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. 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. We have also established that the proposed kinetic proofreading mechanism in which the topoisomerase catalyzes strand passage only after repeatedly encountering a DNA segment, does not hold for type II topoisomerases, thereby ruling out the kinetic proof reading model for below equilibrium topology simplification. We have proposed an alternative model of non-equilibrium topology simplification in which the preferential relaxation of supercoiled DNA arises from preferential binding of type II topoisomerases to supercoiled substrates, i.e., topology-dependent binding. To test this model we first developed and validated an assay to measure DNA topology-dependent protein binding. Using this assay, we have established that DNA topology significantly influences the binding of type II topoisomerases to DNA, consistent with the premise of our model. Furthermore, in collaboration with Neil Osheroff at Vanderbilt University Medical School, we have demonstrated that the differences in the influence of DNA topology on type II topoisomerase binding from different organisms (yeast or bacteria) correlates with the degree of non-equilibrium topology simplification, providing the first measured correlation between a proposed mechanism and the degree of non-equilibrium simplification for enzymes from different organisms. In collaboration with Stephen Levene at the University of Texas at Dallas we are currently extending these measurements to investigate preferential binding to knotted and linked DNA molecules, which are also preferentially unknotted and unlinked by 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. 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. 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. We employed the single-molecule supercoil relaxation assay to characterize the molecular consequences of a unique mitochondrial topoisomerase I specific inhibitor, Lamellarin-D, a natural product derived from a mollusk. Binding of the inhibitor to the topoisomerase results in DNA topology-dependent changes in the rate of supercoil relaxation and inhibition of religation. We have recently extended these measurements to compare the mechanistic consequences of nuclear topoisomerase I inhibition by four different inhibitors corresponding to three classes of compounds. The single-molecule results reveal significant topology-dependent changes in the effects of the inhibitors on the topoisomerase. These measurements also provide a complete kinetic characterization of inhibitor binding to topoisomerases engaged in relaxation of defined topological states, which have implications for the mechanism and efficacy of inhibition in vivo. Finally, we have also characterized two human mitochondrial topoisomerase variants associated with single nucleotide polymorphisms that are correlated with certain cancer types in the NCI cancer data base, and a point-mutant of mitochondrial topoisomerase associated with mitochondrial disease. The single-molecule measurements revealed specific alterations in the activity of these topoisomerase mutants at relatively high supercoiling levels that were not detectable in conventional biochemical assays of topoisomerase activity. 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 goals include the development of a new optical trap and magnetic tweezers instrument combined with single-molecule fluorescence detection, incorporating single-molecule detection into the magnetic tweezers instrument, and developing a set of tools and approaches to directly monitor type II inhibition by antibiotics and chemotherapeutic compounds at the single-molecule level. Longer terms goals include performing the single-molecule assays in eukaryotic or prokaryotic cell extract to more closely recapitulate the processes occurring in the cellular context.
| 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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