The intertwining of the two strands of a DNA helix poses a number of topological problems by catalyzing changes in DNA topology through a concerted mechanism of DNA strand breakage and rejoining. This process is accompanied by the formation of covalent enzyme-DNA intermediates, which conserve the energy of the broken phosphodiester linkages. A number of therapeutically important drugs, including the potent antineoplastic agent camptothecin, reversibly stabilize these covalent complexes. Camptothecin specifically targets eukaryotic DNA topoisomerase 1, which is highly conserved in terms of its primary amino acid sequence, mechanism of action and drug sensitivity. The cytotoxicity of camptothecin is S-phase dependent, resulting from the double-strand DNA breaks produced by the collision of DNA replication forks with the drug-stabilized enzyme-DNA adducts. However, little is known about the mechanisms involved in the processing and repair of these potentially lethal lesions, or the signaling pathways required for drug- induced cell killing. This application proposes to define the pathway of camptothecin-induced cell lethality, by screening for yeast and human gene products whose overexpression in the yeast Saccharomyces cerevisiae protects these cells from drug-mediated cell death. Since the phenotypic consequences of camptothecin treatment are faithfully reiterated in yeast, the cellular processes involved in converting the drug-stabilized complexes into lethal lesions can be experimentally addressed in this genetically tractable system. The subsequent identification of these high copy suppressor (HCS) genes and their cellular functions, both in yeast and in mammalian cells, will elucidate athe cellular processes required for camptothecin-induced DNA damage and apoptosis. The ability of these genes to suppress related mechanisms of cell death will also be examined in yeast and mammalian cells expressing lethal DNA topoisomerase 1 mutants that mimic the cytotoxic action of camptothecin. These studies will further our understanding of the mechanism of drug-induced lethality, and will also lead to greater understanding of how normal cellular functions can be perturbed to a cause cell death. As several camptothecin analogs have been entered into clinical trials for the treatment of ovarian, breast, colon and non small cell lung cancers, the characterization of HCS gene functions will have much broader applications in the design and development of new therapeutics.
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