic sites and anticancer drugs, and 4) to determine whether abasic sites function as topoisomerase II poisons in vivo. The information generated by this study should greatly increase our understanding of how topoisomerase II-targeted agents stimulate enzyme-mediated DNA cleavage and ultimately cause cell death. Drosophila and yeast will serve as the primary research models for this study. The Drosophila and yeast enzymes are the most well characterized type II topoisomerases and yeast allows a degree of genetic manipulation that is unmatched by any other eukaryotic system. The proposed studies will take advantage of several recently developed assay systems. The mechanism by which abasic sites enhance enzyme-mediated DNA cleavage will be analyzed by a variety of biochemical, kinetic, and genetic approaches. The recognition of abasic sites by topoisomerase II will be characterized by determining how the enzyme scans DNA for this lesion and by defining the structural features of this lesion that are required to alter enzyme activity. Relationships between abasic sites and anticancer agents will be defined by mapping the interaction domain of these lesions on topoisomerase II relative to that of drugs. Finally, the physiological role of abasic sites as topoisomerase II poisons will be characterized by determining whether these lesions induce topoisomerase II-mediated cell death or mutagenesis.