DNA topoisomerases catalyze changes in DNA topology through a concerted mechanism of DNA strand breakage and rejoining. The transient cleavage of the DNA backbone is accompanied by the formation of a covalent enzyme-DNA intermediate. These intermediates are reversibly stabilized by a number of therapeutically important drugs, including the antitumor agent camptothecin (Cpt), which specifically targets eukaryotic DNA topoisomerase l. This enzyme plays an important role in DNA replication, recombination and transcription and is highly conserved among eukaryotes. This is reflected in similarities in enzyme structure, function and sensitivity to Cpt. The cytotoxic activity of Cpt is S-phase specific, presumably resulting from the interaction of DNA replication forks with the drug-stabilized enzyme- DNA cleavable complex. However, the exact nature of the molecular interactions required for the formation of these cleavable complexes and their subsequent conversion into lethal events remains unknown. This application proposes to define the specific molecular interactions required for Cpt-induced lethality in the yeast Saccharomyces cerevisiae. The restoration of Cpt sensitivity to yeast cells expressing either yeast or human DNA topoisomerase l, suggest that the processes involved in converting the drug stabilized complex into lethal lesions could be experimentally addressed in this genetically tractable system. Specifically, the enzyme domains and amino acid residues required for a productive interaction with Cpt and DNA will be defined. Two classes of DNA topoisomerase l mutants, one exhibiting resistance to Cpt and the other exhibiting a camptothecin-independent lethal phenotype (top1-L), will be assessed for changes in enzyme structure, DNA cleavage and Cpt binding. Second site mutations, that suppress these phenotypes, will also be characterized. Two genetic screens will also be undertaken to identify mutations in genes other than TOP1 that (1) suppress Cpt toxicity and (2) suppress the top1-lethal phenotype. Subsequent identification of these genes and their in vivo function(s) will elucidate the molecular interactions required for drug-induced cell death. These studies will also lead to a greater understanding of how normal cellular functions can be perturbed to cause cell death. As Cpt analogues are currently being developed as therapeutic agents for the treatment of lung, breast, ovarian and colon cancers, these results will have much broader application in the design and development of new therapeutics.
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