Research in Progress Currently, there are three main ongoing projects in the lab: 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 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. Previously we have shown that a mechanism based on a sharp bend imposed on the DNA by the topoisomerase cannot explain the extent of non-equilibrium simplification, and cannot explain the differences in non-equilibrium simplification among different type II topoisomerases (bacterial, human, yeast). We have recently completed testing two alternative models of topology simplification. The models postulate either a kinetic proofreading mechanism in which the topoisomerase catalyzes strand passage only after repeatedly encountering a DNA segment, or a mechanism in which the topoisomerase specifically recognizes DNA in a hooked juxtaposition geometry. Using magnetic tweezers we measured the unlinking of two DNA strands wrapped around each other a specific number of times under a controlled force. By measuring the rate of strand passage by a type II topoisomerase as a function of the imposed geometry and force and performing Monte-Carlo simulations to obtain the distribution of DNA configurations for each condition, we were able to test both models. The data indicate that type II topoisomerases can catalyze DNA strand-transfer with each collision of two DNA segments, thereby ruling out the kinetic proof reading model. Furthermore, preliminary evidence suggests that DNA unlinking rates are not highly correlated with the degree of hookedness of the two strands. Further tests, currently underway, will allow us to unambiguously determine the validity of the hooked juxtaposition model in describing the activity of type II topoisomerases. A second aspect of the interaction between type II topoisomerases and their DNA substrates concerns the diverse topological activities exhibited by type II topoisomerases that share a common mechanism. These activities include the symmetric relaxation of positively and negatively supercoiled DNA by most type II topoisomerases, the introduction of negative supercoils by DNA gyrase, and the asymmetric relaxation of negative and positive supercoils by some type II enzymes. These differences in activity are believed to arise from differences in the C-terminal domains (CTDs), but the molecular basis underling these variations in activity have not been elucidated. We have produced a series of CTD mutants of E. coli Topoisomerase IV (Topo IV). We are employing a combination of ensemble and single molecule assays to test the effects of these mutations on the substrate selectivity. In collaboration with Neil Osheroff at Vanderbilt University, we have investigated the mechanism of chiral sensing by human type II topoisomerase (hTopo II). Employing a single-molecule magnetic-tweezers based supercoil relaxation assay, we compared the chiral discrimination activity of hTopo II with that of E. coli Topo IV. Both enzymes preferentially relax positive supercoils. Despite this functional similarity, the two enzymes employ different mechanisms to achieve chiral discrimination. For the bacterial enzyme there is a dramatic difference in the processivity of positive verses negative supercoil relaxation. In contrast chiral discrimination by the human enzyme is achieved by changes in relaxation rate rather than processivity, which we have shown is remarkably high.These results combined with the tension dependence of the relaxation rate indicate that capture of the second DNA segment (the transfer segment) is the rate determining step in the strand passage reaction of human type II topoisomerase, and by extension all type II topoisomerases. 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. The ability of single-molecule techniques to measure the activity of a single enzyme or enzyme complex in real time is well suited to the study of such complicated processes in which multiple activities may occur over multiple time scales. In collaboration with Mihaly Kovacs at Etovos University, Hungry, we are using single-molecule measurements of DNA unwinding to elucidate the kinetics and step size of RecQ helicase 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. The third related project, in collaboration with Yves Pommier in NCI, is focused on the mechanisms of supercoil relaxation by human type IB topoisomerases, and in the effects of chemotherapy agents that inhibit type IB topoisomerases. Type IB topoisomerases are essential enzymes that relax over wound (positively supercoiled) DNA generated ahead of the replication machinery during DNA synthesis. Type IB topoisomerases are also important chemotherapy targets. Potent chemotherapy agents that specifically inhibit type IB topoisomerases are currently in clinical use and additional agents are in development. We are using single-molecule magnetic-tweezers based assays to measure the activity of individual type IB topoisomerases and the effects of chemotherapy agents on the activity. These experiments provide molecular level details of the supercoil relaxation process and how it is influenced by the degree of DNA supercoiling, the tension on the DNA, and the presence of specific chemotherapy agents. These measurements provide an unprecedented level of detail concerning how these important enzymes work and are inhibited by chemotherapy agents. We recently demonstrated that the human nuclear Topoisomerase IB is remarkably insensitive to the effects of twist or torque on the DNA. This observation, combined with the first direct measurement of the cleavage kinetics at the single-molecule level, allowed us to formulate a comprehensive model for the complete relaxation and religation process catalyzed by type IB topoisomerases. This model reveals a hitherto unobserved intermediate state in the relaxation cycle, and provides a mechanistic framework for the action of inhibitors. We are currently using this model to interpret the affects of three inhibitors representing different inhibition mechanisms. 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. Future research goals: Our immediate goal is the completion of the ongoing projects in the lab. Longer term goals include the development of a new optical trap and magnetic tweezers instrument combined with single-molecule fluorescence detection.

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Vecchiarelli, Anthony G; Neuman, Keir C; Mizuuchi, Kiyoshi (2014) A propagating ATPase gradient drives transport of surface-confined cellular cargo. Proc Natl Acad Sci U S A 111:4880-5
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