ANTI TOPOISOMERASE DRUG ACTION IN YEAST

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DNA topoisomerases are the targets for a wide range of clinically useful anti-cancer drugs. Although the basic biochemical reaction pathway for the topoisomerases is fairly well understood, there is still little information available regarding the mechanism of action of anti- topoisomerase drugs in generating the stable covalent that is necessary for cell killing. In addition to topoisomerase poisons, there has been recent interest in drugs that inhibit topoisomerase II (Top2), but do not stabilize a covalent complex. An important drug in this category is dexrazoxane (ICRF-187) which is used clinically to prevent doxorubicin cardiotoxicity. Recent experiments have demonstrated that ICRF-187 has an unexpected mechanism of cell killing, with the ability to convert Top2 into a poison even though it does not stabilize covalent complexes. Studies are proposed to understand the biochemical mechanism of action of ICRF-187. A combination of genetic and biochemical approaches will be used to further unravel how both catalytic inhibitors such as ICRF-187 and Top2 poisons such as etoposide block the enzyme's progression through its normal reaction pathway. Yeast is used as a model system for analyzing the effects of Top2 mutants, and for over-expressing the mutant proteins . The research strategy involves the construction of novel topoisomerase mutants, including mutants that are hypertensive to anti- topoisomerase drugs. The mutant proteins are purified, and their biochemical characteristics are used to infer changes in protein: drug interactions. A new tool has been developed understand how drugs trap covalent complexes: a mutant in human topoisomerase II alpha that mimics the action of Top2 poisons. Additionally, work on Top2 mutants that confer resistance to etoposide has led to the development of a new system for investigating whether mutations in topoisomerases play a role in acquired clinical drug resistance. Finally, recent experiments indicating that topoisomerase I and II may be an important determinant in cell killing by other DNA damaging agents has led to studies of how cells may modulate topoisomerases following DNA damage. This work is designed to provide insight into the biochemical mechanisms of anti- topoisomerase drug action, and may be useful in the development of new and more effective topoisomerase inhibitors.