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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要 IA 型拓扑异构酶在生命的三个王国中普遍存在，并在在复制、转录、重组等重要细胞过程中保持正确的 DNA 拓扑结构， PI 的研究活动为此类提供了开创性的生化和结构发现。重要的基因组调控因子，并继续解决IA型催化机制的关键问题拓扑异构酶并提供对其功能和调控相互作用的新见解。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应对 IA 型抗生素耐药性带来的严峻的全球健康挑战。拓扑异构酶通过切割DNA中的单链来催化负超螺旋DNA的松弛。缠绕下的双链 DNA 并将互补 DNA 单链穿过之前的断裂处断裂链的关系改变DNA拓扑结构的大酶的分子机制。 DNA进出DNA协调运动所需的构象变化 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 DNA 超螺旋和 R 环稳定化过程中可能出现的情况细菌 TOP1 的这一重要功能是通过 TOP1 与 RNA 的直接相互作用来促进的。我们已对聚合酶进行了表征并发现它是分枝杆菌中内源毒素的目标。在未来的研究中，我们将创建新的 TOP1 突变体，在距活性位点并研究其对体内松弛活性和体外与 DNA 相互作用的影响将进一步研究催化效率降低的突变体，以确定突变是否受到影响。我们将捕获门的打开-关闭动力学和 DNA 链通道。 TOP1-DNA 复合物可能代表 X 射线晶体学催化循环的不同阶段并通过单分子分析测量门的打开-关闭动力学。结合其他配体，包括 RNA，IA 型拓扑异构酶已进化为包括 TOP1 和 TOP3。生命三个王国中的酶，对 DNA 和 RNA 底物具有双重活性。人类 TOP3B 的拓扑异构酶活性已被证明是神经发育和神经发育所必需的酶还参与 R 环抑制和基因组稳定性。我们正在模拟 R 环的 RNA 相互作用。 IA 型拓扑异构酶通过分子动力学模拟确定 DNA 和 RNA 底物如何可以通过酶构象和残基的变化来进行差异调节。发起了研究来鉴定可用于分离功能突变或小分子探针区分体内 DNA 和 RNA 拓扑异构酶活性的研究工具。 RNA拓扑异构酶活性和调控将对该领域产生重要而持久的影响。