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。体外实验表明确实如此，但其机制仍然是个谜。一种针对这种拓扑简化的机制提出，拓扑异构酶在结合时诱导 DNA 急剧弯曲，这将有利于断开连接而不是连接。使用原子力显微镜 (AFM)，我们测量了来自不同生物体（人类、酵母和大肠杆菌）的三种 II 型拓扑异构酶结合所产生的 DNA 弯曲角度。测量的弯曲角度不支持拓扑简化的弯曲角度模型。这是一个重要的发现，因为迄今为止关于弯曲角度模型的实验证据是模棱两可的。我们还为蛋白质 DNA 复合物的 AFM 图像开发了新的定量分析方法。其中包括基于图像处理的 AFM 图像 DNA 弯曲角度分析，与当前使用的手动方法相比，该分析速度更快且不易产生伪影。 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。为了测试这些模型，我们使用磁性镊子来测量两条 DNA 链在受控力下相互缠绕特定次数的脱开情况。通过测量链通过速率与施加的几何形状和力的关系，并执行蒙特卡罗模拟以获得每种条件下 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证实或反驳 II 型拓扑异构酶非平衡拓扑简化的两个竞争模型。 II 型拓扑异构酶与其 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不同拓扑活性。这些活性包括大多数 II 型拓扑异构酶对正超螺旋 DNA 的对称松弛、DNA 旋转酶引入负超螺旋的作用以及某些 II 型酶对负超螺旋和正超螺旋的不对称松弛。这些活性差异被认为是由 C 末端结构域 (CTD) 的差异引起的，但这些活性差异的分子基础尚未阐明。我们已经生产了一系列大肠杆菌拓扑异构酶 IV (Topo IV) 的 CTD 突变体。我们正在采用整体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。我们还与范德比尔特大学的 Neil Osheroff 合作，研究人类 II 型拓扑异构酶 (hTopo II) 的手性传感机制。通过使用磁力镊子测量 hTopo II 对单个 DNA 分子的松弛，我们确定该酶的手性辨别机制是由于正超螺旋 DNA 和负超螺旋 DNA 之间的盐依赖性松弛速​​率差异所致。这是令人惊讶的，因为大肠杆菌 Topo IV 的手性辨别是由持续性而不是弛豫率的差异引起的。 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。我们使用单分子荧光技术来测量 RecQ 解旋酶单独和在 Topo III 存在下的解旋动力学和步长。这些实验和将为使用单分子操作和单分子可视化技术的组合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。 在第三个项目中，我们与圣路易斯华盛顿大学的 Gregory Goldberg 合作，采用单分子 TIRF 来研究单基质金属蛋白酶 (MMP) 在胶原蛋白消化过程中的运动。 MMP 在生理胶原蛋白加工途径中发挥着重要作用，包括组织重塑、伤口愈合和细胞迁移。然而，由于胶原蛋白基质和 MMP 运动的复杂性，MMP 与胶原蛋白相互作用的机制细节一直难以研究。通过以高空间和时间分辨率跟踪分离的胶原纤维上的单个 MMP，我们可以表征基质上 MMP 的运动，以及这种运动如何与蛋白水解活性耦合。这种方法为这一类重要的酶提供了详细的机制信息。我们首次观察到胶原纤维上单个 MMP 的复杂运动，并开发了一个全面的定量模型，描述这种运动如何与胶原纤维的蛋白水解耦合。我们发现MMPs在胶原蛋白上的运动是有偏差和受阻扩散的，MMPs在胶原蛋白上存在间隔1微米的结合热点，并且MMPs在胶原蛋白上的运动被持续1秒的长时间停顿打断。这些结果是出乎意料的，为 MMP 与胶原蛋白的相互作用提供了前所未有的见解，同时凸显了单分子方法测量复杂生物分子过程的独特能力。初步测量和综合建模已经完成，我们正在撰写第一篇手稿。 此外，我们开发了处理单分子痕迹扩散的新方法，适用于扩散轨迹的任何单分子分析。 MMP 跟踪的未来工作将集中于提高跟踪的时间和空间分辨率，以及通过使用量子点标签或氮空位纳米金刚石标签来延长单个轨迹的持续时间。 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。长期目标包括开发结合单分子荧光检测功能的新型光阱和磁镊仪器。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。该仪器将开辟其他研究领域，包括观察超螺旋 DNA 动力学的可能性。作为朝这个方向迈出的第一步，我们正在将磁镊仪器与单分子荧光检测功能相结合。