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: 10253797
• 负责人: Keir Neuman
• 金额: $ 120.39万
• 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/10253797
• 关键词: Aging; Antibiotics; Bacteria; Binding; Binding Proteins; Biological Assay; Bloom Syndrome; C-terminal; Cell Death; Cell division; Chromosomal Instability; Chromosomes; Cleaved cell; Collaborations; Complex; Computers; DNA; DNA Repair; Development; Electron Transport Complex III; Elements; Engineering; Enzymes; Equilibrium; Exhibits; Fluorescence; Genetic Recombination; Genome Stability; Geometry; Goals; Homologous Gene; Human; In Vitro; Incidence; Individual; Link; Magnetism; Maintenance; Malignant Neoplasms; Maryland; Measurement; Measures; Mechanics; Mediating; Meiosis; Methanosarcina; Methods; Microscope; Modality; Modeling; Molecular; Molecular Probes; Monitor; Motion; Multienzyme Complexes; Organism; Orthologous Gene; Pathway interactions; Plants; Play; Poison; Positioning Attribute; Process; Proteins; Reaction; Reporting; Research; Resolution; Role; SPO11 gene; Single-Stranded DNA; Sister; Site; Superhelical DNA; Techniques; Tertiary Protein Structure; Testing; Time; Topoisomerase; Topoisomerase II; Topoisomerase III; Type I DNA Topoisomerases; Universities; Visualization; Work; base; chemotherapy; enzyme activity; enzyme model; enzyme structure; experimental study; follow-up; helicase; in vivo; inhibitor/antagonist; instrument; instrumentation; interest; molecular dynamics; molecular scale; mutant; novel; preference; preservation; prevent; repair enzyme; replication factor A; simulation; single molecule; small molecule inhibitor; temporal measurement

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. In particular, we have a longstanding interest in the complex interplay between DNA topology and the binding and activity of type II topoisomerases. 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 continue to experimentally test mechanistic models for below equilibrium topology simplification. Another aspect of topology-dependent activity of type II topoisomerases is their ability to distinguish the chirality of the supercoiling. We completed a collaborative project with Anthony Maxell of the John Innes Center in the UK investigating the activity and topological selection by topoisomerase VI, which is a type IIb topoisomerase. The type IIb enzymes are structurally related to the type IIa enzymes, but they lack a key element (the C-terminal gate) that is believed to contribute to the directionality of the type IIa enzymes. We used a combination of single-molecule and ensemble methods to probe the strand passage mechanism of this topoisomerase VI from Methanosarcina Mazei. We discovered that Topo VI is a preferential decatenase, i.e., it preferentially removes intermolecular links associated with linked DNA rather than intramolecular links associated with supercoiled DNA. We identified that Topo VI exhibits a strong preference for passing DNA strands juxtaposed with geometries that are more favored in linked DNA molecules than supercoiled DNA molecules. Our findings from this topoisomerase IIb have expanded our understanding of type II topoisomerase activities and may have important ramifications for the topoisomerase VI enzymes from plants in addition to the closely related human enzyme Spo11 that is involved in generating DNA breaks during meiosis. 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. We recently demonstrated that RecQ helicase can remove single stranded binding protein (SSB) from single-stranded DNA and we elucidated the molecular mechanism through which this process is mediated. This work contributes to our understanding of the putative role of SSB or the eukaryotic homolog replication protein A (RPA) plays in the activity within the complex of a RecQ helicase, Topoisomerase III, and SSB or RPA. The third project involves the molecular mechanism of topoisomerase IA activity. In the last reporting period we achieved the long-sought goal of directly observing the opening and closing of type IA enzymes as they reversibly cleave and religate a singe DNA strand. Reversible opening of a protein mediated gap in the DNA associated with reversible opening of a gate in the protein had been postulated since the very earliest models of this enzyme were developed. For the past several decades this gate opening had never been resolved. By applying force directly on the protein mediated gate we were able to slow down the reaction sufficiently to directly observe the gate opening dynamics. We are following up on this discovery by investigating the human enzymes topoisomerase III and Topoisomerase III along with their accessory domains that have been predicted to alter the gate dynamics, though this has not been experimentally verified. We are also testing the effects of point mutants on the gate dynamics of the bacterial enzymes topoisomerase I and topoisomerase III. We are simultaneously probing the molecular motions of these type IA topoisomerases via molecular dynamics simulations which allow us to relate the force-dependent motions we observe with the single-molecule measurements to the molecular scale motions of the protein. The fourth project involves the role of DNA topology on the identification and repair of DNA damage. We recently established that a single mismatched base in 6 kb of DNA will preferentially localize the tip of a plectoneme at the mismatch. This experimental finding was theoretically extended in collaboration with John Marko at Northwestern University. These experimental and theoretical results indicate that supercoiling of DNA can contribute to the localization and identification of mismatches or other DNA damage by repair enzymes that recognize sharply bent DNA with a flipped-out base, both of which are favored when the damaged site is localized at the tip of a plectoneme in supercoiled DNA. We have further extending these results via multiscale simulations of DNA containing mismatches in collaboration with Siddhartha Das in the Mechanical Engineering department at the University of Maryland. 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. We have recently added a total internal reflection microscope modality to the magnetic tweezers instrument that permits single-molecule fluorescence measurements in conjunction with single-molecule manipulation via the magnetic tweezers.

