FUNCTIONAL STUDIES OF TOPOISOMERASE I

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The overall goal of this project is to improve the effectiveness of camptothecin through a clearer understanding of the cellular roles of topoisomerase I. Camptothecins represent an important new class of drugs with activity in a wide variety of malignancies. While camptothecins specifically induce topoisomerase I-linked DNA strand breaks, this event is not sufficient to cause cell death and the parameters that modulate the cytotoxic effects of these protein-linked DNA strand breaks are poorly understood. In this regard, interactions between topoisomerase I and other proteins are likely to be important in determining the cytotoxic effects of camptothecin. Prior work from this laboratory and others led to the identification of topoisomerase I-binding proteins that are involved in RNA synthesis and processing. The studies proposed in this project will determine the biological relevance of interactions between topoisomerase I and two specific proteins implicated in RNA metabolism, nucleolin and a novel arginine- serine protein. In Specific Aim 1 the interaction between nucleolin and topoisomerase I will be investigated biochemically using recombinant proteins. In particular, studies in this Aim are designed to establish whether nucleolin is a helicase and to determine the effects of topoisomerase I on in vitro rRNA processing mediated by nucleolin. Specific Aim 2 involves a genetic approach to understanding interactions between yeast topoisomerase I and nucleolin orthologues with regard to rRNA synthesis and processing. The role of a nucleolin orthologue in the nucleocytoplasmic transport of topoisomerase I and in camptothecin- mediated cytotoxicity will be investigated in this Aim. Specific Aim 3 focuses on a novel arginine-serine protein that interacts with topoisomerase I in vitro and in a two-hybrid assay. Experiments in this Aim are designed to confirm preliminary data suggesting that this topoisomerase I-binding protein is involved in mRNA synthesis or splicing. Collectively, these studies should provide information valuable not only for understanding the cellular role of topoisomerase I but also for elucidating the cytotoxic mechanisms of camptothecin.