Structure, Mechanism and Interactions of Type IA Topoisomerases

IA型拓扑异构酶的结构、机制和相互作用

• 批准号: 10093404
• 负责人: Yuk-Ching Tse-Dinh
• 金额: $ 20.92万
• 依托单位: FLORIDA INTERNATIONAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-01 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10093404
• 关键词: Active Sites; Address; Affect; Antibiotic Resistance; Antibiotics; Bacterial DNA Topoisomerase I; Biochemical; Biological Assay; Cell physiology; Cleaved cell; Complementary DNA; Complex; DNA; DNA-Directed RNA Polymerase; Drug resistance; Enzymes; Face; Future; Genetic Transcription; Genome; Genome Stability; Genus Mycobacterium; Goals; Human Activities; In Vitro; Infection; Investigation; Knowledge; Life; Ligands; Measures; Modeling; Molecular; Molecular Conformation; Movement; Mutation; Play; RNA; Regulation; Relaxation; Replication-Associated Process; Research; Research Activity; Role; Seminal; Single-Stranded DNA; Structure; Superhelical DNA; TOP1 gene; Topoisomerase; Topoisomerase III; Toxin; X-Ray Crystallography; global health; in vivo; insight; molecular dynamics; mutant; neurodevelopment; new therapeutic target; pathogenic bacteria; prevent; recombinational repair; single molecule; small molecule; targeted treatment; tool

Project Summary/Abstract Type IA topoisomerases are ubiquitous in the three kingdoms of life, and play critically important roles in maintaining proper DNA topology during the vital cellular processes of replication, transcription, recombination, and repair. The PI’s research activities have provided seminal biochemical and structural findings for this class of essential genome regulator, and continue to address key questions on the catalytic mechanism of type IA topoisomerases and provide new insights into their functional and regulatory interactions. This information is needed to utilize type IA topoisomerases present in every bacterial pathogen as a novel therapeutic target for finding new antibiotics to help face our serious global health challenge of antibiotic resistance. Type IA topoisomerases catalyze the relaxation of negatively supercoiled DNA by cleaving a single DNA strand in the underwound duplex DNA and passing the complementary DNA single strand through the break before religation of the cleaved strand to change the DNA topology. The molecular mechanism of the large enzyme conformational changes that are required for the coordinated movement of the passing DNA in and out of the DNA gate is the critical barrier for elucidating how bacterial TOP1 can relax negatively supercoiled DNA with high efficiency to prevent hypernegative DNA supercoiling and R-loop stabilization that can arise during transcription. This important function of bacterial TOP1 is facilitated by the direct TOP1 interaction with RNA polymerase that we have characterized and found to be targeted by endogenous toxin in mycobacteria. For future studies, we will create new TOP1 mutants perturbed in interdomain interactions at a distance from the active site and investigate the effect on the in vivo relaxation activity and in vitro interactions with DNA substrate. Mutants with reduced catalytic efficiency will be further studied to determine if the mutations affected the gate opening-closing dynamics and DNA strand passage. We will capture new structural conformations of the TOP1-DNA complex that may represent different stages of the catalytic cycle with X-ray crystallography and measure the gate opening-closing dynamics with single molecule assays. Structural studies will also incorporate other ligands including RNA. Type IA topoisomerases have evolved to include TOP1 and TOP3 enzymes in all three kingdoms of life that possess dual activities on both DNA and RNA substrates. The RNA topoisomerase activity of human TOP3B has been shown to be required for neurodevelopment and the enzyme is also involved in R-loop suppression and genome stability. We are modeling the RNA interaction of type IA topoisomerases with molecular dynamics simulations to determine how the DNA and RNA substrate may be accommodated differentially by change in enzyme conformation and interacting residues. We have initiated studies to identify a separation of function mutation or small molecule probe that can be used to distinguish between the DNA and RNA topoisomerase activity in vivo. Such research tools for study of cellular RNA topoisomerase activity and regulation will have an important and lasting impact on the field.

项目摘要/摘要 IA型拓扑异构酶在生命的三个王国中无处不在，并且在至关重要的角色中起着至关重要的作用 在重复，转录，重组的重要细胞过程中，保持适当的DNA拓扑结构 和维修。 PI的研究活动为此提供了半生物化学和结构性发现 基本基因组调节剂，并继续解决有关IA型催化机制的关键问题 拓扑异构酶并为其功能和调节性相互作用提供新的见解。此信息是 需要利用每种细菌病原体中存在的IA型拓扑异构酶作为一种新的治疗靶点 寻找新的抗生素，以帮助面对我们对抗生素耐药性的严重全球健康挑战。类型IA 拓扑异构酶通过在裂解单个DNA链中催化否定性超螺旋DNA的放松 内部的双链DNA并通过完整的DNA单链穿过休息 切割链的宗教改变DNA拓扑。大酶的分子机制 传递DNA进出的协调运动所需的构象变化 DNA门是阐明细菌top1如何放松超涂层的DNA的关键障碍 高效率以防止高性DNA超螺旋和R环稳定，可能会在 转录。细菌TOP1的这一重要功能得到了与RNA的直接TOP1相互作用的支持 我们已经表征并发现由内源性毒素在分枝杆菌中靶向的聚合酶。为了 未来的研究，我们将创建新的Top1突变体 主动部位并研究对体内弛豫活动的影响以及与DNA的体外相互作用 基材。催化效率降低的突变体将进一步研究，以确定突变是否影响 门开口的动力学和DNA链通道。我们将捕获新的结构构象 X射线晶体学的Top1-DNA复合物可能代表催化循环的不同阶段 并用单分子测定测量栅极开放闭合动力学。结构研究也将 结合包括RNA在内的其他配体。 IA型拓扑异构酶已经发展为包括TOP1和TOP3 在生命的所有三个王国中，都有在DNA和RNA底物上都具有双重活性的酶。 RNA 人类top3b的拓扑异构酶活性已被证明是神经发育和 酶还参与R环抑制和基因组稳定性。我们正在建模RNA相互作用 带有分子动力学模拟的IA型拓扑异构酶，以确定DNA和RNA底物如何 酶会议和相互作用残差的变化可能会有所不同。我们有 发起的研究以确定功能突变或小分子探针的分离 区分体内的DNA和RNA拓扑异构酶活性。研究细胞研究的研究工具 RNA拓扑异构酶的活性和调节将对该领域产生重要而持久的影响。