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 was the case, but the mechanism remains a mystery. One proposed mechanism for this topology simplification posits that the topoisomerase induces a sharp bend in the DNA on binding, which would favor unlinking over linking. Using atomic force microscopy (AFM), we have measured the DNA bend angle imposed by the binding of three type II topoisomerases from different organisms (Human, Yeast, and E. coli). The measured bend angles do not support the bend angle model of topology simplification. This is an important finding as the experimental evidence to date has been equivocal concerning the bend angle model. We have also developed new quantitative analysis methodologies for AFM images of protein DNA complexes. These include an image-processing based analysis of the DNA bend angle from AFM images that is faster and less prone to artifact than the currently used manual methods. Currently, we are 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. To test these models we are using magnetic tweezers to measure 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 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 can test both models. We anticipate that these experiments will unambiguously confirm or refute the two competing models for non-equilibrium topology simplification by 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 are also investigating the mechanism of chiral sensing by human type II topoisomerase (hTopo II). 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 fluorescence techniques to measure the unwinding 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. Several 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. 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 capabilities. This instrument will permit simultaneous measurements of the activity and composition of multi-enzyme complexes interacting with a single DNA molecule. This instrument will open up other areas of research including the possibility of observing the dynamics of supercoiled DNA. As a first step in this direction, we are in the process of combining a magnetic tweezers instrument with single-molecule fluorescence detection capabilities.

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Bharti, Sanjay Kumar; Sommers, Joshua A; Awate, Sanket et al. (2018) A minimal threshold of FANCJ helicase activity is required for its response to replication stress or double-strand break repair. Nucleic Acids Res 46:6238-6256
Brahmachari, Sumitabha; Dittmore, Andrew; Takagi, Yasuharu et al. (2018) Defect-facilitated buckling in supercoiled double-helix DNA. Phys Rev E 97:022416
Seol, Yeonee; Neuman, Keir C (2018) Combined Magnetic Tweezers and Micro-mirror Total Internal Reflection Fluorescence Microscope for Single-Molecule Manipulation and Visualization. Methods Mol Biol 1665:297-316
Dittmore, Andrew; Brahmachari, Sumitabha; Takagi, Yasuharu et al. (2017) Supercoiling DNA Locates Mismatches. Phys Rev Lett 119:147801
Zhang, Hongliang; Seol, Yeonee; Agama, Keli et al. (2017) Distribution bias and biochemical characterization of TOP1MT single nucleotide variants. Sci Rep 7:8614
Hu, Longhua; Vecchiarelli, Anthony G; Mizuuchi, Kiyoshi et al. (2017) Brownian ratchet mechanisms of ParA-mediated partitioning. Plasmid 92:12-16
Harami, Gábor M; Seol, Yeonee; In, Junghoon et al. (2017) Shuttling along DNA and directed processing of D-loops by RecQ helicase support quality control of homologous recombination. Proc Natl Acad Sci U S A 114:E466-E475
Ashley, Rachel E; Dittmore, Andrew; McPherson, Sylvia A et al. (2017) Activities of gyrase and topoisomerase IV on positively supercoiled DNA. Nucleic Acids Res 45:9611-9624
Mills, Maria; Harami, Gábor M; Seol, Yeonee et al. (2017) RecQ helicase triggers a binding mode change in the SSB-DNA complex to efficiently initiate DNA unwinding. Nucleic Acids Res 45:11878-11890
Hu, Longhua; Vecchiarelli, Anthony G; Mizuuchi, Kiyoshi et al. (2017) Brownian Ratchet Mechanism for Faithful Segregation of Low-Copy-Number Plasmids. Biophys J 112:1489-1502

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