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。我们最近的测量表明，II型拓扑异构酶可以在两个DNA片段的每次碰撞中催化DNA链转移，从而排除动力学预测阅读模型。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。 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的优先放松是由II型拓扑异构酶与更多超涂层底物的优先结合产生的，即拓扑依赖性的结合。 为了测试该模型，我们首先开发了一种新的测定法，以测量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法。使用此测定，我们已经确定了DNA拓扑对II型拓扑异构酶与DNA的结合的重要影响，这与我们模型的前提一致。弛豫过程的热力学建模表明，依赖拓扑的结合有助于非平衡弛豫过程，但我们尚未达到与非平衡松弛的实验测量的定量一致性。 正在进行进一步的实验和理论工作，以解决剩余的定量差异。 尽管如此，这项工作导致了一种新型且非常通用的新蛋白-DNA结合测定法的发展，并发现了II型拓扑异构酶的新型作用方式。 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。最近，我们采用了单分子超高释放测定法来表征独特的线粒体拓扑异构酶I特异性抑制剂Lamellarin-D的分子后果，这是一种源自软体动物的天然产物。抑制剂与拓扑异构酶的结合会导致DNA拓扑依赖性的超属宽松率变化和宗教抑制。 我们最近扩展了这些测量值，以比较与三类化合物相对应的四种不同抑制剂的核拓扑异构酶I抑制作用的机理后果。 单分子的结果揭示了抑制剂对拓扑异构酶的影响的显着依赖性变化。 这些测量值还提供了抑制剂结合与拓扑异构体的抑制剂结合的完整动力学表征，这对定义的拓扑状态放松，这对体内抑制的机制和功效具有影响。 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。