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: 7734958
• 负责人: Keir Neuman
• 金额: $ 124.12万
• 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/7734958
• 关键词: Aging; Antibiotics; Area; Atomic Force Microscopy; Bacteria; Binding; Biological Assay; Biological Models; C-terminal; Cell Death; Cell division; Chromosomal Instability; Chromosomes; Communication; Complex; Coupled; DNA; DNA Gyrase; DNA Structure; DNA Topoisomerase IV; DNA reverse gyrase; Decompression Sickness; Detection; Development; Disruption; Enzymes; Equilibrium; Escherichia coli; Exhibits; Family; Fluorescence; Fluorescence Resonance Energy Transfer; Future; Goals; Human; Image; Imagery; In Vitro; Incidence; Individual; Kinetics; Link; Magnetism; Maintenance; Malignant Neoplasms; Measurement; Measures; Mechanics; Modeling; Molecular; Monitor; Motion; Multienzyme Complexes; Mutation; Nucleotides; Organism; Pathway interactions; Personal Satisfaction; Play; Point Mutation; Poison; Population; Process; Protein Isoforms; Range; Reaction; Relaxation; Research; Role; Series; Sister; Superhelical DNA; System; Techniques; Testing; Time; Topoisomerase; Topoisomerase II; Topoisomerase III; Variant; analog; base; chemotherapy; cofactor; helicase; in vivo; inhibitor/antagonist; instrument; instrumentation; interest; molecular domain; nanoscale; optical traps; polypeptide; prevent; research study; simulation; single molecule; size; small molecule

Summary of Research in Progress Currently, there are two 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 these enzymes preferentially unlink rather than link DNA. However the mechanism by which an enzyme that acts locally on the scale of nanometers can determine the global linking topology of micron sized DNA molecules remains a mystery. One proposed mechanism suggests that unlinking may be favored over linking if the topoisomerase induces a sharp bend in the DNA on binding. We are currently using atomic force microscopy to directly image type II topoisomerases bound to DNA. From these measurements we hope to extract the induced bend angle, which in combination with Monte Carlo simulations of DNA molecules with a given bend angle, will allow us to determine if the topoisomerase induced bending model can explain the observed unlinking/linking asymmetry. 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 reaction mechanism. Specifically, we are studying the molecular basis underlying the range of activities catalyzed by different isoforms of type II topoisomerases. 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 poorly conserved C-terminal domains (CTDs), but the molecular basis underling these variations in activity have not been elucidated. We have produced a series of specific mutations in the CTD region of Topoisomerase IV. We are employing a combination of ensemble and single molecule assays to test the effects of these mutations on the substrate selectivity and processivity of topoisomerase IV. We are also investigating the role of the CTD linker on the activity of Topoisomerase IV. The second project is focused on extending single-molecule techniques to dissect the detailed mechanisms underlying multi-enzyme complex formation and activity. Helicases of the RecQ family and topoisomerase III have been shown to functionally and physically interact in organisms ranging from bacteria to humans. Disruption of the interaction between the two enzymes leads to severe chromosome instability, however the mechanisms underlying their interaction, and the specific activity of the coupled enzyme remain unclear. Analysis of the coupled enzyme system is complicated by the fact that both the helicase and the topoisomerase individually modify the structure of DNA, and these activities must be distinguished from the activity of the coupled enzymes. 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. Following the activity of a single enzyme or multi-enzyme complex over time can reveal transient phenomena, fluctuations in activity, and the presence of enzyme sub-populations or enzymatic states, all of which are obscured by the averaging inherent in traditional ensemble measurements. In one project, we are investigating the activity of reverse gyrase, a unique topoisomerase from hyperthermophilic bacteria that is comprised of a helicase and a topoisomerase on a single polypeptide. Reverse gyrase serves as a model system in which to study the interaction of a helicase and a topoisomerase. Through the concerted activity of the two domains, reverse gyrase promotes the positive supercoiling (over-winding) of DNA, however the mechanism underlying this activity remains speculative. Single-molecule experiments will allow us to probe the details of the supercoiling reaction, and in conjunction with non-hydrolysable ATP analogs and point mutations, will allow us to determine the molecular basis for communication between the helicase and topoisomerase domains. In the second project are using single-molecule fluorescence techniques, primarily fluorescence resonance energy transfer (FRET), to measure the binding kinetics of RecQ helicase and Topo III form E. coli in isolation and in the presence of a variety of DNA substrates and nucleotide cofactors. These experiments and the experimental techniques employed will pave the way for more complex 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. 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. The activity of the complex can be determined from the mechanical changes in the DNA, or from the motion of the complex along the DNA strand. The composition of the complex can be determined from multicolor fluorescence detection and localization. This instrument will open up other areas of research including the possibility of observing the dynamics of supercoiled DNA.

