Single-molecule measurements of DNA topology and topoisomerases

National Heart, Lung, And Blood Institute

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Abstract

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. 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 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 in 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. These projects have been enabled by the continued development of magnetic tweezers instruments that afford high spatial and temporal resolution measurements of the topology of individual DNA molecules. 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, and a separate total internal reflection dark-field scattering modality to the magnetic tweezers instruments that permit single-molecule fluorescence measurements and high temporal-spatial resolution tracking of nm scale gold particles acting as local probes of displacement and torque, in conjunction with single-molecule manipulation.