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Keir C. Neuman - One of the best experts on this subject based on the ideXlab platform.
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The dynamic interplay between DNA topoisomerases and DNA Topology
Biophysical Reviews, 2016Co-Authors: Yeonee Seol, Keir C. NeumanAbstract:Topological properties of DNA influence its structure and biochemical interactions. Within the cell, DNA Topology is constantly in flux. Transcription and other essential processes, including DNA replication and repair, not only alter the Topology of the genome but also introduce additional complications associated with DNA knotting and catenation. These topological perturbations are counteracted by the action of topoisomerases, a specialized class of highly conserved and essential enzymes that actively regulate the topological state of the genome. This dynamic interplay among DNA Topology, DNA processing enzymes, and DNA topoisomerases is a pervasive factor that influences DNA metabolism in vivo. Building on the extensive structural and biochemical characterization over the past four decades that has established the fundamental mechanistic basis of topoisomerase activity, scientists have begun to explore the unique roles played by DNA Topology in modulating and influencing the activity of topoisomerases. In this review we survey established and emerging DNA Topology-dependent protein–DNA interactions with a focus on in vitro measurements of the dynamic interplay between DNA Topology and topoisomerase activity.
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A robust assay to measure DNA Topology-dependent protein binding affinity.
Nucleic acids research, 2014Co-Authors: Tamara R. Litwin, Maria Solà, Ian J. Holt, Keir C. NeumanAbstract:DNA structure and Topology pervasively influence aspects of DNA metabolism including replication, transcription and segregation. However, the effects of DNA Topology on DNA-protein interactions have not been systematically explored due to limitations of standard affinity assays. We developed a method to measure protein binding affinity dependence on the Topology (topological linking number) of supercoiled DNA. A defined range of DNA topoisomers at equilibrium with a DNA binding protein is separated into free and protein-bound DNA populations using standard nitrocellulose filter binding techniques. Electrophoretic separation and quantification of bound and free topoisomers combined with a simple normalization procedure provide the relative affinity of the protein for the DNA as a function of linking number. Employing this assay we measured Topology-dependent DNA binding of a helicase, a type IB topoisomerase, a type IIA topoisomerase, a non-specific mitochondrial DNA binding protein and a type II restriction endonuclease. Most of the proteins preferentially bind negatively supercoiled DNA but the details of the Topology-dependent affinity differ among proteins in ways that expose differences in their interactions with DNA. The Topology-dependent binding assay provides a robust and easily implemented method to probe topological influences on DNA-protein interactions for a wide range of DNA binding proteins.
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below equilibrium DNA Topology simplification by type ii topoisomerases can be explained by DNA linking number dependent binding affinity
Biophysical Journal, 2013Co-Authors: Tamara R. Litwin, Susanta S Sarkar, Ashley H Hardin, Keir C. NeumanAbstract:Topoisomerases modify and regulate the Topology of cellular DNA and are essential for successful cell division and other DNA metabolism processes. Type I topoisomerases are ATP-independent and cut one strand of DNA in order to relax supercoils. Type II topoisomerases are ATP-dependent and pass a duplex DNA segment through a transient double-stranded cut in a second DNA segment to change levels of supercoils, knots, or catenanes. Whereas type I topoisomerases simplify DNA Topology towards thermal equilibrium, type II enzymes can simplify DNA Topology to levels below thermal equilibrium. This obeys thermodynamic laws because ATP is hydrolyzed in the process, but the mechanism by which type II topoisomerases acting on the nanometer scale are able to detect and simplify the global Topology of DNA on a much larger scale is not yet known. We propose and test a model to explain non-equilibrium Topology simplification by type II topoisomerases in which the enzyme affinity increases in proportion with the DNA linking number. E. Coli Topo IV has a higher binding affinity for negatively supercoiled DNA than for relaxed DNA, but the general relationship between linking number and affinity has not been established for type II topoisomerases. To determine the linking number dependent-binding affinity and to test the validity of the proposed model, we developed a method to measure the relative biding affinity of type II topoisomerases to different DNA topoisomers. The results of these measurements, in conjunction with computer simulations, permit a sensitive test of the proposed non-equilibrium Topology simplification model.