正在进行的研究摘要 目前，实验室中有两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。 在体外表明，这些酶优先脱链，而不是链接DNA。 然而，在纳米范围内局部作用的酶可以确定微米大小的DNA分子的全局链接拓扑的机制仍然是一个谜。 提出的一种机制表明，如果拓扑异构酶在结合时诱导DNA急剧弯曲，则可能有利于连接链接。 我们目前正在使用原子力显微镜直接成像与DNA结合的II型拓扑异构酶。 从这些测量值中，我们希望提取诱导的弯曲角，这些弯曲角将与给定弯曲角的蒙特卡洛模拟与DNA分子的模拟结合使用，将使我们能够确定拓扑异构酶诱导的弯曲模型是否可以解释观察到的连接/链接/链接不对称。 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反应机制的II型拓扑异构酶所表现出的多种拓扑活性。 具体而言，我们正在研究由II型拓扑异构酶不同同工型催化的活性范围的分子基础。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保守的C末端结构域（CTD）的差异引起的，但是尚未阐明这些活性差异的分子基础。 我们在拓扑异构酶IV的CTD区域中产生了一系列特异性突变。 我们正在使用集合和单分子测定的组合来测试这些突变对拓扑异构酶IV的底物选择性和加工性的影响。我们还正在研究CTD接头在拓扑异构酶IV活性中的作用。 第二个项目的重点是扩展单分子技术，以剖析多酶复合物形成和活性的详细机制。 RECQ家族和拓扑异构酶III的解旋酶已显示在功能和物理上在细菌到人类的生物体中在物理上相互作用。 两种酶之间相互作用的破坏会导致严重的染色体不稳定，但是它们相互作用的机制以及耦合酶的特定活性仍不清楚。 偶联酶系统的分析使得解旋酶和拓扑异构酶单独修改DNA的结构，并且必须将这些活性与耦合酶的活性区分开来，这使得偶联酶系统的分析变得复杂。 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。随着时间的流逝，遵循单个酶或多酶复合物的活性可以揭示瞬时现象，活性波动以及酶亚群或酶状状态的存在，所有这些状态都因传统合奏测量中固有的平均而掩盖了所有这些。 在一个项目中，我们正在研究反向回旋酶的活性，这是一种来自热粒细菌的独特拓扑异构酶，该蛋白酶由单个多肽上由解旋酶和拓扑异构酶组成。 反向回旋酶是研究解旋酶和拓扑异构酶相互作用的模型系统。 通过这两个结构域的一致活性，反向回旋酶促进了DNA的阳性超螺旋（过度偏波），但是该活性的基础机制仍然投机。 单分子实验将使我们能够探测超串联反应的细节，并与不可用的ATP类似物和点突变结合使用，这将使我们能够确定解旋酶和拓扑酶和拓扑酶域之间通信的分子基础。 在第二个项目中，使用单分子荧光技术，主要是荧光共振能传递（FRET），以分离RecQ解旋酶和Topo III形成大肠杆菌的结合动力学，并在各种DNA底物和核苷酸辅助因子的情况下进行。 这些实验和所采用的实验技术将为更复杂的实验铺平道路，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缔合状态。 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。 复合物的活性可以从DNA的机械变化或沿DNA链的络合物运动中确定。 复合物的组成可以通过多色荧光检测和定位来确定。 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。