The long-term goal of our research program is to provide a biochemical and molecular blueprint for the action of anti-cancer drugs that target DNA topoisomerase 2 (Top2). Top2 carries out changes in DNA structure needed for efficient transcription, replication, and DNA repair. These enzymes introduce transient double strand breaks in DNA through a protein/DNA covalent intermediate termed the cleavage complex. This intermediate allows cells to catalyze changes in DNA conformation without the dangers of frank DNA double strand breaks. Anti-cancer drugs such as etoposide and doxorubicin generate elevated levels of cleavage complexes, leading to cytotoxicity. The biochemical details of trapping Top2 covalent complexes remain poorly understood. We devised a variety of approaches for developing novel reagents for studying Top2/drug interactions. One key strategy is to isolate and characterize mutants in eukaryotic Top2 with altered drug sensitivity. Classically, studying drug/protein interactions is facilitated by the identification of drug resistant mutant proteins. However, this approach has proven unsatisfactory for topoisomerases, since any mutations that reduce enzyme activity typically confer high levels of drug resistance. We have adopted two alternate approaches. First, we devised screens for the identification of drug hypersensitive alleles of Top2. This approach used ectopic expression of human Top2 enzymes (both Top2? and Top2? isoforms have been functionally expressed in yeast) with screening for alleles that conferred elevated levels of sensitivity to etoposide. We identified a large number of mutants with greatly elevated levels of sensitivity to etoposide. In our proposed experiments, we will carry out biochemical analyses of the hypersensitive alleles. One class of mutant alleles that is of great interest is mutants in the Top2 ATPase domain. Current models suggest that etoposide interacts with the protein at the protein/DNA interface. Therefore, the mutants we have identified in the ATPase domain likely regulate progression through the catalytic cycle rather than directly affect drug/protein interactions. How the enzyme regulates progression through the catalytic cycle, and how this progression affects drug binding and action remains poorly understood. A second approach is based on recently identified mutants of yeast Top2 that require cells be proficient in DNA repair for viability. Biochemical analysis of the purified mutant proteins demonstrated elevated levels of drug independent DNA cleavage. Therefore, the mutant Top2 mimics the action of Top2 poisons and are termed self-poisoning Top2 mutants. We expanded this observation to human Top2, and have identified several self-poisoning mutations in human Top2?. We hypothesize that these mutants will also have elevated levels of drug independent DNA cleavage. Biochemical and structural analysis of these mutants will lend unique insights into the processes of DNA cleavage and religation by human Top2 proteins. Taken together, our work will provide unique reagents and insights into the action of anti-cancer drugs that target Top2.
Etoposide and doxorubicin are anti-cancer drugs that damage DNA through an enzyme named DNA topoisomerase II. This enzyme is a precisely coordinated machine, and etoposide and doxorubicin cause the machine to fail in ways that promote the killing of cancer cells. The experiments proposed will more precisely define how these drugs interfere with DNA topoisomerase II and may lead to more active and safer chemotherapeutic agents.
