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Nicholas R. Cozzarelli - One of the best experts on this subject based on the ideXlab platform.
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the effect of ionic conditions on the conformations of supercoiled dna ii equilibrium catenation
Journal of Molecular Biology, 1997Co-Authors: Valentin V. Rybenkov, Alexander Vologodskii, Nicholas R. CozzarelliAbstract:We studied the equilibrium formation of DNA catenanes to assess the conformational properties of supercoiled DNA as a function of ionic conditions and supercoiling density. Catenanes were formed by cyclizing linear DNA with long cohesive ends in the presence of supercoiled molecules. The efficiency of the catenation depends on the distance between opposing segments of DNA in the interwound Superhelix. The fraction of cyclizing molecules that becomes topologically linked with the supercoiled DNA is the product of the concentration of the supercoiled DNA and a proportionality constant, B, that depends on conformations of supercoiled DNA. In parallel with these experimental studies, we calculated the values of B using Monte Carlo simulations of the equilibrium distribution of DNA conformations. There were no adjustable parameters in the calculations because all three parameters of the DNA model, bending and torsional elasticity of DNA and DNA effective diameter, specifying intersegment interactions, were known from independent studies. We found very good agreement between measured and simulated values of B for all the ionic conditions and DNA Superhelix densities studied; the discrepancy was less than a factor of 2 over the 200-fold variation in B. The value of B decreases nearly exponentially with increasing superhelicity, this dependence being especially strong at low salt concentration. The dependence of B on the concentration of NaCl, MgCl2, and spermidine can be described with good accuracy in terms of changes of the DNA effective diameter. We found no indication of Superhelix collapse under any ionic conditions studied. We discuss, in light of these results, the biological importance of the effect of DNA supercoiling on the unlinking of the products of DNA replication. # 1997 Academic Press Limited
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the effect of ionic conditions on the conformations of supercoiled dna i sedimentation analysis
Journal of Molecular Biology, 1997Co-Authors: Valentin V. Rybenkov, Alexander Vologodskii, Nicholas R. CozzarelliAbstract:We studied the conformations of supercoiled DNA as a function of superhelicity and ionic conditions by determining its sedimentation coefficient both experimentally and by calculation. To cancel out unknown parameters from both calculations and experiments, we determined the ratio of the sedimentation coefficient, s, to that of open circular DNA, soc. Calculations of the sedimentation coefficient were based on direct solution of the Burgers-Oseen problem for an equilibrium set of DNA conformations generated for each condition by the Metropolis Monte Carlo procedure. There were no adjustable parameters in the Monte Carlo simulations because all three parameters of the DNA model used, bending and torsional elasticity of DNA and DNA effective diameter specifying electrostatic interactions, were known from independent data. The good agreement between measured and calculated values of s/soc allowed us to interpret the sedimentation results in terms of DNA conformations, with particular emphasis on the marked effect of ionic conditions. As NaCl concentration decreases, s/soc increases because the Superhelix becomes less regular and more compact. In the presence of just 10 mM MgCl2, supercoiled DNA adopts essentially the same set of conformations as in moderate to high concentrations of NaCl. Our simulations showed that s is a strong function of the Superhelix branching frequency. At near physiological ionic conditions, there are about four branches in the 7 kb DNA molecule used in this work. We found no indication of Superhelix collapse in any ionic conditions even remotely approaching physiological ones. For all ionic conditions studied, we conclude that the electrostatic interaction of DNA segments specified by the DNA effective diameter is the primary determinant of supercoiled DNA conformations. # 1997 Academic Press Limited
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effect of supercoiling on the juxtaposition and relative orientation of dna sites
Biophysical Journal, 1996Co-Authors: Alexander Vologodskii, Nicholas R. CozzarelliAbstract:There are many proteins that interact simultaneously with two or more DNA sites that are separated along the DNA contour. These sites must be brought close together to form productive complexes with the proteins. We used Monte Carlo simulation of supercoiled DNA conformations to study the effect of supercoiling and DNA length on the juxtaposition of DNA sites, the angle between them, and the branching of the interwound Superhelix. Branching decreases the probability of juxtaposition of two DNA sites but increases the probability of juxtaposition of three sites at branch points. We found that the number of Superhelix branches increases linearly with the length of DNA from 3 to 20 kb. The simulations showed that for all contour distances between two sites, the juxtaposition probability in supercoiled DNA is two orders of magnitude higher than in relaxed DNA. Supercoiling also results in a strong asymmetry of the angular distribution of juxtaposed sites. The effect of supercoiling on site-specific recombination and the introduction of supercoils by DNA gyrase is discussed in the context of the simulation results.
