Structure, Mechanism and Interactions of Type IA Topoisomerases

项目摘要/摘要 IA型拓扑异构酶广泛存在于生命的三大王国中，并在 在复制、转录、重组等重要细胞过程中保持适当的DNA拓扑 并进行修复。PI的研究活动为这门课提供了开创性的生化和结构发现 ，并继续解决有关IA型催化机制的关键问题 并对它们的功能和调控相互作用提供了新的见解。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峻的全球健康挑战。IA类型 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 缠绕双链DNA并使互补DNA单链穿过之前的断裂 裂解链的宗教化以改变DNA拓扑。大分子酶的分子机制 通过的DNA进出的协调运动所需的构象变化 DNA门是阐明细菌TOP1如何松弛负超螺旋DNA的关键障碍 高效率防止超负DNA超卷曲和R环稳定 抄写。细菌TOP1的这一重要功能是通过TOP1与RNA的直接相互作用而促进的 聚合酶，我们已经确定并发现它是分枝杆菌内源性毒素的靶标。为 在未来的研究中，我们将创造新的TOP1突变体，这些突变体在距离 活性部位及对体内松弛活性和体外与DNA相互作用的影响 底物。将对催化效率降低的突变进行进一步研究，以确定突变是否影响 门的启闭动力学和DNA链通道。我们将捕获新的结构构象 用X射线结晶学研究可能代表催化循环不同阶段的TOP1-DNA复合体 并用单分子检测法测定了闸门的开闭动力学。结构研究也将 加入包括RNA在内的其他配体。IA型拓扑异构酶已进化为包括TOP1和TOP3 在所有三个生命王国中，在DNA和RNA底物上都具有双重活性的酶。核糖核酸 人类TOP3B的拓扑异构酶活性被证明是神经发育所必需的，并且 酶还参与了R环的抑制和基因组的稳定。我们正在对RNA相互作用进行建模 用分子动力学模拟确定DNA和RNA底物的IA型拓扑异构酶 可以通过酶构象和相互作用残基的变化来不同地调节。我们有 启动研究以确定功能突变或小分子探针的分离，可用于 区分DNA和RNA体内的拓扑异构酶活性。这样的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和持久的影响。

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.