TOPOISOMERASE I STRUCTURE AND REGULATION

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This proposal is designed to enhance the current understanding of the interaction between DNA topoisomerase I and inhibitory antineoplastic drugs. Knowledge of factors involved in the interaction between the enzyme and inhibitors such as camptothecin would be of benefit in the clinical application of these agents. The studies proposed in Aim l are an extension of prior work in which a novel mechanism of cellular resistance to camptothecin was identified, involving a point mutation at position 361 in topoisomerase I. Preliminary studies suggest that this mutation confers resistance to the effects of camptothecin on the enzyme, and that additional mutations in this region impair the DNA cleavage step of the topo 1 catalytic cycle. These findings will be confirmed and extended by the use of oligonucleotide cleavage assays and gelshift assays performed with bacterially expressed mutant proteins. Additional studies will evaluate the effects of these mutations on camptothecin binding and on the inhibitory activity of noncamptothecin topoisomerase T poisons such as Hoechst dye 33342 and actinomycin D. Since the cellular response to topoisomerase I inhibition may be influenced by proteins associating with the enzyme, Specific Aim 2 is designed to identify such proteins. Preliminary work using affinity chromatography with an immobilized topoisomerase I fusion protein has identified several putative binding proteins, and microsequencing has identified one of these proteins as nucleolin. Studies in this Aim are designed to confirm this finding and to localize the site of binding to topoisomerase I by the use of deletion mutants. Additional studies will attempt to identify other binding proteins and to investigate the effects of these proteins on topoisomerase I function and on the interaction between topoisomerase I and inhibitors such as camptothecin.