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: 9354111
• 负责人: Keir Neuman
• 金额: $ 113.41万
• 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/9354111
• 关键词: Accelerometer; Aging; Antibiotics; Bacteria; Binding; Binding Proteins; Biochemical; Biological Assay; Bloom Syndrome; Cell Death; Cell Extracts; Cell division; Chromosomal Instability; Chromosomes; Clinical; Collaborations; Complex; Computers; DNA; DNA Topoisomerases; DNA biosynthesis; Databases; Detection; Development; Enzymes; Equilibrium; Eukaryotic Cell; Fluorescence; Genetic Recombination; Genome Stability; Geometry; Goals; Human; Imagery; In Vitro; Incidence; Individual; Kinetics; Link; Magnetism; Maintenance; Malignant Neoplasms; Measurement; Measures; Mediating; Mitochondria; Mitochondrial Diseases; Modeling; Molecular; Monitor; Multienzyme Complexes; National Heart, Lung, and Blood Institute; National Institute of Diabetes and Digestive and Kidney Diseases; Natural Products; Nuclear; Organism; Orthologous Gene; Pathway interactions; Plasmids; Play; Poison; Population; Positioning Attribute; Process; Prokaryotic Cells; Reading; Relaxation; Research; Resolution; Role; Single Nucleotide Polymorphism; Sister; Superhelical DNA; System; Techniques; Tertiary Protein Structure; Testing; Texas; Time; Topoisomerase; Topoisomerase I inhibition; Topoisomerase II; Topoisomerase III; Type I DNA Topoisomerases; United States National Institutes of Health; Universities; Variant; Work; Yeasts; base; cancer type; chemotherapy; follow-up; helicase; in vivo; inhibitor/antagonist; instrument; instrumentation; interest; lamellarin D; medical schools; mutant; novel; optical traps; prevent; research study; segregation; single molecule; small molecule inhibitor; temporal measurement; tool; wound

Research in Progress Currently, there are Four 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. 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. We have also established that the proposed kinetic proofreading mechanism in which the topoisomerase catalyzes strand passage only after repeatedly encountering a DNA segment, does not hold for type II topoisomerases, thereby ruling out the kinetic proof reading model for below equilibrium topology simplification. We have proposed an alternative model of non-equilibrium topology simplification in which the preferential relaxation of supercoiled DNA arises from preferential binding of type II topoisomerases to supercoiled substrates, i.e., topology-dependent binding. To test this model we first developed and validated an assay to measure DNA topology-dependent protein binding. Using this assay, we have established that DNA topology significantly influences the binding of type II topoisomerases to DNA, consistent with the premise of our model. Furthermore, in collaboration with Neil Osheroff at Vanderbilt University Medical School, we have demonstrated that the differences in the influence of DNA topology on type II topoisomerase binding from different organisms (yeast or bacteria) correlates with the degree of non-equilibrium topology simplification, providing the first measured correlation between a proposed mechanism and the degree of non-equilibrium simplification for enzymes from different organisms. In collaboration with Stephen Levene at the University of Texas at Dallas we are currently extending these measurements to investigate preferential binding to knotted and linked DNA molecules, which are also preferentially unknotted and unlinked by 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. In collaboration with Mihaly Kovacs at Etovos University, Hungry, we are using single-molecule measurements of DNA unwinding and unlinking to elucidate the detailed of RecQ helicase activity 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. Working towards the overarching goal of understanding the mechanistic