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: 10699696
• 负责人: Keir Neuman
• 金额: $ 156.77万
• 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/10699696
• 关键词: 2019-nCoV; Aging; Antibiotics; Bacteria; Binding; Biochemical; Bloom Syndrome; C-terminal; COVID-19 pandemic; Cell Death; Cell division; Chromosomal Instability; Chromosomes; Clinical; Collaborations; Complement; Complex; DNA; DNA Repair; DNA Topoisomerases; DNA topoisomerase II alpha; Data; Dependence; Development; Electron Transport Complex III; Elements; Engineering; Enzyme Inhibition; Enzymes; Equilibrium; Florida; Fluorescence; Frequencies; Genetic Recombination; Goals; Hand; Human; In Vitro; Incidence; Individual; International; Left; Link; Location; Magnetism; Maintenance; Malignant Neoplasms; Maps; Maryland; Measurement; Measures; Mechanics; Methanosarcina; Methods; Modality; Modeling; Molecular; Monitor; Motion; Multienzyme Complexes; National Institute of Diabetes and Digestive and Kidney Diseases; Nucleotides; Organism; Orthologous Gene; Pathway interactions; Play; Poison; Poisoning; Process; Proteins; Quality Control; RNA Helicase; RNA Processing; RNA-Directed RNA Polymerase; Relaxation; Research; Resolution; Role; Single-Stranded DNA; Sister; Site; Structure; Superhelical DNA; Techniques; Tertiary Protein Structure; Testing; Time; Topoisomerase; Topoisomerase II; Topoisomerase III; Torque; Universities; Work; Yang; base; biophysical model; chemotherapy; college; enzyme activity; experimental study; helicase; homologous recombination; in vivo; inhibitor; instrument; instrumentation; interest; molecular dynamics; molecular scale; next generation sequencing; plasmid DNA; preference; preservation; prevent; professor; repair enzyme; response; simulation; single molecule; small molecule inhibitor; temporal measurement

Research in Progress 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 topos 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 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. This has been demonstrated In vitro, but the mechanism remains a mystery. In a new project in collaboration with Professor Siddhartha Das in the Department of Mechanical Engineering at the University of Maryland College Park, we are using a combination of single-molecule DNA relaxation measurements and molecular dynamics simulations to test the hooked-juxtaposition model of type II topoisomerase unlinking activity. This model suggests that the non-equilibrium topology simplification by type IIA topoisomerases arises from preferential passage of DNA segments that are juxtaposed in a hooked configuration in which the two strands are sharply bent towards each other. We can directly control the degree of this hooked bending and measure how this influences the rate of strand passage in single-molecule experiments combined with MD simulations, which will provide the first experimental test of this hypothesis. Another aspect of topology-dependent activity of type II topoisomerases is their ability to distinguish the chirality of supercoiling. We completed a collaborative project with Anthony Maxell of the John Innes Center in the UK investigating the activity and topological selection of 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 chirally sensitive preferential decatenase, i.e., it preferentially removes intermolecular links associated with linked DNA rather than intramolecular links associated with supercoiled DNA, and it displays a 2-fold preference in relaxing positive supercoiling and the associated left handed links. To complement the single-molecule approaches, we developed next generation sequencing based approaches to probe topoisomerases-DNA interactions. The in vitro approach provides nucleotide resolution mapping of topoisomerase binding, and cleavage site location and frequency. By varying the topology of the DNA plasmids we can quantitatively map the dependence of binding and cleavage site preferences and absolute cleavage levels. Furthermore, we can determine how clinically important topoisomerase poisons alter the cleavage site selection and cleavage levels and how these respond to DNA topology. An ongoing effort is combining the extensive cleavage site data with biophysical modeling to define the mechanisms governing the weak but distinct cleavage site preferences of type II topoisomerases. In another new project in collaboration with Neil Osheroff at Vanderbilt University, we are directly monitoring the poisoning of type II topoisomerases by antibiotics, including fluroquinolone derivatives, at the single-molecule level. We can directly measure the transient poisoning of the topoisomerase during ATP driven strand passage, which allows us to determine the on-rate and off-rate of the poison interacting with the active topoisomerase and how these rates are influenced by the topology, torque, and force on the DNA. 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. 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. Working towards the overarching goal of understanding the mechanistic basis for the chromosome maintenance activities of the RecQ-Topo III complex, we dissected the functional roles of specific conserved protein domains in both the bacterial RecQ and in the human ortholog, Blooms syndrome helicase (BLM). We recently demonstrated how specific domains in RecQ, and accessory protein factors associated with BLM, orient the helicases to promote dissolution of D-loops, early homologous recombination intermediates that are specifically regulated by these helicases. This work, and related work demonstrating how RecQ helicase selectively unwinds D-loops containing regions of low homology while preserving legitimate recombination intermediates, contributes to our understanding of how RecQ helicases perform quality control over the homologous recombination process. The third project involves the molecular mechanism of topoisomerase IA activity. We previously directly observed the opening and closing of type IA enzymes as they reversibly cleave and religate a single DNA strand during their catalytic cycle. We are currently investigating the human enzymes topoisomerase III and III along with their accessory domains that have been predicted to alter the gate dynamics. In collaboration with the Pommier lab in NCI and the Yang lab in NIDDK, we studied the structural and biochemical basis for DNA and RNA processing by topo III alone and in complex with its accessory factor TDRD3. In collaboration with Yuk-Ching Tse-Dinh at Florida International University, we are conducting structure function measurements of the gate dynamics of the bacterial type IA enzymes to elucidate the critical structural features that govern gate dynamics and performing molecular dynamics simulations to relate the motions we observe experimentally to the molecular scale motions of the proteins. The fourth project involves the role of DNA topology on the identification and repair of DNA damage. We previously established that a single mismatched base in 6 kb of DNA will preferentially localize at the tip of a plectoneme in supercoiled DNA. We have recently extended these results to include negatively supercoiled DNA via multiscale simulations of DNA containing mismatches in collaboration with Siddhartha Das in the Mechanical Engineering department at the University of Maryland. Experimental and computational 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. The fifth project involves determining the mechanism and mechanisms of inhibition of SARS COV-2 RNA helicase (NSP13) and RNA dependent RNA polymerase (NSP12) through single-molecule measurements of enzyme activity and inhibition. These projects have been enabled by the continued development of magnetic tweezers instruments that afford high spatial and temporal resolution measurements of DNA topology. The ongoing development and improvement of this magnetic tweezers instrument represents a sustained research endeavor. We have recently added a total internal reflection fluorescence (TIRF) modality to the magnetic tweezers instrument that permits single-molecule fluorescence measurements in conjunction with single-molecule manipulation.