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Below-Equilibrium DNA Topology Simplification by Type II Topoisomerases can be Explained by DNA Linking Number-Dependent Binding Affinity
Biophysical Journal, 2013Co-Authors: Tamara R. Litwin, Susanta S Sarkar, Ashley H Hardin, Keir C. NeumanAbstract:Topoisomerases modify and regulate the Topology of cellular DNA and are essential for successful cell division and other DNA metabolism processes. Type I topoisomerases are ATP-independent and cut one strand of DNA in order to relax supercoils. Type II topoisomerases are ATP-dependent and pass a duplex DNA segment through a transient double-stranded cut in a second DNA segment to change levels of supercoils, knots, or catenanes. Whereas type I topoisomerases simplify DNA Topology towards thermal equilibrium, type II enzymes can simplify DNA Topology to levels below thermal equilibrium. This obeys thermodynamic laws because ATP is hydrolyzed in the process, but the mechanism by which type II topoisomerases acting on the nanometer scale are able to detect and simplify the global Topology of DNA on a much larger scale is not yet known. We propose and test a model to explain non-equilibrium Topology simplification by type II topoisomerases in which the enzyme affinity increases in proportion with the DNA linking number. E. Coli Topo IV has a higher binding affinity for negatively supercoiled DNA than for relaxed DNA, but the general relationship between linking number and affinity has not been established for type II topoisomerases. To determine the linking number dependent-binding affinity and to test the validity of the proposed model, we developed a method to measure the relative biding affinity of type II topoisomerases to different DNA topoisomers. The results of these measurements, in conjunction with computer simulations, permit a sensitive test of the proposed non-equilibrium Topology simplification model.
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Single-molecule measurements of DNA Topology and topoisomerases.
The Journal of biological chemistry, 2010Co-Authors: Keir C. NeumanAbstract:Topological properties of DNA influence its mechanical and biochemical interactions. Genomic DNA is maintained in a state of topological homeostasis by topoisomerases and is subjected to mechanical stress arising from replication and segregation. Despite their fundamental roles, the effects of Topology and force have been difficult to ascertain. Developments in single-molecule manipulation techniques have enabled precise control and measurement of the Topology of individual DNA molecules under tension. This minireview provides an overview of these single-molecule techniques and illustrates their unique capabilities through a number of specific examples of single-molecule measurements of DNA Topology and topoisomerase activity.
Alexander Vologodskii - One of the best experts on this subject based on the ideXlab platform.
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Bridged DNA Circles: A New Model System To Study DNA Topology
Macromolecules, 2012Co-Authors: Alexander VologodskiiAbstract:We demonstrate the assembly of two bridged circular DNA molecules. The circles are specially engineered plasmid DNAs a few thousands base pairs in length connected by the synthetic double-stranded linker. The construct represents a very convenient system for studying DNA Topology and its enzymatic transformations, since at thermodynamic equilibrium the circles have comparable probability to adopt two different topological states and the exchange between the states is an intramolecular reaction. We obtained the equilibrium distribution of these states, in which the circles form the simplest link or remain unlinked, by opening and closing the long sticky ends made in one of the bridged circles. Separation of the linked and unlinked circles by gel electrophoresis allowed us to determine the fraction of linked circles. The obtained value is in very good agreement with the results of computer simulation.
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Theoretical models of DNA Topology simplification by type IIA DNA topoisomerases.
Nucleic acids research, 2009Co-Authors: Alexander VologodskiiAbstract:It was discovered 12 years ago that type IIA topoisomerases can simplify DNA Topology--the steady-state fractions of knots and links created by the enzymes are many times lower than the corresponding equilibrium fractions. Though this property of the enzymes made clear biological sense, it was not clear how small enzymes could selectively change the Topology of very large DNA molecules, since Topology is a global property and cannot be determined by a local DNA-protein interaction. A few models, suggested to explain the phenomenon, are analyzed in this review. We also consider experimental data that both support and contravene these models.
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SURVEY AND SUMMARY Theoretical models of DNA Topology simplification by type IIA DNA topoisomerases
2009Co-Authors: Alexander VologodskiiAbstract:It was discovered 12 years ago that type IIA topoisomerases can simplify DNA Topology—the steadystate fractions of knots and links created by the enzymes are many times lower than the corresponding equilibrium fractions. Though this property of the enzymes made clear biological sense, it was not clear how small enzymes could selectively change the Topology of very large DNA molecules, since Topology is a global property and cannot be determined by a local DNA–protein interaction. A few models, suggested to explain the phenomenon, are analyzed in this review. We also consider experimental data that both support and contravene these models.
Craig L. Peterson - One of the best experts on this subject based on the ideXlab platform.
