Eukaryotic Topoisomerase I

描述（申请人提供）：拟议研究的总体目标是从分子上详细了解人类拓扑异构酶I如何发挥作用以控制细胞中DNA的拓扑状态，以及酪氨酰-DNA磷酸二酯酶（Tdp 1）如何修复拓扑异构酶I-DNA共价复合物，当拓扑异构酶在某些类型的DNA损伤后以及在抗癌药物存在下未能重新连接DNA时，喜树碱生物化学和X射线晶体学的组合将被用来研究伴随DNA结合的拓扑异构酶的构象变化，并阐明拓扑异构酶I再连接反应的机制。一组GST融合到各种人类拓扑异构酶I片段将被用来检查拓扑异构酶与HMG 1和沃纳综合征（WRN）蛋白的相互作用。一些假设将被测试有关的功能意义的人拓扑异构酶I和WRN之间的相互作用。的亲核试剂的身份和其他保守的活性位点残基在Tdp 1中观察到的最近解决的酶的晶体结构的功能将通过定点诱变进行研究。将使用在切割时提供更好离去基团的合成底物来鉴定当Tdp 1切割其DNA-蛋白质底物时充当使离去基团质子化的一般酸的催化残基。一组具有不同DNA结构和不同长度的拓扑异构酶I衍生肽的底物将在动力学实验中进行测试，以确定Tdp 1切割的最佳底物。最后，将开发一种系统来研究通过诱导切割但不能重新连接DNA的“毒性”拓扑异构酶I产生的拓扑异构酶I-DNA共价复合物的体内命运。鉴定沿着这些共价复合物的修复途径的中间体将提供关于在Tdp 1切割之前复合物的蛋白质和DNA部分如何加工的重要见解。有关Tdp 1结构和功能的信息可能会导致开发Tdp 1抑制剂，可用于与喜树碱联合抗癌药物治疗方案。

DESCRIPTION (provided by applicant): The overall goals of the proposed research are to understand in molecular detail how human topoisomerase I functions to control the topological state of the DNA in the cell, and how tyrosyl-DNA phosphodiesterase (Tdp1) repairs the topoisomerase I-DNA covalent complexes that arise when the topoisomerase fails to religate the DNA after certain kinds of DNA damage and in the presence of the anticancer drug, camptothecin. A combination of biochemistry and X-ray crystallography will be used to study the conformational changes in the topoisomerase that accompany DNA binding and to elucidate the mechanism of the topoisomerase I religation reaction. A panel of GST fusions to various human topoisomerase I fragments will be employed to examine interactions of the topoisomerase with the HMG1 and Werner syndrome (WRN) proteins. A number of hypotheses will be tested concerning the functional significance of the interaction between human topoisomerase I and WRN. The identity of the nucleophile and the functions of other conserved active site residues in Tdp1 observed in the recently-solved crystal structure of the enzyme will be investigated by site-directed mutagenesis. Synthetic substrates that provide better leaving groups upon cleavage will be employed to identify the catalytic residue that acts as a general acid to protonate the leaving group when Tdp1 cleaves its DNA-protein substrate. A panel of substrates with different DNA structures and different lengths of topoisomerase I-derived peptides will be tested in kinetic experiments to determine the optimal substrate for Tdp1 cleavage. Finally, a system to study the in vivo fate of the topoisomerase I-DNA covalent complexes produced by the induction of a "toxic" topoisomerase I that cleaves, but fails to religate the DNA will be developed. The identification of intermediates along the pathway for the repair of these covalent complexes will provide important insights regarding how both the protein and the DNA moieties of the complexes are processed prior to cleavage by Tdp1. Information gained on the structure and function of Tdp1 may lead to the development of inhibitors of Tdp1 that could be used in combination anticancer drug therapy regimens with camptothecin.