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conformational and thermodynamic properties of supercoiled dna
Journal of Molecular Biology, 1992Co-Authors: Alexander Vologodskii, Stephen D Levene, K V Klenin, Maxim D Frankkamenetskii, Nicholas R. CozzarelliAbstract:Abstract We used Monte Carlo simulations to investigate the conformational and thermodynamic properties of DNA molecules with physiological levels of supercoiling. Three parameters determine the properties of DNA in this model: Kuhn statistical length, torsional rigidity and effective double-helix diameter. The chains in the simulation resemble strongly those observed by electron microscopy and have the conformation of an interwound Superhelix whose axis is often branched. We compared the geometry of simulated chains with that determined experimentally by electron microscopy and by topological methods. We found a very close agreement between the Monte Carlo and experimental values for writhe, Superhelix axis length and the number of superhelical turns. The computed number of Superhelix branches was found to be dependent on Superhelix density, DNA chain length and double-helix diameter. We investigated the thermodynamics of supercoiling and found that at low Superhelix density the entropie contribution to Superhelix free energy is negligible, whereas at high Superhelix density, the entropie and enthalpic contributions are nearly equal. We calculated the effect of supercoiling on the spatial distribution of DNA segments. The probability that a pair of DNA sites separated along the chain contour by at least 50 nm are juxtaposed is about two orders of magnitude greater in supercoiled DNA than in relaxed DNA. This increase in the effective local concentration of DNA is not strongly dependent on the contour separation between the sites. We discuss the implications of this enhancement of site juxtaposition by supercoiling in the context of protein-DNA interactions involving multiple DNA-binding sites.
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Conformational and Thermodynamic Properties of Supercoiled DNA
Journal of molecular biology, 1992Co-Authors: Alexander Vologodskii, Stephen D Levene, K V Klenin, Maxim D. Frank-kamenetskii, Nicholas R. CozzarelliAbstract:We used Monte Carlo simulations to investigate the conformational and thermodynamic properties of DNA molecules with physiological levels of supercoiling. Three parameters determine the properties of DNA in this model: Kuhn statistical length, torsional rigidity and effective double-helix diameter. The chains in the simulation resemble strongly those observed by electron microscopy and have the conformation of an interwound Superhelix whose axis is often branched. We compared the geometry of simulated chains with that determined experimentally by electron microscopy and by topological methods. We found a very close agreement between the Monte Carlo and experimental values for writhe, Superhelix axis length and the number of superhelical turns. The computed number of Superhelix branches was found to be dependent on Superhelix density, DNA chain length and double-helix diameter. We investigated the thermodynamics of supercoiling and found that at low Superhelix density the entropic contribution to Superhelix free energy is negligible, whereas at high Superhelix density, the entropic and enthalpic contributions are nearly equal. We calculated the effect of supercoiling on the spatial distribution of DNA segments. The probability that a pair of DNA sites separated along the chain contour by at least 50 nm are juxtaposed is about two orders of magnitude greater in supercoiled DNA than in relaxed DNA. This increase in the effective local concentration of DNA is not strongly dependent on the contour separation between the sites. We discuss the implications of this enhancement of site juxtaposition by supercoiling in the context of protein-DNA interactions involving multiple DNA-binding sites.
Alexander Vologodskii - One of the best experts on this subject based on the ideXlab platform.
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dynamics of site juxtaposition in supercoiled dna
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Jing Huang, Tamar Schlick, Alexander VologodskiiAbstract:Juxtaposition kinetics between specific sites in supercoiled DNA is investigated at close to physiological ionic conditions by Brownian dynamics simulations. At such conditions, supercoiled DNA is interwound, and the probability of spatial site juxtaposition is much higher than in relaxed DNA. We find, however, that supercoiling does not correspondingly increase the rate of juxtaposition at these physiological conditions. An explanation to this unexpected finding emerges on analysis of the juxtaposition dynamics. We note that although a particular site i1 in supercoiled DNA is often in close proximity (juxtaposed) to another site i2, the change of i2 occurs very slowly and depends largely on internal slithering of opposite segments of the DNA Superhelix. Such slithering results in long correlations between successive values of i2; these correlations increase the average time of juxtaposition between two DNA sites. Random collisions between sites located on different Superhelix branches—although increasing in importance with DNA size—contribute less substantially to site juxtaposition at high salt than slithering for DNA up to 6 kb in length.
