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: 7594380
• 负责人: Keir Neuman
• 金额: $ 166.76万
• 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/7594380
• 关键词: Aging; Antibiotics; Atomic Force Microscopy; Bacteria; Binding; Biological Assay; Biological Models; Cell Death; Cell division; Cells; Chromosomal Instability; Chromosomes; Communication; Complex; Coupled; DNA; DNA Structure; DNA reverse gyrase; Decompression Sickness; Development; Disruption; Enzymes; Equilibrium; Escherichia coli; Family; Fluorescence; Fluorescence Resonance Energy Transfer; Frequencies; Future; Goals; Human; Image; Imagery; In Vitro; Incidence; Individual; Kinetics; Lasers; Light; Link; Magnetism; Maintenance; Malignant Neoplasms; Measurement; Measures; Modeling; Molecular; Monitor; Multienzyme Complexes; Nucleotides; Optics; Organelles; Organism; Oxygen; Pathway interactions; Personal Satisfaction; Play; Point Mutation; Poison; Population; Process; Proteins; Range; Rate; Reaction; Relative (related person); Research; Role; Rotation; Sampling; Singlet Oxygen; Sister; System; Techniques; Testing; Time; Topoisomerase; Topoisomerase II; Topoisomerase III; Type I DNA Topoisomerases; United States National Institutes of Health; analog; base; chemotherapy; cofactor; design; design and construction; helicase; human TOP1 protein; in vivo; inhibitor/antagonist; instrument; instrumentation; interest; nanometer; nanoscale; optical traps; polypeptide; prevent; research study; simulation; single molecule; singlet state; size; small molecule; sperm cell

Summary of Research in Progress Since arriving at the NIH at the end of February 2007, I have been concerned principally with setting up the lab and ordering parts for the single molecule instrumentation we are building. In addition to the ongoing process of designing and building single-molecule optical and magnetic manipulation instruments, we are pursing two projects. The first project 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 indeed 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 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. The second project concerns the longstanding problem of optically induced damage in optical trapping experiments. Optical traps provide a means of non-invasively manipulating micron sized objects with light. The objects that can be manipulated include single bacterial or spermatozoa, the organelles of larger cells or organisms, and micro-spheres used as handles to manipulate single molecules of DNA or single proteins. Optical traps can be configured to apply controlled forces ranging from one to over a hundred piconewtons and to measure displacements on the order of one nanometer or less. One limiting drawback of optical trapping experiments is that the sample is damaged by the laser light used to produce the trap. Whereas optically induced damage occurs in all optical trapping experiments, it is of particular concern for in vivo measurements. The precise origin of optically induced damage has not been identified, however the most likely candidate is singlet oxygen generated by the trapping laser light. To test this hypothesis and to explore the possibility of eliminating optically induced damage in optical traps, we are testing the damage induced by long wavelength trapping light. The rational is that by trapping with laser light that does not have enough energy to excite molecular oxygen to the singlet state, optically induced damage will be dramatically reduced or eliminated. We have chosen Escherichia coli (E. coli) cells as a model system in which to quantify optically induced damage. Single E. coli cells are held in the optical trap and their flagellar rotation frequency is determined from the scattered laser light. The accumulation of optically induced damage leads to a decrease in the flagellar rotation frequency. We will test the relative rate of damage at the most commonly used wavelength (1064 nm) and at a wavelength corresponding to an energy less than that necessary to excite single oxygen (>1300 nm). Future Research Plans In addition to completing the above projects, our immediate goal is to finish the design and construction of an optical trap, a magnetic tweezers and a single-molecule fluorescence instrument. These instruments will be employed to pursue the longer term goals of the lab. Initially we will focus on the interaction of type I topoisomerases with helicases. 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. Initially, we will use two complementary approaches to investigate different aspects of the interactions between helicases and topoisomerases. In one project, we will investigate 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 will use 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.

正在进行的研究摘要 自2007年2月底到达NIH以来，我一直担心建立实验室并为我们正在建造的单分子仪器订购零件。 除了持续设计和构建单分子光学和磁性操纵仪器的过程外，我们还在追求两个项目。 第一个项目涉及II型拓扑异构酶放松DNA拓扑的能力，至平衡值以下。 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，这些酶的确优先脱链，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。 我们目前正在使用与DNA结合的II型原子力显微镜进行图像。 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。 第二个项目涉及光学诱捕实验中光学诱发损伤的长期问题。 光学陷阱提供了一种非侵入性操纵微米大小的物体的方法。 可以操纵的物体包括单细菌或精子，大细胞或生物的细胞器以及用作操纵DNA或单个蛋白质单分子的手柄的微球。 可以将光学陷阱配置为施加从一个到一百多个piconewton的受控力，并在一个纳米或更小的订单上测量位移。 光学诱捕实验的一个限制缺点是，样品被用于产生陷阱的激光损坏。 尽管在所有光学诱捕实验中都会发生光学诱导的损伤，但体内测量特别关注。 尚未确定光学诱导损伤的确切起源，但是最有可能的候选者是诱捕激光产生的单线氧。 为了检验这一假设并探索消除光学陷阱中光学诱导损伤的可能性，我们正在测试长波长陷阱光引起的损害。 理性的是，通过用激光捕获，没有足够的能量来激发分子氧对单线状态，将大大减少或消除光学诱导的损伤。 我们选择了大肠杆菌（大肠杆菌）细胞作为模型系统，在其中量化光学诱导的损伤。 单个大肠杆菌细胞保持在光学陷阱中，其鞭毛旋转频率由散射的激光确定。 光学诱发损伤的积累导致鞭毛旋转频率的下降。 我们将在最常用的波长（1064 nm）和对应于对应的能量的波长下测试相对的损伤速率，而造成单个氧气（> 1300 nm）所必需的能量。 未来的研究计划 除了完成上述项目外，我们的近期目标是完成光学陷阱，磁性镊子和单分子荧光仪器的设计和构建。 这些工具将被用来追求实验室的长期目标。 最初，我们将专注于I型拓扑异构酶与解旋酶的相互作用。 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性，但是，其相互作用的机制以及耦合酶的特定活性仍然不清楚。 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。 最初，我们将使用两种互补方法来研究解旋酶和拓扑异构酶之间相互作用的不同方面。 在一个项目中，我们将研究反向回旋酶的活性，这是一种来自热粒细菌的独特的拓扑异构酶，该蛋白酶由单个多肽上的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。 通过两个结构域的一致活性，反向回旋酶促进了DNA的正涂层（绕组），但是这种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，将使用主要是荧光共振能传递（FRET）的单分子荧光技术，以分离RECQ解旋酶的结合动力学和TOPO III形成大肠杆菌的结合动力学，并在各种DNA底物和核苷酸辅助剂中存在。 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。