STRUCTURE/FUNCTION STUDIES OF TYPE I TOPOISOMERASES

I 型拓扑异构酶的结构/功能研究

• 批准号: 2189816
• 负责人: Alfonso Mondragon
• 金额: $ 17.79万
• 依托单位: NORTHWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 1998-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2189816
• 关键词: DNA; DNA topoisomerases; X ray crystallography; chemical models; conformation; crystallization; enzyme activity; enzyme mechanism; enzyme structure; enzyme substrate; enzyme substrate complex; high performance liquid chromatography; molecular dynamics; mutant; nucleic acid chemical synthesis; oligonucleotides; physical model; protein purification; site directed mutagenesis; structural biology; synthetic nucleotide

DNA topoisomerases are ubiquitous proteins that control and maintain the topological state of DNA in the cell. They are involved in replication, transcription, and genetic recombination. Their role in DNA metabolism has made the important targets of novel chemotherapeutic and antibiotic agents. In Escherichia coli, two type I DNA topoisomerases have been identified and characterized: DNA topoisomerase I and DNA topoisomerase III. Both proteins work by forming a transient phosphotyrosine bond with DNA that allows another strand or duplex to pass through the break. We have identified, characterized, and elucidated the atomic structures of a 67 kDa fragment of E. coli DNA topoisomerase I. The structure suggests an enzyme-bridged mechanism of action for these type of proteins and poses new questions about their detailed catalytic mechanisms and their interactions with DNA. The long term goal of this proposal is to understand the mechanism of action of prokaryotic type I enzymes in atomic detail. The specific aims of the proposal are: a) To refine the atomic structure of the 67 kDa fragment of E. coli DNA topoisomerase I to 1.8 A; b) To study the interactions between DNA topoisomerase I and DNA; c) To study the chemical and structural determinants of catalytic function by a combination of site-directed mutagenesis, high resolution x-ray analysis, and biochemical and physicochemical studies; d) To solve the structure of E. coli DNA topoisomerase III; and e) To compare the structures of the two enzyme to identify the features important for their similarities and differences. Our work on E. coli DNA topoisomerase I will be largely guided by the recently solve structure of a 67kDa fragment of the enzyme in our laboratory. The crystals of the 67 kDa fragment diffract to 1.8 A and will allow us to obtain a very detailed atomic model on which to base our studies of DNA-protein interactions and the atomic catalytic mechanism. Crystals of the intact E. coli DNA topoisomerase III have already been obtained, and they diffract to at least 3.0 A. The structure determination by x-ray crystallography is in progress.

DNA拓扑异构酶是无处不在的蛋白质，可以控制和维持 细胞中DNA的拓扑状态。 他们参与复制， 转录和遗传重组。 它们在DNA代谢中的作用 已经成为新型化学治疗和抗生素的重要目标 代理商。 在大肠杆菌中，两种I型DNA拓扑异构酶已经 鉴定和表征：DNA拓扑异构酶I和DNA拓扑异构酶 iii。 两种蛋白质都通过与 DNA允许另一链或双链体通过断裂。 我们 已经确定，表征和阐明了 大肠杆菌DNA拓扑异构酶的67 kDa片段I。结构表明 这些类型的蛋白质和 提出了有关其详细催化机制及其的新问题 与DNA相互作用。 该提议的长期目标是了解 原子细节中核I型酶的作用。 具体目标 该提案的内容是：a）完善67 kDa的原子结构 大肠杆菌DNA拓扑异构酶I至1.8 a的片段； b）研究 DNA拓扑异构酶I和DNA之间的相互作用； c）研究 催化功能的化学和结构决定因素 位置定向诱变的组合，高分辨率X射线分析， 以及生化和物理化学研究； d）解决结构 大肠杆菌DNA拓扑异构酶III； e）比较 两种酶确定对其相似性很重要的特征 和差异。 我们在大肠杆菌DNA拓扑异构酶上的工作很大程度上将是 在酶的67kDa碎片的最近求解结构的指导下 在我们的实验室。 67 kDa片段的晶体衍射至1.8 a并将允许我们获得一个非常详细的原子模型 基于我们对DNA-蛋白质相互作用和原子催化的研究 机制。 完整的大肠杆菌DNA拓扑异构酶III的晶体具有 已经获得了，它们衍射至至少3.0 A. 通过X射线晶体学确定结构正在进行中。