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: 8939762
• 负责人: Keir Neuman
• 金额: $ 81.49万
• 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/8939762
• 关键词: Aging; Agreement; Antibiotics; Bacteria; Bacterial DNA Topoisomerase II; Binding; Biological Assay; Biological Factors; Cell Death; Cell division; Chromosomal Instability; Chromosomes; Clinical; Collaborations; Complex; Computers; DNA; DNA Topoisomerases; DNA biosynthesis; DNA-Binding Proteins; Detection; Development; Enzymes; Equilibrium; Fluorescence; Geometry; Goals; Human; Imagery; In Vitro; Incidence; Individual; Kinetics; Link; Magnetism; Maintenance; Malignant Neoplasms; Measurement; Measures; Mitochondria; Modeling; Molecular; Monitor; Multienzyme Complexes; Nuclear; Organism; Pathway interactions; Play; Poison; Population; Positioning Attribute; Process; Protein Binding; Reading; Relaxation; Research; Resolution; Role; Sister; Superhelical DNA; Techniques; Testing; Thermodynamics; Time; Topoisomerase; Topoisomerase I inhibition; Topoisomerase II; Topoisomerase III; Torque; Type I DNA Topoisomerases; Universities; Work; Yeasts; base; chemotherapy; helicase; in vivo; inhibitor/antagonist; instrument; instrumentation; interest; lamellarin D; novel; optical traps; prevent; research study; 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. Our recent measurements 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. More recently we proposed an alternative model of non-equilibrium topology simplification in which the preferential relaxation of supercoiled DNA arises from preferential binding of the type II topoisomerases to more supercoiled substrates, i.e., a topology-dependent binding. To test this model we first developed a novel assay to measure DNA topology-dependent protein binding. We have validated this assay with a number of DNA binding proteins. Using this assay, we have identified a significant influence of DNA topology on the binding of type II topoisomerases to DNA, consistent with the premise of our model. Thermodynamic modeling of the relaxation process suggests that the topology-dependent binding contributes to the non-equilibrium relaxation process, but we have not as of yet achieved quantitative agreement with experimental measurements of non-equilibrium relaxation. Further experimental and theoretical work is ongoing to resolve the remaining quantitative discrepancies. Nonetheless, this work has led to the development of a novel and very general new protein-DNA binding assay and has uncovered a novel mode of action of 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. More recently, we have employed the single-molecule supercoil relaxation assay to characterize the molecular consequences of a unique mitochondrial topoisomerase I specific inhibitor, Lamellarin-D, a natural product derived from a mollusk. Binding of the