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SWI/SNF Chromatin Remodeling Requires Changes in DNA Topology
Molecular cell, 2001Co-Authors: Igor M. Gavin, Peter J. Horn, Craig L. PetersonAbstract:ySWI/SNF complex belongs to a family of enzymes that use the energy of ATP hydrolysis to remodel chromatin structure. Here we examine the role of DNA Topology in the mechanism of ySWI/SNF remodeling. We find that the ability of ySWI/SNF to enhance accessibility of nucleosomal DNA is nearly eliminated when DNA Topology is constrained in small circular nucleosomal arrays and that this inhibition can be alleviated by topoisomerases. Furthermore, we demonstrate that remodeling of these substrates does not require dramatic histone octamer movements or displacement. Our results suggest a model in which ySWI/SNF remodels nucleosomes by using the energy of ATP hydrolysis to drive local changes in DNA twist.
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swi snf chromatin remodeling requires changes in DNA Topology
Molecular Cell, 2001Co-Authors: Igor M. Gavin, Peter J. Horn, Craig L. PetersonAbstract:ySWI/SNF complex belongs to a family of enzymes that use the energy of ATP hydrolysis to remodel chromatin structure. Here we examine the role of DNA Topology in the mechanism of ySWI/SNF remodeling. We find that the ability of ySWI/SNF to enhance accessibility of nucleosomal DNA is nearly eliminated when DNA Topology is constrained in small circular nucleosomal arrays and that this inhibition can be alleviated by topoisomerases. Furthermore, we demonstrate that remodeling of these substrates does not require dramatic histone octamer movements or displacement. Our results suggest a model in which ySWI/SNF remodels nucleosomes by using the energy of ATP hydrolysis to drive local changes in DNA twist.
Tamara R. Litwin - One of the best experts on this subject based on the ideXlab platform.
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A robust assay to measure DNA Topology-dependent protein binding affinity.
Nucleic acids research, 2014Co-Authors: Tamara R. Litwin, Maria Solà, Ian J. Holt, Keir C. NeumanAbstract:DNA structure and Topology pervasively influence aspects of DNA metabolism including replication, transcription and segregation. However, the effects of DNA Topology on DNA-protein interactions have not been systematically explored due to limitations of standard affinity assays. We developed a method to measure protein binding affinity dependence on the Topology (topological linking number) of supercoiled DNA. A defined range of DNA topoisomers at equilibrium with a DNA binding protein is separated into free and protein-bound DNA populations using standard nitrocellulose filter binding techniques. Electrophoretic separation and quantification of bound and free topoisomers combined with a simple normalization procedure provide the relative affinity of the protein for the DNA as a function of linking number. Employing this assay we measured Topology-dependent DNA binding of a helicase, a type IB topoisomerase, a type IIA topoisomerase, a non-specific mitochondrial DNA binding protein and a type II restriction endonuclease. Most of the proteins preferentially bind negatively supercoiled DNA but the details of the Topology-dependent affinity differ among proteins in ways that expose differences in their interactions with DNA. The Topology-dependent binding assay provides a robust and easily implemented method to probe topological influences on DNA-protein interactions for a wide range of DNA binding proteins.
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below equilibrium DNA Topology simplification by type ii topoisomerases can be explained by DNA linking number dependent binding affinity
Biophysical Journal, 2013Co-Authors: Tamara R. Litwin, Susanta S Sarkar, Ashley H Hardin, Keir C. NeumanAbstract:Topoisomerases modify and regulate the Topology of cellular DNA and are essential for successful cell division and other DNA metabolism processes. Type I topoisomerases are ATP-independent and cut one strand of DNA in order to relax supercoils. Type II topoisomerases are ATP-dependent and pass a duplex DNA segment through a transient double-stranded cut in a second DNA segment to change levels of supercoils, knots, or catenanes. Whereas type I topoisomerases simplify DNA Topology towards thermal equilibrium, type II enzymes can simplify DNA Topology to levels below thermal equilibrium. This obeys thermodynamic laws because ATP is hydrolyzed in the process, but the mechanism by which type II topoisomerases acting on the nanometer scale are able to detect and simplify the global Topology of DNA on a much larger scale is not yet known. We propose and test a model to explain non-equilibrium Topology simplification by type II topoisomerases in which the enzyme affinity increases in proportion with the DNA linking number. E. Coli Topo IV has a higher binding affinity for negatively supercoiled DNA than for relaxed DNA, but the general relationship between linking number and affinity has not been established for type II topoisomerases. To determine the linking number dependent-binding affinity and to test the validity of the proposed model, we developed a method to measure the relative biding affinity of type II topoisomerases to different DNA topoisomers. The results of these measurements, in conjunction with computer simulations, permit a sensitive test of the proposed non-equilibrium Topology simplification model.
