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.

项目总结/摘要 IA型拓扑异构酶普遍存在于生命的三个王国中，并且在生物学中起着至关重要的作用。 在复制、转录、重组的重要细胞过程中维持适当的DNA拓扑结构， 和修复。PI的研究活动为这门课提供了开创性的生化和结构发现 重要基因组调节因子的研究，并继续解决IA型催化机制的关键问题 拓扑异构酶和提供新的见解，他们的功能和监管的相互作用。该信息 需要利用存在于每种细菌病原体中的IA型拓扑异构酶作为新的治疗靶点， 寻找新的抗生素来帮助我们应对抗生素耐药性这一严重的全球健康挑战。IA型 拓扑异构酶通过切割DNA中的单个DNA链来催化负超螺旋DNA的松弛 下缠绕双链体DNA，并使互补DNA单链通过断裂， 切割链的重新连接以改变DNA拓扑结构。大酶的分子机制 构象变化，所需的协调运动的通过DNA进出的 DNA门是阐明细菌TOP1如何松弛负超螺旋DNA的关键屏障， 高效率地防止超负性DNA超螺旋和R环稳定化， 转录。细菌TOP 1的这一重要功能是通过TOP 1与RNA的直接相互作用来促进的 聚合酶，我们已经表征并发现其被分枝杆菌中的内源性毒素靶向。为 在未来的研究中，我们将创造新的TOP1突变体，这些突变体在结构域间相互作用中受到干扰， 活性位点，并研究对体内松弛活性和体外与DNA相互作用的影响 衬底将进一步研究催化效率降低的突变体，以确定受影响的突变是否 门开关动力学和DNA链通过。我们将捕捉新的结构构象 TOP 1-DNA复合物可能代表了X射线晶体学催化循环的不同阶段 并通过单分子测定来测量门的打开-关闭动力学。结构研究还将 并掺入其他配体，包括RNA。IA型拓扑异构酶已经进化为包括TOP 1和TOP 3 在所有三个生命王国中的酶，对DNA和RNA底物具有双重活性。的RNA 人TOP 3B的拓扑异构酶活性已被证明是神经发育所需的， 酶还参与R环抑制和基因组稳定性。我们正在模拟 IA型拓扑异构酶与分子动力学模拟，以确定如何DNA和RNA底物 可以通过改变酶的构象和相互作用的残基来进行差异调节。我们有 启动研究，以确定分离的功能突变或小分子探针，可用于 区分体内DNA和RNA拓扑异构酶活性。这种研究工具用于研究细胞 RNA拓扑异构酶的活性和调控将对该领域产生重要而持久的影响。