Single-molecule measurements of DNA topology and topoisomerases

DNA 拓扑和拓扑异构酶的单分子测量

• 批准号: 8557906
• 负责人: Keir Neuman
• 金额: $ 80.57万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8557906
• 关键词: Affect; Aging; Antibiotics; Bacteria; Bacterial DNA Topoisomerase II; Biological Assay; C-terminal; Cell Death; Cell division; Chromosomal Instability; Chromosomes; Clinical; Collaborations; Complex; Computers; DNA; DNA Gyrase; DNA Topoisomerase IV; DNA Topoisomerases; DNA biosynthesis; Data; Dependence; Detection; Development; Discrimination; Enzymes; Equilibrium; Escherichia coli; Exhibits; Fluorescence; Goals; Human; Imagery; In Vitro; Incidence; Individual; Kinetics; Link; Magnetism; Maintenance; Malignant Neoplasms; Measurement; Measures; Modeling; Molecular; Monitor; Multienzyme Complexes; Mutation; Nuclear; Organism; Pathway interactions; Play; Poison; Population; Positioning Attribute; Process; Reaction; Reading; Relaxation; Research; Resolution; Role; Series; Sister; Superhelical DNA; Techniques; Testing; Time; Topoisomerase; Topoisomerase II; Topoisomerase III; Torque; Type I DNA Topoisomerases; Universities; Variant; Work; Yeasts; base; chemotherapy; helicase; in vivo; inhibitor/antagonist; instrument; instrumentation; interest; molecular domain; mutant; optical traps; prevent; research study; simulation; single molecule; small molecule; wound

Research in Progress Currently, there are three main ongoing projects in the lab: The first project is focused on elucidating mechanistic details of the interaction between type II topoisomerases and DNA. One aspect of this interaction concerns the ability of type II topoisomerases to relax the topology of DNA to below equilibrium values. In vivo these topoisomerases are responsible for unlinking replicated chromosomes prior to cell division. Since even a single link between sister chromosomes can prevent division and induce cell death, it is important that these enzymes preferentially unlink rather than link DNA molecules. In vitro it was shown that this is the case, but the mechanism remains a mystery. Previously we have shown that a mechanism based on a sharp bend imposed on the DNA by the topoisomerase cannot explain the extent of non-equilibrium simplification, and cannot explain the differences in non-equilibrium simplification among different type II topoisomerases (bacterial, human, yeast). We have recently completed testing two alternative models of topology simplification. The models postulate either a kinetic proofreading mechanism in which the topoisomerase catalyzes strand passage only after repeatedly encountering a DNA segment, or a mechanism in which the topoisomerase specifically recognizes DNA in a hooked juxtaposition geometry. Using magnetic tweezers we measured the unlinking of two DNA strands wrapped around each other a specific number of times under a controlled force. By measuring the rate of strand passage by a type II topoisomerase as a function of the imposed geometry and force and performing Monte-Carlo simulations to obtain the distribution of DNA configurations for each condition, we were able to test both models. The data indicate that type II topoisomerases can catalyze DNA strand-transfer with each collision of two DNA segments, thereby ruling out the kinetic proof reading model. Furthermore, preliminary evidence suggests that DNA unlinking rates are not highly correlated with the degree of hookedness of the two strands. Further tests, currently underway, will allow us to unambiguously determine the validity of the hooked juxtaposition model in describing the activity of type II topoisomerases. A second aspect of the interaction between type II topoisomerases and their DNA substrates concerns the diverse topological activities exhibited by type II topoisomerases that share a common