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Below-Equilibrium DNA Topology Simplification by Type II Topoisomerases can be Explained by DNA Linking Number-Dependent Binding Affinity
Biophysical Journal, 2013Co-Authors: Tamara R. Litwin, Susanta S Sarkar, Ashley H Hardin, Keir C. NeumanAbstract:Topoisomerases modify and regulate the Topology of cellular DNA and are essential for successful cell division and other DNA metabolism processes. Type I topoisomerases are ATP-independent and cut one strand of DNA in order to relax supercoils. Type II topoisomerases are ATP-dependent and pass a duplex DNA segment through a transient double-stranded cut in a second DNA segment to change levels of supercoils, knots, or catenanes. Whereas type I topoisomerases simplify DNA Topology towards thermal equilibrium, type II enzymes can simplify DNA Topology to levels below thermal equilibrium. This obeys thermodynamic laws because ATP is hydrolyzed in the process, but the mechanism by which type II topoisomerases acting on the nanometer scale are able to detect and simplify the global Topology of DNA on a much larger scale is not yet known. We propose and test a model to explain non-equilibrium Topology simplification by type II topoisomerases in which the enzyme affinity increases in proportion with the DNA linking number. E. Coli Topo IV has a higher binding affinity for negatively supercoiled DNA than for relaxed DNA, but the general relationship between linking number and affinity has not been established for type II topoisomerases. To determine the linking number dependent-binding affinity and to test the validity of the proposed model, we developed a method to measure the relative biding affinity of type II topoisomerases to different DNA topoisomers. The results of these measurements, in conjunction with computer simulations, permit a sensitive test of the proposed non-equilibrium Topology simplification model.
Patrick Forterre - One of the best experts on this subject based on the ideXlab platform.
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DNA Topology and the thermal stress response, a tale from mesophiles and hyperthermophiles
BioEssays : news and reviews in molecular cellular and developmental biology, 2000Co-Authors: Purificación López-garcía, Patrick ForterreAbstract:Summary During heat shock and cold shock, plasmid DNA supercoiling changes transiently both in mesophilic bacteria and in hyperthermophilic archaea, despite a different overall Topology (negative supercoiling versus relaxation to positive supercoiling). Transient changes in DNA supercoiling might be essential to generate the stress response, but they could also be a consequence of the physical effects of temperature on cellular components. Indeed, both appear intertwined. Comparison of the mechanisms acting in the two biological systems suggests that the dependence on temperature of the activity of different DNA topoisomerases, as well as of protein binding, are key factors for the control of DNA Topology during stress, which may in turn be relevant for the expression of stress-induced genes. BioEssays 22:738‐746, 2000. fl 2000 John Wiley & Sons, Inc.
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Control of DNA Topology during thermal stress in hyperthermophilic archaea: DNA topoisomerase levels, activities and induced thermotolerance during heat and cold shock in Sulfolobus.
Molecular microbiology, 1999Co-Authors: Purificación López-garcía, Patrick ForterreAbstract:Summary Plasmid Topology varies transiently in hyperthermophilic archaea during thermal stress. As in mesophilic bacteria, DNA linking number (Lk) increases during heat shock and decreases during cold shock. Despite this correspondence, plasmid DNA Topology and proteins presumably involved in DNA topological control in each case are different. Plasmid DNA in hyperthermophilic archaea is found in a topological form from relaxed to positively supercoiled in contrast to the negatively supercoiled state typical of bacteria, eukaryotes and mesophilic archaea. We have analysed the regulation of DNA topological changes during thermal stress in Sulfolobus islandicus (kingdom Crenarchaeota), which harbours two plasmids, pRN1 and pRN2. In parallel with plasmid topological variations, we analysed levels of reverse gyrase, topoisomerase VI (Topo VI) and the small DNA-binding protein Sis7, as well as topoisomerase activities in crude extracts during heat shock from 808 Ct o 85‐878C, and cold shock from 808 Ct o 658C. Quantitative changes in reverse gyrase, Topo VI and Sis7 were not significant. In support of this, inhibition of protein synthesis in S. islandicus during shocks did not alter plasmid topological dynamics, suggesting that an increase in topoisomerase levels is not needed for control of DNA Topology during thermal stress. A reverse gyrase activity was detected in crude extracts, which was strongly dependent on the assay temperature. It was inhibited at 658C, but was greatly enhanced at 858C. However, the intrinsic reverse gyrase activity did not vary with heat or cold shock. These results suggest that the control of DNA Topology during stress in Sulfolobus relies primarily on the physical effect of temperature on topoisomerase activities and on the geometry of DNA itself. Additionally, we have detected an enhanced thermoresistance of reverse gyrase activities in cultures subject to prolonged heat shock (but not cold shock). This acquired thermotolerance at the enzymatic level is abolished when cultures are treated with puromycin, suggesting a requirement for protein synthesis.
