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.

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