DNA LESIONS AS ENDOGENOUS TOPOISOMERASE POISONS

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DESCRIPTION (As Adapted From the Investigator's Abstract): Topoisomerase II is an essential enzyme that is required for proper chromosome structure and segregation and plays important roles in DNA replication and recombination. Beyond its critical cellular functions, topoisomerase II is the primary target for some of the most active and widely prescribed drugs used for the treatment of human cancers. These agents elicit their cytotoxic effects by a mechanism that is markedly different than that of other drugs. Rather than inhibiting the catalytic activity of topoisomerase II, anticancer drugs targeted to the enzyme dramatically increase levels of covalent topoisomerase II-cleaved DNA complexes that are normal, but fleeting, catalytic intermediates. When the resulting topoisomerase II-associated double-stranded DNA breaks are present in high concentrations, they generate mutations, chromosomal translocations, and trigger cell death pathways. Because topoisomerase II-targeted anticancer drugs convert this dispensable enzyme into a potent physiological toxin, they are referred to as topoisomerase II poisons. Although topoisomerase II is one of the most important targets for cancer chemotherapy, there is compelling circumstantial evidence that the enzyme also has the potential to trigger the disease. Together with the unique mechanism of action of topoisomerase II poisons, this suggests that topoisomerase II-targeted drugs may represent exogenous counterparts of cellular components that induce DNA recombination, mutagenesis, or cell death pathways. Previous results form this laboratory indicated that abasic sites, the most commonly formed lesions in DNA, stimulate topoisomerase II-mediated double-stranded DNA cleavage with a potency that is greater than 1000-fold higher than that of etoposide, one of the most widely prescribed anticancer drugs in clinical use. Therefore, the ultimate goals of the proposal are to further define interactions between topoisomerase II and DNA damage and to determine whether DNA lesions function in vivo as endogenous topoisomerase II poisons. The specific aims of this proposal are to: 1) further define the spectrum of DNA damage that alters the catalytic function of type II topoisomerases; 2) define the mechanism by which DNA lesions enhance topoisomerase II-mediated DNA cleavage; 3) determine whether abasic intermediates generated by base excision repair can trigger the formation of permanent topoisomerase II-mediated double-stranded DNA breaks; and 4) determine whether DNA lesions act as topoisomerase II poisons in the cell. The primary enzymological model for this study will be human topoisomerase II alpha and beta. Physiological studies will employ human cell lines and yeast (Saccharomyces cerevisiae). Cellular DNA damage will be induced by a combination of chemical and genetic approaches.