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。 特别是，我们对DNA拓扑与II型拓扑异构酶的结合和活性之间的复杂相互作用具有长期的兴趣。 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。 我们继续通过实验测试以下平衡拓扑简化的机械模型。 II型拓扑异构酶拓扑依赖性活性的另一个方面是它们区分超涂层手性的能力。 我们与英国John Innes Center的Anthony Maxell完成了一个合作项目，调查了Topoisomerase VI的活动和拓扑选择，这是IIB型topoisomerase。 IIB类型酶在结构上与IIA型酶相关，但是它们缺乏关键元素（C端门），该元素被认为有助于IIA型酶的方向性。 我们使用了单分子和集合方法的组合来探测该拓扑异构酶VI的链通过机理，从甲虫菌Mazei探测。 我们发现TOPO VI是一种优先的deTANEPASE，即，它优先删除与链接的DNA相关的分子间链接，而不是与超螺旋DNA相关的分子内链接。我们确定TOPO VI表现出对传递与几何形状并列的DNA链的强烈偏爱，这些DNA链比超涂的DNA分子在链接的DNA分子中更受青睐。 我们从该拓扑异构体IIB中的发现扩展了我们对II型拓扑异构酶活性的理解，除了与在减麻一年度期间引起的DNA断裂相关的人类酶SPO11外，还可能对植物的拓扑异构酶VI酶产生重要的影响。 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。 我们最近证明，RECQ解旋酶可以从单链DNA中去除单链结合蛋白（SSB），并阐明了该过程介导的分子机制。 这项工作有助于我们理解SSB或真核同源性复制蛋白A（RPA）在RECQ解旋酶，拓扑酶III和SSB或RPA中的活性中发挥作用。 第三个项目涉及拓扑异构酶IA活性的分子机制。 在最后一个报告时期，我们实现了长期以来的目标，即直接观察IA型酶的开放和关闭，因为它们可逆地裂开并宗教为单身DNA链。 自从开发出最早的该酶模型以来，已经假定了与蛋白质中元素开放相关的DNA中蛋白质介导的间隙的可逆开口。 在过去的几十年中，这个大门的开口从未得到解决。 通过直接在蛋白质介导的栅极上施加力，我们能够充分降低反应，以直接观察栅极开口动力学。 我们正在通过研究人类酶拓扑异构酶III和拓扑异构酶III及其附件域，这些酶及其辅助域被预测会改变栅极动力学，尽管尚未经过实验验证，我们正在跟踪这一发现。 我们还正在测试点突变体对细菌酶拓扑异构酶I和拓扑异构酶III的栅极动力学的影响。 我们同时通过分子动力学模拟探测这些类型IA拓扑异构酶的分子运动，这使我们能够将观察到的力依赖性运动与单分子测量值与蛋白质的分子尺度运动联系起来。 第四个项目涉及DNA拓扑在识别和修复DNA损伤方面的作用。 我们最近确定，在6 kb的DNA中的单个不匹配的碱基将优先在不匹配处定位plectoneme的尖端。 从理论上讲，这一实验发现与西北大学的约翰·马克（John Marko）合作扩展了。 这些实验性和理论结果表明，DNA的超涂层可以通过修复酶对不匹配或其他DNA损伤的定位和鉴定，而修复酶识别具有翻转碱基的急剧弯曲的DNA，当受损的位点位于超涂层DNA中的Plectoneme尖端时，这两者都受到青睐。 我们通过与马里兰大学机械工程系的Siddhartha Das合作的DNA的多尺度模拟进一步扩展了这些结果。 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。 我们最近在磁性镊子仪器中添加了总内反射显微镜模态，该仪器允许通过磁镊子结合单分子操作单分子荧光测量。