Single-molecule measurements of DNA topology and topoisomerases

National Heart, Lung, And Blood Institute

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Abstract

Research in Progress Currently, there are Five 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. 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 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.