FUNCTIONAL STUDIES OF TOPOISOMERASE I

这个项目的总体目标是提高 通过更清楚地了解喜树碱在细胞中的作用 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。而当 喜树碱特异性诱导拓扑异构酶I连接的DNA链 中断时，此事件不足以导致细胞死亡和 调节这些蛋白相关的细胞毒性效应的参数 人们对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关系可能是重要的 在确定喜树碱的细胞毒性效应时。之前的工作来自 这个实验室和其他实验室发现了拓扑异构酶 参与RNA合成和加工的i结合蛋白。 本项目中提出的研究将确定生物 拓扑异构酶I与两种特异性拓扑异构酶相互作用的相关性 与RNA代谢有关的蛋白质，核仁素和一种新的精氨酸- 丝氨酸蛋白质。在特定目标1中，核仁素和核素之间的相互作用 拓扑异构酶I的生化研究将使用重组 蛋白质。特别是，这一目标的研究旨在建立 核仁素是否是解旋酶，并确定其对 拓扑异构酶I在核仁介导的体外rRNA加工中的作用。 特定目标2涉及一种理解相互作用的遗传学方法 酵母拓扑异构酶I和核仁同系物之间的关系 RRNA的合成和加工。核仁素同源异构体在 拓扑异构酶I在喜树碱中的核质转运 为达到这一目的，我们将研究其介导的细胞毒性。特定目标 3重点研究了一种新的精氨酸-丝氨酸蛋白，它与 拓扑异构酶I在体外和双杂交试验中。在这方面的实验 AIM旨在证实初步数据表明这一点 拓扑异构酶I结合蛋白参与mRNA合成或 拼接。总而言之，这些研究应该提供信息 不仅对理解拓扑异构酶的细胞作用有价值 也是为了阐明喜树碱的细胞毒性机制。

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.