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: 8149475
• 负责人: Keir Neuman
• 金额: $ 81.35万
• 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/8149475
• 关键词: Binding; Chromosomal Instability; DNA; Diffusion; Digestion; Discrimination; Measurement; Quantum Dots; Topoisomerase; Topoisomerase III; single molecule

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 was the case, but the mechanism remains a mystery. One proposed mechanism for this topology simplification posits that the topoisomerase induces a sharp bend in the DNA on binding, which would favor unlinking over linking. Using atomic force microscopy (AFM), we have measured the DNA bend angle imposed by the binding of three type II topoisomerases from different organisms (Human, Yeast, and E. coli). The measured bend angles do not support the bend angle model of topology simplification. This is an important finding as the experimental evidence to date has been equivocal concerning the bend angle model. We have also developed new quantitative analysis methodologies for AFM images of protein DNA complexes. These include an image-processing based analysis of the DNA bend angle from AFM images that is faster and less prone to artifact than the currently used manual methods. Currently, we are 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. To test these models we are using magnetic tweezers to measure 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 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 can test both models. We anticipate that these experiments will unambiguously confirm or refute the two competing models for non-equilibrium topology simplification by 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 are also investigating the mechanism of chiral sensing by human type II topoisomerase (hTopo II). By measuring the relaxation of individual DNA molecules by hTopo II with magnetic tweezers, we determined that the mechanism of chiral discrimination by this enzyme is due to a salt-dependent difference in relaxation rates between positively and negatively supercoiled DNA. This was surprising given that chiral discrimination by E. coli Topo IV results from differences in processivity rather than relaxation rate. 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. We are using single-molecule fluorescence techniques to measure the unwinding kinetics and step size of RecQ helicase alone and in the presence of Topo III. These experiments and the 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. In the third project, a collaboration with Gregory Goldberg at Washington University St. Louis, we employed single-molecule TIRF to study the motion of single matrix metalloproteinases (MMPs) during the digestion of collagen. MMPs play an important role in physiological collagen processing pathways including tissue remodeling, wound healing and cell migration. However, the mechanistic details of MMP interactions with collagen have been refractory to study due to the complex nature of the collagen substrate and the motion of the MMPs. By tracking individual MMPs on isolated collagen fibers with high spatial and temporal resolution we could characterize the motion of the MMP on the substrate, and how this motion is coupled to proteolytic activity. This approach has provided detailed mechanistic information for this important class of enzymes. We have for the first time observed the complex motion of individual MMPs on collagen fibers and have developed a comprehensive quantitative model describing how this motion is coupled to proteolysis of the collagen fiber. We found that the motion of MMPs on collagen is both biased and hindered diffusion, that there are binding hot-spots for MMPs on collagen spaced 1 micron apart, and that the motion of MMPs on collagen is interrupted by long pauses of duration 1 second. These results were unanticipated and provide unprecedented insight into the interaction of MMPs with collagen while highlighting the unique capabilities of single-molecule methods to measure complex biomolecular processes. The initial measurements and comprehensive modeling are complete and we are writing the first manuscript. Furthermore, we developed new methodologies to treat diffusion in single-molecule traces, which are applicable to any single-molecule analysis of diffusion trajectories. Future work on the MMP tracking will be focused on improving the temporal and spatial resolution of the tracking in addition to extending the duration of individual trajectories through the use of quantum dot labels, or nitrogen vacancy nano-diamond labels. 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 capabilities. This instrument will permit simultaneous measurements of the activity and composition of multi-enzyme complexes interacting with a single DNA molecule. This instrument will open up other areas of research including the possibility of observing the dynamics of supercoiled DNA. As a first step in this direction, we are in the process of combining a magnetic tweezers instrument with single-molecule fluorescence detection capabilities.

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，但这种机制仍然是一个谜。该拓扑简化的一种提出的机制认为，拓扑异构酶在结合上诱导DNA急剧弯曲，这将有利于链接而不是链接。使用原子力显微镜（AFM），我们通过来自不同生物体（人类，酵母和大肠杆菌）的三种II型拓扑异构酶的结合来测量DNA弯曲角。测得的弯曲角不支持拓扑简化的弯曲角模型。这是一个重要发现，因为迄今为止的实验证据是关于弯曲角模型的模棱两可的。我们还为蛋白DNA复合物的AFM图像开发了新的定量分析方法。这些包括对AFM图像的DNA弯曲角度的基于图像处理的分析，该分析比当前使用的手动方法更快且易于伪像。 目前，我们正在测试两种拓扑简化的替代模型。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。为了测试这些模型，我们使用磁镊子来测量在受控力下相互包裹的两个DNA链的未链接。通过测量链条通道的速率作为施加的几何形状和力的函数，并执行蒙特卡洛模拟以获得每种条件的DNA构型的分布，我们可以测试这两个模型。我们预计，这些实验将明确确认或反驳两个竞争模型，以简化II型拓扑异构酶的非平衡拓扑模型。 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。我们生产了一系列的E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·奥塞罗夫（Neil Osheroff）合作，我们还研究了人类II型拓扑异构酶（HTOPO II）的手性传感机制。通过用磁镊子测量HTOPO II对单个DNA分子的松弛，我们确定该酶的手性歧视机制是由于盐依赖的盐依赖性差异呈阳性和负面层面的DNA之间的弛豫率差异。鉴于大肠杆菌IV的手性歧视是由于加工性差异而不是放松率而导致的。 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。我们正在使用单分子荧光技术来单独测量RECQ解旋酶的放松动力学和步骤大小，并且在TOPO III的存在下。这些实验以及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单个酶和复合物的关联的道路。 在第三个项目中，与华盛顿大学圣路易斯的Gregory Goldberg合作，我们采用了单分子TIRF来研究胶原蛋白消化过程中单基质金属蛋白酶（MMP）的运动。 MMP在生理胶原蛋白加工途径中起重要作用，包括组织重塑，伤口愈合和细胞迁移。然而，由于胶原蛋白底物的复杂性质和MMP的运动，MMP与胶原蛋白相互作用的机械细节已难以理解。通过在具有高空间和时间分辨率的孤立胶原纤维上跟踪单个MMP，我们可以表征MMP在底物上的运动，以及该运动如何与蛋白水解活性耦合。这种方法为这类酶提供了详细的机械信息。我们首次观察到单个MMP在胶原蛋白纤维上的复杂运动，并开发了一个全面的定量模型，描述了该运动如何与胶原纤维的蛋白水解结合。我们发现，MMP在胶原蛋白上的运动既有偏置又受阻的扩散，因此在胶原蛋白间隔1微米的胶原蛋白上，MMP具有结合热点，并且MMP在胶原蛋白上的运动被持续时间1秒的长时间停顿。这些结果是意想不到的，并提供了对MMP与胶原蛋白相互作用的前所未有的见解，同时突出了单分子方法的独特能力来测量复杂的生物分子过程。最初的测量和全面的建模是完整的，我们正在编写第一个手稿。 此外，我们开发了新的方法来处理单分子轨迹的扩散，这些方法适用于任何扩散轨迹的单分子分析。除了通过使用量子点标签或氮空位纳米二角标记，未来在MMP跟踪上的工作将集中在跟踪的时间和空间分辨率上。 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能力。该仪器将允许同时测量与单个DNA分子相互作用的多酶复合物的活性和组成。该仪器将开放其他研究领域，包括观察超螺旋DNA的动力学的可能性。作为朝这个方向的第一步，我们正在将磁性镊子仪器与单分子荧光检测能力相结合。