FUNCTIONAL STUDIES OF TOPOISOMERASE I

该项目的总体目标是提高 喜树碱通过更清楚地了解细胞的作用， 拓扑异构酶I 喜树碱代表了一类重要的新的 在多种恶性肿瘤中具有活性的药物。 而 喜树碱特异性诱导拓扑异构酶I连接的DNA链 但是，这一事件不足以导致细胞死亡， 调节这些蛋白质相关的细胞毒性作用的参数 对DNA链断裂知之甚少。 在这方面，互动 拓扑异构酶I和其他蛋白质之间的联系可能是重要的 在确定喜树碱的细胞毒性作用。 以前的工作从 这个实验室和其他实验室鉴定了拓扑异构酶， 参与RNA合成和加工的I-结合蛋白。 该项目中提出的研究将确定生物学 拓扑异构酶I与两种特异性 参与RNA代谢的蛋白质，核仁素和一种新的精氨酸- 丝氨酸蛋白 在具体目标1中，核仁素与 拓扑异构酶I将使用重组 proteins. 特别是，本目标的研究旨在建立 核仁素是否是解旋酶，并确定 拓扑异构酶I对核仁素介导的体外rRNA加工的影响 具体目标2涉及到一个遗传学的方法来理解相互作用 酵母拓扑异构酶I和核仁素直向同源物之间 rRNA合成和加工。 核仁素直向同源物在 拓扑异构酶I和喜树碱的核质转运- 介导的细胞毒性将在此目的进行研究。 具体目标 3集中在一种新的丝氨酸-丝氨酸蛋白质， 拓扑异构酶I在体外和在双杂交测定中。 在这方面的实验 目的是确认初步数据表明， 拓扑异构酶I结合蛋白参与mRNA合成，或 拼接 总的来说，这些研究应该提供信息， 不仅对于理解拓扑异构酶的细胞作用， 同时也为阐明喜树碱的细胞毒作用机制提供了理论依据。

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.