mechanism. These activities include the symmetric relaxation of positively and negatively supercoiled DNA by most type II topoisomerases, the introduction of negative supercoils by DNA gyrase, and the asymmetric relaxation of negative and positive supercoils by some type II enzymes. These differences in activity are believed to arise from differences in the C-terminal domains (CTDs), but the molecular basis underling these variations in activity have not been elucidated. We have produced a series of CTD mutants of E. coli Topoisomerase IV (Topo IV). We are employing a combination of ensemble and single molecule assays to test the effects of these mutations on the substrate selectivity. In collaboration with Neil Osheroff at Vanderbilt University, we have investigated the mechanism of chiral sensing by human type II topoisomerase (hTopo II). Employing a single-molecule magnetic-tweezers based supercoil relaxation assay, we compared the chiral discrimination activity of hTopo II with that of E. coli Topo IV. Both enzymes preferentially relax positive supercoils. Despite this functional similarity, the two enzymes employ different mechanisms to achieve chiral discrimination. For the bacterial enzyme there is a dramatic difference in the processivity of positive verses negative supercoil relaxation. In contrast chiral discrimination by the human enzyme is achieved by changes in relaxation rate rather than processivity, which we have shown is remarkably high. These results combined with the tension dependence of the relaxation rate indicate that capture of the second DNA segment (the transfer segment) is the rate determining step in the strand passage reaction of human type II topoisomerase, and by extension all type II topoisomerases. The second project is focused the mechanisms underlying multi-enzyme complex activity. RecQ helicases and topoisomerase III have been shown to functionally and physically interact in organisms ranging from bacteria to humans. Disruption of this interaction leads to severe chromosome instability, however the specific activity of the enzyme complex is unclear. Analysis of the complex is complicated by the fact that both the helicase and the topoisomerase individually modify DNA. The ability of single-molecule techniques to measure the activity of a single enzyme or enzyme complex in real time is well suited to the study of such complicated processes in which multiple activities may occur over multiple time scales. In collaboration with Mihaly Kovacs at Etovos University, Hungry, we are using single-molecule measurements of DNA unwinding to elucidate the kinetics and step size of RecQ helicase alone and in the presence of Topo III. These experiments will pave the way for experiments in which the activity and the association state of single enzymes and complexes will be assayed simultaneously using a combination of single molecule manipulation and single molecule visualization techniques. The third related project, in collaboration with Yves Pommier in NCI, is focused on the mechanisms of supercoil relaxation by human type IB topoisomerases, and in the effects of chemotherapy agents that inhibit type IB topoisomerases. Type IB topoisomerases are essential enzymes that relax over wound (positively supercoiled) DNA generated ahead of the replication machinery during DNA synthesis. Type IB topoisomerases are also important chemotherapy targets. Potent chemotherapy agents that specifically inhibit type IB topoisomerases are currently in clinical use and additional agents are in development. We are using single-molecule magnetic-tweezers based assays to measure the activity of individual type IB topoisomerases and the effects of chemotherapy agents on the activity. These experiments provide molecular level details of the supercoil relaxation