basis for the chromosome maintenance activities of the RecQ-Topo III complex, we have recently dissected the functional roles of specific and conserved protein domains in both the bacterial RecQ and in the human ortholog, Blooms syndrome helicase. We identified a novel DNA geometry-dependent binding mode of RecQ helicases mediated by a specific domain. We further establish the importance of this domain for proper resolution of recombination intermediates both in vitro and in vivo. In follow up work, we have determined the mechanism through which RecQ unwinds DNA and how this mechanism leads to the coordinated binding of key accessory domains involved in preserving genomic stability. 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. We 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. Finally, we have also characterized two human mitochondrial topoisomerase variants associated with single nucleotide polymorphisms that are correlated with certain cancer types in the NCI cancer data base, and a point-mutant of mitochondrial topoisomerase associated with mitochondrial disease. The single-molecule measurements revealed specific alterations in the activity of these topoisomerase mutants at relatively high supercoiling levels that were not detectable in conventional biochemical assays of topoisomerase activity. The fourth project is a collaboration with Kiyoshi Mizuuchi in NIDDK and Jian Liu in NHLBI at the NIH to determine the mechanism of plasmid segregation by the bacterial Par system through a combination of experimental and theoretical approaches. 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 goals include the development of a new optical trap and magnetic tweezers instrument combined with single-molecule fluorescence detection, incorporating single-molecule detection into the magnetic tweezers instrument, and developing a set of tools and approaches to directly monitor type II inhibition by antibiotics and chemotherapeutic compounds at the single-molecule level. Longer terms goals include performing the single-molecule assays in eukaryotic or prokaryotic cell extract to more closely recapitulate the processes occurring in the cellula

正在进行的研究 目前，实验室中有四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。我们已经表明，基于拓扑异构酶对DNA施加的急转弯的机制无法解释非平衡简化的程度。我们还确定，所提出的动力学校对机制，其中拓扑异构酶仅在反复遇到DNA片段后才催化链条通过，而对于II型Type type topoisomerases却不成立，从而排除了平衡拓扑以下的动力学验证校验校验模型。 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型拓扑异构酶与超螺旋底物的优先结合（即拓扑依赖性结合）产生的。为了测试该模型，我们首先开发并验证了一种测定DNA拓扑依赖性蛋白结合的测定法。使用该测定法，我们已经确定DNA拓扑会显着影响II型拓扑异构酶与DNA的结合，这与我们模型的前提一致。此外，在与范德比尔特大学医学院的尼尔·奥斯格罗夫（Neil Osheroff）合作，我们证明，DNA拓扑对不同生物体（酵母或细菌）与II型拓扑异构酶结合的影响的差异与非平衡拓扑的程度相关，从而简化了不平衡的范围，而不是在不平衡的范围中，而不是在不平衡的情况下进行了依赖的范围。有机体。在达拉斯德克萨斯大学的斯蒂芬·莱文（Stephen Levene）合作，我们目前正在扩展这些测量值，以研究与打结和连接的DNA分子的优先结合，这些分子也优先通过II型拓扑异构酶链接并取消了链接。 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；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。与Hungry Etovos University的Mihaly Kovacs合作，我们正在使用DNA放松和UNINK的单分子测量值，以阐明单独的RECQ解旋酶活性的详细信息以及在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。 朝着理解RECQ-TOPO III复合物染色体维持活动的机理基础的总体目标努力，我们最近剖析了细菌RECQ和人类直系同源物（Blooms Blooms综合酶）中特定和保守蛋白质结构域的功能作用。我们确定了由特定结构域介导的RECQ解旋酶的新型DNA几何依赖性结合模式。我们进一步确定了该领域对于正确解析重组中间体的重要性。在后续工作中，我们确定了RECQ解开DNA的机制，以及该机制如何导致与维护基因组稳定性有关的关键附件域的协调结合。 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。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Lamellarrin-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。最后，我们还表征了两种与单核苷酸多态性相关的人类线粒体拓扑酶变体，这些变体与NCI癌症数据库中的某些癌症类型相关，以及与线粒体疾病相关的线粒体拓扑酶的点突出。单分子测量结果揭示了这些拓扑异构酶突变体在相对较高的超涂层水平上的活性变化，而在拓扑异构酶活性的常规生化测定中无法检测到。 第四个项目是与NIH的NHLBI的Kiyoshi Mizuuchi合作，通过实验方法和理论方法结合使用细菌PAR系统来确定质粒分离的机制。 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： 我们的直接目标包括开发新的光学陷阱和磁性镊子仪器，结合了单分子荧光检测，将单分子检测掺入磁性镊子仪器中，并开发了一套工具和方法，以直接监测抗生素和化学疗法化合物在单分子级上直接监测II型II型抑制作用。较长的术语目标包括在真核或原核细胞提取物中执行单分子测定，以更加紧密地概括在细胞中发生的过程