Eukaryotic Topoisomerase I

描述(申请人提供)：拟议研究的总体目标是了解分子细节，了解人类拓扑异构酶I如何控制细胞中DNA的拓扑状态，以及酪氨酰-DNA磷酸二酯酶(Tdp1)如何修复拓扑异构酶I-DNA共价复合体，该复合体是在某些DNA损伤后拓扑异构酶未能与DNA结合时以及在抗癌药物喜树碱存在的情况下产生的。生物化学和X射线结晶学的结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的反应机理。一组与各种人类拓扑异构酶I片段的GST融合将被用来检测拓扑异构酶与HMG1和Werner综合征(WRN)蛋白的相互作用。关于人类拓扑异构酶I和WRN之间相互作用的功能意义，将检验一些假说。在最近解开的酶的晶体结构中观察到的Tdp1中的亲核性和其他保守活性位点残基的功能将通过定点突变进行研究。当Tdp1裂解其DNA-蛋白质底物时，提供更好的裂解留下基团的合成底物将被用来识别催化残基，当Tdp1裂解其DNA-蛋白质底物时，催化残基作为普通酸质子化离开基团。一组具有不同DNA结构和不同长度的拓扑异构酶I衍生多肽的底物将进行动力学实验，以确定切割Tdp1的最佳底物。最后，将建立一个系统来研究拓扑异构酶I-DNA共价复合体在体内的命运，该复合体是由一种“有毒的”拓扑异构酶I诱导产生的，这种拓扑异构酶I可以裂解DNA，但不能结合DNA。沿着这些共价复合体修复途径的中间体的鉴定将为了解在被Tdp1切割之前复合体的蛋白质和DNA部分是如何处理的提供重要的见解。关于Tdp1结构和功能的信息可能导致Tdp1抑制剂的开发，这些抑制剂可以用于喜树碱和抗癌药物的联合治疗方案。

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.