process and how it is influenced by the degree of DNA supercoiling, the tension on the DNA, and the presence of specific chemotherapy agents. These measurements provide an unprecedented level of detail concerning how these important enzymes work and are inhibited by chemotherapy agents. We recently demonstrated that the human nuclear Topoisomerase IB is remarkably insensitive to the effects of twist or torque on the DNA. This observation, combined with the first direct measurement of the cleavage kinetics at the single-molecule level, allowed us to formulate a comprehensive model for the complete relaxation and religation process catalyzed by type IB topoisomerases. This model reveals a hitherto unobserved intermediate state in the relaxation cycle, and provides a mechanistic framework for the action of inhibitors. We are currently using this model to interpret the affects of three inhibitors representing different inhibition mechanisms. These projects have been enabled by the development of a unique magnetic tweezers instrument that affords high spatial and temporal resolution measurements of DNA topology combined with real-time computer control and position stabilization. The ongoing development and improvement of this magnetic tweezers instrument represents a sustained research endeavor. Future research goals: Our immediate goal is the completion of the ongoing projects in the lab. Longer term goals include the development of a new optical trap and magnetic tweezers instrument combined with single-molecule fluorescence detection.

正在进行的研究 目前，实验室中有三个主要正在进行的项目： 第一个项目的重点是阐明II型拓扑异构酶与DNA之间相互作用的机理细节。这种相互作用的一个方面涉及II型拓扑异构酶将DNA拓扑拓扑的能力降低到平衡值以下。在体内，这些拓扑异构酶负责在细胞分裂之前与重复的染色体联系。由于即使是姊妹染色体之间的单一联系也可以防止分裂并诱导细胞死亡，因此重要的是，这些酶优先链接而不是链接DNA分子。在体外，这表明是这种情况，但是机制仍然是一个谜。 以前，我们已经表明，基于拓扑异构酶对DNA施加的尖锐弯曲的机制无法解释非平衡简化的程度，并且无法解释不同II型拓扑异构酶（细菌，人类，人类，酵母）之间非平衡简化的差异。我们最近完成了测试拓扑简化的两个替代模型。这些模型假设是一种动力学校对机制，在该机制中，拓扑异构酶仅在反复遇到DNA节段后才催化链段，或者在其中特定识别挂钩并置几何形状中的DNA的机制。使用磁镊子，我们测量了两个DNA链在受控力下互相包裹的两个DNA链。通过测量II型拓扑异构酶的链条通过的速率，这是强加的几何形状和力的函数，并执行蒙特卡罗模拟，以获得每种条件的DNA构型的分布，我们能够测试这两个模型。数据表明，II型拓扑异构酶可以在两个DNA段的每个碰撞中催化DNA链转移，从而排除动力学校验阅读模型。此外，初步证据表明，DNA未链接速率与两条链的钩度程度不高度相关。目前正在进行的进一步测试将使我们能够明确确定钩置模型在描述II型拓扑异构酶活性时的有效性。 II型拓扑异构酶及其DNA底物之间相互作用的第二个方面涉及具有共同机制的II型拓扑异构酶所表现出的各种拓扑活动。这些活性包括大多数II型拓扑异构酶对阳性和负面的DNA的对称性放松，通过DNA陀螺酶引入负超高以及通过某些II型II型酶对负和阳性超级辅助的不对称松弛。人们认为这些活性的差异是由C末端结构域（CTD）的差异引起的，但是尚未阐明这些活性变化的分子基础。我们已经产生了一系列大肠杆菌拓扑异构酶IV（TOPO IV）的CTD突变体。我们正在采用集合和单分子测定的组合来测试这些突变对底物选择性的影响。与范德比尔特大学（Vanderbilt University）的尼尔·奥斯格罗夫（Neil Osheroff）合作，我们研究了人类II型拓扑异构酶（HTOPO II）的手性传感机制。 我们采用基于单分子磁性 - 磁铁的超级涂层弛豫测定法，我们将HTOPO II的手性歧视活性与大肠杆菌TOPO IV的手性歧视活性进行了比较。 这两种酶都优先放松阳性超元。尽管这种功能相似性，但这两种酶采用了不同的机制来实现手性歧视。 对于细菌酶，正经文的加工性存在巨大差异。 相比之下，人类酶的手性歧视是通过放松率的变化而不是加工性来实现的，我们已经证明这非常高。 这些结果结合了弛豫率的张力依赖性，表明捕获第二个DNA段（转移段）是人类II型拓扑异构酶的链传递反应的速率确定步骤，并且通过扩展所有II型拓扑异构酶。 第二个项目集中于多酶复合活性的基础机制。 RECQ解旋酶和拓扑异构酶III已显示在功能和物理上在细菌到人类的生物中相互作用。这种相互作用的破坏会导致严重的染色体不稳定性，但是酶复合物的特定活性尚不清楚。对复合物分析的分析使解旋酶和拓扑异构酶单独修饰DNA的事实变得复杂。单分子技术实时测量单个酶或酶复合物的活性的能力非常适合研究此类复杂过程，在这些过程中可能会在多个时间尺度上发生多种活动。与Hungry Etovos大学的Mihaly Kovacs合作，我们正在使用放弃的DNA的单分子测量值，以阐明单独的RECQ解旋酶的动力学和步骤大小，并且在Topo III的存在下。这些实验将为实验铺平道路，在这种实验中，将使用单分子操纵和单分子可视化技术的组合同时测定单个酶和复合物的活性和关联状态。 第三个相关项目与NCI中的Yves Pommier合作，重点介绍了人类IB IB拓扑异构酶的超级套弛豫的机制，以及抑制IB型拓扑异构酶的化学疗法剂的作用。 IB型拓扑异构酶是必不可少的酶，在DNA合成过程中在复制机械前产生的伤口（正涂成阳性的）DNA松弛。 IB型拓扑异构酶也是重要的化学疗法靶标。 目前，专门抑制IB型拓扑异构酶的有效化学疗法剂目前正在临床中，并且正在开发其他药物。 我们正在使用基于单分子磁性 - 磁性分子的测定法来测量单个IB型拓扑异构酶的活性以及化学疗法剂对活性的影响。 这些实验提供了超副液弛豫过程的分子水平细节，以及它如何受到DNA超螺旋的影响，DNA上的张力以及特定化学疗法剂的存在。这些测量结果提供了前所未有的细节水平，涉及这些重要酶如何起作用并被化学疗法剂抑制。 我们最近证明，人核拓扑异构酶IB对扭曲或扭矩对DNA的影响非常不敏感。该观察结果与单分子水平上的裂解动力学的第一个直接测量相结合，使我们能够为由IB型topoisomerases催化的完整放松和宗教过程制定综合模型。 该模型在弛豫周期中揭示了迄今未观察到的中间状态，并为抑制剂作用提供了机械框架。 我们目前正在使用此模型来解释代表不同抑制机制的三种抑制剂的影响。 通过开发独特的磁性镊子仪器来实现这些项目，该仪器提供了对DNA拓扑的高空间和时间分辨率测量，并结合了实时计算机控制和位置稳定。 这种磁性镊子仪器的持续发展和改进代表了一项持续的研究努力。 未来的研究目标： 我们的直接目标是完成实验室正在进行的项目。长期目标包括开发新的光学陷阱和磁性镊子仪器，并结合单分子荧光检测。