Eukaryotic Topoisomerase I

描述（由申请人提供）：拟议研究的总体目标是从机制和结构-功能的角度了解人类拓扑异构酶I如何管理体内DNA的拓扑结构，以及细胞如何修复拓扑异构酶I在DNA上的共价复合物中停滞时发生的损伤。这种复合物是由拓扑异构酶I在几种类型的DNA损伤附近以及在用抗癌药物喜树碱治疗后裂解而产生的。可能的修复途径的考虑将包括酶，酪氨酰-DNA磷酸二酯酶（Tdp 1）的性质的详细分析，和一个内切核酸酶介导的修复假说的测试。拟议的研究将确定拓扑异构酶I的接头域如何调节切割-再连接平衡，以及这种效应是否需要接头与底物DNA之间的直接相互作用。将研究接头在导致染色体缺失的非法重组期间对齐平端连接的DNA中的可能作用。为了深入了解Tdp 1的体内底物的性质，将在结合和切割试验中测试底物的DNA和肽部分中具有修饰的一系列模型结构。已经开发了一种用于分离在将细胞暴露于喜树碱或表达“毒性”拓扑异构酶I之后在体内产生的拓扑异构酶I-DNA共价复合物的方法。该程序将用于鉴定在限制Tdp 1条件下积累的修复途径中间体。初步结果表明，可能参与的核酸内切酶在共价复合物的修复也将被追求。为了更好地了解Tdp 1如何修复拓扑异构酶l相关的DNA损伤，并研究Tdp 1也修复其他类型DNA损伤的可能性，我们将鉴定和表征与Tdp 1相互作用的蛋白质。这些研究在医学上是重要的，因为拓扑异构酶I是多种抗癌药物的靶标，并且因为阐明Tdp 1或其他酶如何修复拓扑异构酶I-DNA共价复合物可能促进可用于联合抗癌药物疗法的新药的开发。最后，表征异二聚体杜氏利什曼原虫拓扑异构酶I的附属结构域将可能促进选择性靶向这种非典型IB型拓扑异构酶用于治疗利什曼病的新药的开发。

DESCRIPTION (provided by applicant): The overall goals of the proposed research are to understand from a mechanistic and structure-function perspective how human topoisomerase I manages the topology of DNA in vivo, and how cells repair the damage that occurs when topoisomerase I becomes stalled in covalent complexes on the DNA. Such complexes result from topoisomerase I cleavage in the vicinity of several types of DNA damage and after treatment with the anticancer drug, camptothecin. A consideration of possible repair pathways will include both a detailed analysis of the properties of the enzyme, tyrosyl-DNA phosphodiesterase (Tdp1), and a test of an endonuclease-mediated repair hypothesis. The proposed studies will determine how the linker domain of topoisomerase I regulates the cleavage-religation equilibrium and whether this effect requires a direct interaction between the linker and the substrate DNA. A possible role for the linker in aligning the DNA for blunt-end ligation during illegitimate recombination leading to chromosomal deletions will be investigated. To gain insights into the nature of the in vivo substrates for Tdp1, a series of model structures with modifications in both the DNA and peptide moieties of the substrate will be tested in binding and cleavage assays. A procedure has been developed for the isolation of the topoisomerase I-DNA covalent complexes generated in vivo after exposing cells to camptothecin or expressing a "toxic" topoisomerase I. This procedure will be used to identify repair pathway intermediates that accumulate under conditions of limiting Tdp1. Preliminary results suggesting the possible involvement of an endonuclease in the repair of covalent complexes will also be pursued. To better understand how Tdp1 repairs topoisomerase l-related DNA damage and to investigate the likely possibility that Tdp1 also repairs other types of DNA damage, we will identify and characterize proteins that interact with Tdp1. These studies are medically important because topoisomerase I is the target of a variety of anticancer drugs and because elucidating how Tdp1 or other enzymes repair topoisomerase I-DNA covalent complexes may promote the development of new drugs that could be used in combination anticancer drug therapies. Finally, characterizing the accessory domains of the heterodimeric Leishmania donovani topoisomerase I will likely facilitate the development of new drugs that selectively target this atypical type IB topoisomerase for the treatment of Leishmaniasis.