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，实验室主要正在进行两个项目： 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。 体外研究表明，这些酶优先断开而不是连接 DNA。 然而，在纳米尺度上局部作用的酶能够确定微米级 DNA 分子的全局连接拓扑的机制仍然是个谜。 一种提出的机制表明，如果拓扑异构酶在结合时诱导 DNA 急剧弯曲，则断开连接可能比连接更有利。 我们目前正在使用原子力显微镜直接对与 DNA 结合的 II 型拓扑异构酶进行成像。 我们希望从这些测量中提取诱导弯曲角度，结合具有给定弯曲角度的 DNA 分子的蒙特卡罗模拟，将使我们能够确定拓扑异构酶诱导弯曲模型是否可以解释观察到的断开/连接不对称性。 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反应机制的 II 型拓扑异构酶所表现出的不同拓扑活性。 具体来说，我们正在研究 II 型拓扑异构酶不同亚型催化的一系列活性的分子基础。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保守性较差的 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。 我们在拓扑异构酶 IV 的 CTD 区域产生了一系列特定突变。 我们正在采用整体和单分子检测的组合来测试这些突变对拓扑异构酶 IV 的底物选择性和持续合成能力的影响。我们还在研究 CTD 连接子对拓扑异构酶 IV 活性的作用。 第二个项目的重点是扩展单分子技术来剖析多酶复合物形成和活性的详细机制。 RecQ 家族的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。 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偶联酶的比活性仍不清楚。 由于解旋酶和拓扑异构酶都单独修饰 DNA 结构，因此偶联酶系统的分析变得复杂，并且必须将这些活性与偶联酶的活性区分开来。 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。随着时间的推移跟踪单一酶或多酶复合物的活性可以揭示瞬态现象、活性波动以及酶亚群或酶状态的存在，所有这些都被传统整体测量中固有的平均所掩盖。 在一个项目中，我们正在研究反向旋转酶的活性，这是一种来自超嗜热细菌的独特拓扑异构酶，由单个多肽上的解旋酶和拓扑异构酶组成。 反向旋转酶作为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协同活动，反向旋转酶促进 DNA 的正超螺旋（过度缠绕），但这种活动背后的机制仍然是推测性的。 单分子实验将使我们能够探索超螺旋反应的细节，并与不可水解的 ATP 类似物和点突变相结合，使我们能够确定解旋酶和拓扑异构酶结构域之间通讯的分子基础。 在第二个项目中，使用单分子荧光技术，主要是荧光共振能量转移 (FRET)，来测量 RecQ 解旋酶和 Topo III 形式大肠杆菌在分离情况下以及在各种 DNA 底物和核苷酸辅因子存在下的结合动力学。 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，在这些实验中，将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。 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。 复合物的活性可以通过 DNA 的机械变化或复合物沿 DNA 链的运动来确定。 复合物的组成可以通过多色荧光检测和定位来确定。 该仪器将开辟其他研究领域，包括观察超螺旋 DNA 动力学的可能性。