The long range goal of the proposed research is a detailed molecular understanding of the relationship between the structure of human topoisomerase I and its role in the cell of providing swivels for DNA replication, RNA synthesis, chromatin assembly and recombination. Based on the impending crystal structure and the use of site-directed mutagenesis, we will investigate the chemistry of the nicking-closing reaction, the structural determinants for the nucleotide bias in the four base pairs upstream of the nicking site, the role of the linker region in topoisomerization, and the mode of action of the potent topoisomerase I poison, camptothecin. A combination of biochemistry and structural analyses will be used to distinguish between the strand- passage and the free-rotation models for the relaxation reaction. The human phosphodiesterase that is believed to be the important for the repair of topo I-mediated damage will be purified and characterized. The cDNA encoding the phosphodiesterase will be cloned and sequenced and the possible involvement of the enzyme in human cancers and susceptibility to camptothecin will be evaluated. Since cytotoxicity in cell culture has been shown to correlate with camptothecin-induced damage following administration of the drug, an assay will be developed to quantitate the level of topoisomerase I-DNA covalent adducts in the cells. Such an assay could be useful in testing new camptothecin derivatives and in evaluating whether individual patient tumors will respond to drug treatment. Finally, our recent discovery that lipophilic amines enhance the cytotoxicity of camptothecin will be pursued to understand the basis of the effect and to determine whether a combination drug regimen might have therapeutic potential in anticancer treatment.
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