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DNA Topology in hyperthermophilic archaea: reference states and their variation with growth phase, growth temperature, and temperature stresses
Molecular microbiology, 1997Co-Authors: Purificación López-garcía, Patrick ForterreAbstract:Summary In order to address the dynamics of DNA Topology in hyperthermophilic archaea, we analysed the topological state of several plasmids recently discovered in Thermococcales and Sulfolobales. All of these plasmids were from relaxed to highly positively super-coiled in vitro, i.e. they exhibited a significant linking excess compared to the negatively supercoiled plasmids from mesophilic organisms (both Archaea and Bacteria). In the two archaeai orders, plasmid linking number (Lk) decreased as growth temperature was lowered from its optimal value, i.e. positively super-coiled plasmids were relaxed whereas relaxed plasmids became negatively supercoiled. Growth temperatures above the optimum correlated with higher positive supercoiling in Sulfolobales (Lk increase) but with relaxation of positive supercoils in Thermococcus sp. GE31. The topological variation of plasmid DNA isolated from cells at different growth phases were found to be species specific in both archaeai orders. In contrast, the direction of topological variation under temperature stress was the same, i.e. a heat shock correlated with an increase in plasmid positive supercoiling, whilst a cold shock induced negative supercoiling. The kinetics of these effects were analysed in Sulfolobales. In both temperature upshift (from 80 to 85C) and downshift (from 80 to 65C), a transient sharp variation of Lk occurred first, and then DNA supercoiling progressively reached levels typical of steady-state growth at the final temperature. These results indicate that DNA Topology can change with physiological states and environmental modifications in hyperthermophilic archaea.
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The unique DNA Topology and DNA topoisomerases of hyperthermophilic archaea
FEMS microbiology reviews, 1996Co-Authors: Patrick Forterre, Agnes Bergerat, Purificacion Lopex-garciaAbstract:Abstract Hyperthermophilic archaea exhibit a unique pattern of DNA topoisomerase activities. They have a peculiar enzyme, reverse gyrase, which introduces positive superturns into DNA at the expense of ATP. This enzyme has been found in all hyperthermophiles tested so far (including Bacteria) but never in mesophiles. Reverse gyrases are formed by the association of a helicase-like domain and a 5′-type I DNA topoisomerase. These two domains might be located on the same polypeptide. However, in the methanogenic archaeon Methanopyrus kandleri , the topoisomerase domain is divided between two subunits. Besides reverse gyrase, Archaea contain other type I DNA topoisomerases; in particular, M. kandleri harbors the only known procaryotic 3′-type I DNA topoisomerase (Topo V). Hyperthermophilic archaea also exhibit specific type II DNA topoisomerases (Topo II), i.e. whereas mesophilic Bacteria have a Topo II that produces negative supercoiling (DNA gyrase), the Topo II from Sulfolobus and Pyrococcus lack gyrase activity and are the smallest enzymes of this type known so far. This peculiar pattern of DNA topoisomerases in hyperthermophilic archaea is paralleled by a unique DNA Topology, i.e. whereas DNA isolated from Bacteria and Eucarya is negatively supercoiled, plasmidic DNA from hyperthermophilic archaea are from relaxed to positively supercoiled. The possible evolutionary implications of these findings are discussed in this review. We speculate that gyrase activity in mesophiles and reverse gyrase activity in hyperthermophiles might have originated in the course of procaryote evolution to balance the effect of temperature changes on DNA structure.
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DNA Topology in Halobacteria
General and Applied Aspects of Halophilic Microorganisms, 1991Co-Authors: Patrick Forterre, Danièle Gadelle, Franck Charbonnier, Mouldy SioudAbstract:Inhibitors of eubacterial and eucaryotic DNA topoisomerases II induce topological changes and/or DNA cleavage in the plasmids of halobacteria. As in eubacteria, novobiocin halts DNA replication and induces positive supercoiling of plasmids in halobacteria. This positive supercoiling is prevented by actinomycin D, indicating that it may be generated by transcription as in eubacteria.