inhibitor to the topoisomerase results in DNA topology-dependent changes in the rate of supercoil relaxation and inhibition of religation. We have recently extended these measurements to compare the mechanistic consequences of nuclear topoisomerase I inhibition by four different inhibitors corresponding to three classes of compounds. The single-molecule results reveal significant topology-dependent changes in the effects of the inhibitors on the topoisomerase. These measurements also provide a complete kinetic characterization of inhibitor binding to topoisomerases engaged in relaxation of defined topological states, which have implications for the mechanism and efficacy of inhibition in vivo. 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，实验室主要正在进行三个项目： 第一个项目的重点是阐明 II 型拓扑异构酶和 DNA 之间相互作用的机制细节。这种相互作用的一个方面涉及 II 型拓扑异构酶将 DNA 拓扑松弛至低于平衡值的能力。在体内，这些拓扑异构酶负责在细胞分裂之前断开复制的染色体的连接。由于即使姐妹染色体之间的单个连接也可以阻止分裂并诱导细胞死亡，因此重要的是这些酶优先断开而不是连接 DNA 分子。体外研究表明确实如此，但其机制仍然是个谜。之前我们已经证明，基于拓扑异构酶对 DNA 施加急剧弯曲的机制无法解释非平衡简化的程度，也无法解释不同 II 型拓扑异构酶（细菌、人类、酵母）之间非平衡简化的差异。我们最近完成了两种拓扑简化替代模型的测试。这些模型假设要么是一种动力学校对机制，其中拓扑异构酶仅在反复遇到 DNA 片段后才催化链通过，要么是一种机制，其中拓扑异构酶特异性识别钩状并置几何结构中的 DNA。我们最近的测量表明，II 型拓扑异构酶可以在两个 DNA 片段每次碰撞时催化 DNA 链转移，从而排除了动力学校对模型。此外，初步证据表明 DNA 解链率与两条链的钩状程度并不高度相关。目前正在进行的进一步测试将使我们能够明确确定钩状并置模型在描述 II 型拓扑异构酶活性方面的有效性。 最近，我们提出了一种非平衡拓扑简化的替代模型，其中超螺旋 DNA 的优先松弛源自 II 型拓扑异构酶与更多超螺旋底物的优先结合，即拓扑依赖性结合。 为了测试这个模型，我们首先开发了一种新的检测方法来测量 DNA 拓扑依赖性蛋白质结合。 我们已经用许多 DNA 结合蛋白验证了该测定。通过这种测定，我们发现了 DNA 拓扑结构对 II 型拓扑异构酶与 DNA 结合的显着影响，这与我们模型的前提一致。弛豫过程的热力学模型表明，拓扑依赖的结合有助于非平衡弛豫过程，但我们尚未与非平衡弛豫的实验测量达成定量一致。 进一步的实验和理论工作正在进行中，以解决剩余的定量差异。 尽管如此，这项工作导致了一种新颖且非常通用的新蛋白质-DNA 结合测定的发展，并揭示了 II 型拓扑异构酶的新作用模式。 第二个项目重点关注多酶复合物活性的机制。 RecQ 解旋酶和拓扑异构酶 III 已被证明在从细菌到人类的生物体中具有功能和物理相互作用。这种相互作用的破坏会导致严重的染色体不稳定，但酶复合物的具体活性尚不清楚。由于解旋酶和拓扑异构酶都单独修饰 DNA，因此对该复合物的分析变得复杂。单分子技术能够实时测量单个酶或酶复合物的活性，非常适合研究在多个时间尺度上可能发生多种活性的复杂过程。我们与 Hungry 埃托沃斯大学的 Mihaly Kovacs 合作，利用 DNA 解旋的单分子测量来阐明单独的 RecQ 解旋酶以及在 Topo III 存在的情况下的动力学和步长。这些实验将为结合单分子操作和单分子可视化技术同时测定单酶和复合物的活性和缔合状态的实验铺平道路。 第三个相关项目是与 NCI 的 Yves Pommier 合作，重点研究人类 IB 型拓扑异构酶的超螺旋松弛机制，以及抑制 IB 型拓扑异构酶的化疗药物的作用。 IB 型拓扑异构酶是在 DNA 合成过程中复制机制之前产生的伤口（正超螺旋）DNA 上松弛的必需酶。 IB 型拓扑异构酶也是重要的化疗靶点。特异性抑制 IB 型拓扑异构酶的有效化疗药物目前已在临床使用，并且其他药物正在开发中。我们使用基于单分子磁镊的检测来测量单个 IB 型拓扑异构酶的活性以及化疗药物对其活性的影响。这些实验提供了超螺旋松弛过程的分子水平细节，以及 DNA 超螺旋程度、DNA 张力和特定化疗药物的存在如何影响它。这些测量提供了关于这些重要酶如何发挥作用以及如何被化疗药物抑制的前所未有的详细信息。我们最近证明，人类核拓扑异构酶 IB 对 DNA 扭曲或扭矩的影响非常不敏感。这一观察结果与首次在单分子水平上直接测量裂解动力学相结合，使我们能够为 IB 型拓扑异构酶催化的完全松弛和再连接过程制定一个综合模型。该模型揭示了松弛周期中迄今为止未观察到的中间状态，并为抑制剂的作用提供了机制框架。最近，我们采用单分子超螺旋弛豫测定来表征独特的线粒体拓扑异构酶 I 特异性抑制剂 Lamellarin-D（一种源自软体动物的天然产物）的分子后果。抑制剂与拓扑异构酶的结合导致超螺旋松弛速率和再连接抑制发生 DNA 拓扑依赖性变化。 我们最近扩展了这些测量，以比较对应于三类化合物的四种不同抑制剂抑制核拓扑异构酶 I 的机制后果。 单分子结果揭示了抑制剂对拓扑异构酶的影响存在显着的拓扑依赖性变化。 这些测量还提供了抑制剂与拓扑异构酶结合的完整动力学特征，该拓扑异构酶参与了定义的拓扑状态的松弛，这对体内抑制的机制和功效具有影响。 这些项目是通过开发一种独特的磁镊仪器来实现的，该仪器可提供 DNA 拓扑的高空间和时间分辨率测量，并结合实时计算机控制和位置稳定。这种磁性镊子仪器的持续开发和改进代表了持续的研究努力。 未来的研究目标： 我们的近期目标是完成实验室正在进行的项目。长期目标包括开发与单分子荧光检测相结合的新型光阱和磁镊仪器。