Type II topoisomerases are ubiquitous enzymes that are required for proper chromosome structure and segregation. In addition, these enzymes play important roles in DNA replication, transcription, and recombination. Type II topoisomerases relax DNA and remove knots and tangles from the genetic material by passing an intact double helix through a transient double-stranded break that they generate in a separate DNA segment. Humans encode two closely related isoforms of the type II enzyme, topoisomerase II1 and topoisomerase II2. Topoisomerase II1 is essential for the survival of proliferating cells and topoisomerase II2 plays critical roles during development. However, since these enzymes generate requisite double-stranded DNA breaks in order to carry out their crucial catalytic functions, they assume a dual persona. While essential to cell survival, they also pose an intrinsic threat to genomic integrity every time they act. Beyond their important physiological functions, topoisomerase II1 and 2 are the primary targets for some of the most active and widely prescribed drugs currently used for the treatment of human cancers. These agents elicit their cytotoxic effects by dramatically increasing levels of covalent topoisomerase II-cleaved DNA complexes that are normal, but fleeting, intermediates in the catalytic DNA strand passage reaction. When the resulting enzyme-associated DNA breaks are present in sufficient concentrations, they trigger cell death. Anticancer drugs that target type II enzymes are referred to as topoisomerase II poisons because they convert these indispensable enzymes to potent physiological toxins that generate DNA damage in treated cells. Although topoisomerase II1 and 2 are important targets for cancer chemotherapy, evidence suggests that they also have the potential to trigger the disease. Leukemias that carry genomic translocations involving the MLL gene at chromosome band 11q23 are associated with exposure to topoisomerase II poisons. Despite the central importance of type II topoisomerases to the cancer problem, interactions between topoisomerase II1/2, DNA, and anticancer drugs (or other enzyme poisons) have not been well characterized. Thus, the specific aims of this proposal are to further delineate the mechanism by which type II topoisomerases carry out their essential catalytic reactions, to define the mechanism by which drugs and other topoisomerase II-active chemicals alter the catalytic function of these enzymes, and to characterize the response of cells to enzyme-generated DNA strand breaks. Research models for this study will be primarily human topoisomerase II1 and 2, but yeast (Saccharomyces cerevisiae) and Chlorella virus topoisomerase II may also be employed for specific experiments. A variety of approaches, including ensemble and single molecule kinetic studies, novel nucleic acid substrates and topoisomerase II-targeted drugs, and cellular studies will be used to address the stated aims of the proposal.
Type II topoisomerases are ubiquitous enzymes that remove knots and tangles from the genetic material by passing an intact double helix through a transient double-stranded break that they generate in a separate DNA segment. These enzymes are targets for important anticancer drugs, but are also believed to trigger to specific types of leukemias.
The aims of this proposal are to determine how type II topoisomerases function, how drugs and other chemicals alter their activities, and how cells respond to the actions of these enzymes.
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