正在进行的研究 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TOPOS放松DNA拓扑的能力，以低于平衡值。在体内，这些拓扑异构酶负责在细胞分裂之前链接复制的染色体。由于姐妹染色体之间的单个联系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。这已经在体外证明了这一点，但这种机制仍然是一个谜。在马里兰州大学公园机械工程系的一项新项目中，我们使用了单分子DNA放松测量和分子动力学模拟的组合来测试II型Tope-Juxtaposition型模型II型topoisomerase-Unindind链接活性。该模型表明，通过IIA型拓扑异构酶简化了非平衡拓扑结构，这是由DNA片段的优先通过，这些DNA片段是在钩状构型中并置的，在钩状构型中，这两条链相互弯曲。我们可以直接控制这种连锁弯曲的程度，并测量这如何影响单分子实验中的链段速率与MD模拟相结合，这将提供该假设的首次实验测试。 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。我们与英国约翰·英恩斯中心（John Innes Center）的安东尼·麦克斯尔（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型拓扑异构酶。 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是一种手性敏感的优先脱发酶，即，它优先消除与链接的DNA相关的分子间链接，而不是与超涂层DNA相关的分子内链接，并且它在放松正面超螺旋和相关的左手链路方面显示了2倍的偏好。 为了补充单分子方法，我们开发了基于下一代测序的方法来探测拓扑异构酶-DNA相互作用。体外方法提供了拓扑异构酶结合以及切割位点的位置和频率的核苷酸分辨率映射。通过改变DNA质粒的拓扑结构，我们可以定量绘制结合和切割位点偏好和绝对切割水平的依赖性。此外，我们可以确定临床上重要的拓扑异构酶毒物如何改变切割位点的选择和切割水平以及这些对DNA拓扑的反应。一项持续的努力是将广泛的裂解位点数据与生物物理建模相结合，以定义管理II型拓扑异构酶的弱但不同的切割位点偏好的机制。 在与范德比尔特大学（Vanderbilt University）的尼尔·奥斯格罗夫（Neil Osheroff）合作的另一个新项目中，我们正在直接监测单分子水平上的抗生素（包括氟喹诺酮衍生物）对II型拓扑异构酶的中毒。我们可以直接测量ATP驱动的链条通道期间拓扑异构酶的瞬时中毒，这使我们能够确定毒药与活性拓扑异构酶相互作用的速率和降低速率，以及这些速率如何受到拓扑，扭矩和对DNA的力的影响。 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。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。 朝着理解RECQ-TOPO III复合物染色体维持活性的机理基础的总体目标努力，我们阐述了特定保守蛋白域在细菌RECQ和人类直系同源物中的功能作用，Blooms综合征综合症酶（BLM）。最近，我们证明了RECQ中的特定结构域以及与BLM相关的辅助蛋白因子如何定向解旋酶以促进D环的溶解，早期的同源重组中间体，这些中间体受这些解旋酶特异性调节。这项工作以及相关的工作证明了RECQ解旋酶如何选择性地放松含有低同源性区域的D环，同时保留合法的重组中间体，这有助于我们理解RECQ解旋酶如何对同源重组过程进行质量控制。 第三个项目涉及拓扑异构酶IA活性的分子机制。以前，我们直接观察到IA型酶在催化周期中可逆地裂解并宗教的开放和关闭。我们目前正在研究人类酶拓扑异构酶III和III及其辅助域，这些辅助域被预测会改变栅极动力学。与NCI的Pommier Lab和NIDDK的Yang Lab合作，我们研究了单独使用Topo III的DNA和RNA处理的结构和生化基础，并与其辅助因子TDRD3进行了复杂化。通过与佛罗里达国际大学的Yuk-Ching Tse-Dinh合作，我们正在对细菌类型IA酶的栅极动力学进行结构函数测量，以阐明控制栅极动力学的关键结构特征，并执行分子动力学模拟，以使我们与蛋白质的分子尺度运动进行实验，以将动力相关联。 第四个项目涉及DNA拓扑在识别和修复DNA损伤方面的作用。我们先前确定，在6 kb的DNA中的单个不匹配的碱基将优先定位在超螺旋DNA中的Plectoneme尖端。最近，我们通过多尺度模拟了含有不匹配的DNA的否定型DNA，该结果将与超构成的DNA合作，并与马里兰州的机械工程系合作。实验和计算结果表明，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尖锐弯曲的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。 第五个项目涉及通过酶活性和抑制作用的单分子测量来确定SARS COV-2 RNA解旋酶（NSP13）和RNA依赖性RNA聚合酶（NSP12）的机制和机制。 通过持续开发磁性镊子仪器的持续开发，这些仪器提供了高空间和时间分辨率的测量。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荧光（TIRF）模态，该仪器允许单分子荧光测量与单分子操作结合使用。