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the effect of ionic conditions on the conformations of supercoiled dna ii equilibrium catenation
Journal of Molecular Biology, 1997Co-Authors: Valentin V. Rybenkov, Alexander Vologodskii, Nicholas R. CozzarelliAbstract:We studied the equilibrium formation of DNA catenanes to assess the conformational properties of supercoiled DNA as a function of ionic conditions and supercoiling density. Catenanes were formed by cyclizing linear DNA with long cohesive ends in the presence of supercoiled molecules. The efficiency of the catenation depends on the distance between opposing segments of DNA in the interwound Superhelix. The fraction of cyclizing molecules that becomes topologically linked with the supercoiled DNA is the product of the concentration of the supercoiled DNA and a proportionality constant, B, that depends on conformations of supercoiled DNA. In parallel with these experimental studies, we calculated the values of B using Monte Carlo simulations of the equilibrium distribution of DNA conformations. There were no adjustable parameters in the calculations because all three parameters of the DNA model, bending and torsional elasticity of DNA and DNA effective diameter, specifying intersegment interactions, were known from independent studies. We found very good agreement between measured and simulated values of B for all the ionic conditions and DNA Superhelix densities studied; the discrepancy was less than a factor of 2 over the 200-fold variation in B. The value of B decreases nearly exponentially with increasing superhelicity, this dependence being especially strong at low salt concentration. The dependence of B on the concentration of NaCl, MgCl2, and spermidine can be described with good accuracy in terms of changes of the DNA effective diameter. We found no indication of Superhelix collapse under any ionic conditions studied. We discuss, in light of these results, the biological importance of the effect of DNA supercoiling on the unlinking of the products of DNA replication. # 1997 Academic Press Limited
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the effect of ionic conditions on the conformations of supercoiled dna i sedimentation analysis
Journal of Molecular Biology, 1997Co-Authors: Valentin V. Rybenkov, Alexander Vologodskii, Nicholas R. CozzarelliAbstract:We studied the conformations of supercoiled DNA as a function of superhelicity and ionic conditions by determining its sedimentation coefficient both experimentally and by calculation. To cancel out unknown parameters from both calculations and experiments, we determined the ratio of the sedimentation coefficient, s, to that of open circular DNA, soc. Calculations of the sedimentation coefficient were based on direct solution of the Burgers-Oseen problem for an equilibrium set of DNA conformations generated for each condition by the Metropolis Monte Carlo procedure. There were no adjustable parameters in the Monte Carlo simulations because all three parameters of the DNA model used, bending and torsional elasticity of DNA and DNA effective diameter specifying electrostatic interactions, were known from independent data. The good agreement between measured and calculated values of s/soc allowed us to interpret the sedimentation results in terms of DNA conformations, with particular emphasis on the marked effect of ionic conditions. As NaCl concentration decreases, s/soc increases because the Superhelix becomes less regular and more compact. In the presence of just 10 mM MgCl2, supercoiled DNA adopts essentially the same set of conformations as in moderate to high concentrations of NaCl. Our simulations showed that s is a strong function of the Superhelix branching frequency. At near physiological ionic conditions, there are about four branches in the 7 kb DNA molecule used in this work. We found no indication of Superhelix collapse in any ionic conditions even remotely approaching physiological ones. For all ionic conditions studied, we conclude that the electrostatic interaction of DNA segments specified by the DNA effective diameter is the primary determinant of supercoiled DNA conformations. # 1997 Academic Press Limited
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effect of supercoiling on the juxtaposition and relative orientation of dna sites
Biophysical Journal, 1996Co-Authors: Alexander Vologodskii, Nicholas R. CozzarelliAbstract:There are many proteins that interact simultaneously with two or more DNA sites that are separated along the DNA contour. These sites must be brought close together to form productive complexes with the proteins. We used Monte Carlo simulation of supercoiled DNA conformations to study the effect of supercoiling and DNA length on the juxtaposition of DNA sites, the angle between them, and the branching of the interwound Superhelix. Branching decreases the probability of juxtaposition of two DNA sites but increases the probability of juxtaposition of three sites at branch points. We found that the number of Superhelix branches increases linearly with the length of DNA from 3 to 20 kb. The simulations showed that for all contour distances between two sites, the juxtaposition probability in supercoiled DNA is two orders of magnitude higher than in relaxed DNA. Supercoiling also results in a strong asymmetry of the angular distribution of juxtaposed sites. The effect of supercoiling on site-specific recombination and the introduction of supercoils by DNA gyrase is discussed in the context of the simulation results.
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conformational and thermodynamic properties of supercoiled dna
Journal of Molecular Biology, 1992Co-Authors: Alexander Vologodskii, Stephen D Levene, K V Klenin, Maxim D Frankkamenetskii, Nicholas R. CozzarelliAbstract:Abstract We used Monte Carlo simulations to investigate the conformational and thermodynamic properties of DNA molecules with physiological levels of supercoiling. Three parameters determine the properties of DNA in this model: Kuhn statistical length, torsional rigidity and effective double-helix diameter. The chains in the simulation resemble strongly those observed by electron microscopy and have the conformation of an interwound Superhelix whose axis is often branched. We compared the geometry of simulated chains with that determined experimentally by electron microscopy and by topological methods. We found a very close agreement between the Monte Carlo and experimental values for writhe, Superhelix axis length and the number of superhelical turns. The computed number of Superhelix branches was found to be dependent on Superhelix density, DNA chain length and double-helix diameter. We investigated the thermodynamics of supercoiling and found that at low Superhelix density the entropie contribution to Superhelix free energy is negligible, whereas at high Superhelix density, the entropie and enthalpic contributions are nearly equal. We calculated the effect of supercoiling on the spatial distribution of DNA segments. The probability that a pair of DNA sites separated along the chain contour by at least 50 nm are juxtaposed is about two orders of magnitude greater in supercoiled DNA than in relaxed DNA. This increase in the effective local concentration of DNA is not strongly dependent on the contour separation between the sites. We discuss the implications of this enhancement of site juxtaposition by supercoiling in the context of protein-DNA interactions involving multiple DNA-binding sites.
Jörg Langowski - One of the best experts on this subject based on the ideXlab platform.
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Kinetics of intrachain reactions of supercoiled DNA: Theory and numerical modeling
The Journal of Chemical Physics, 2001Co-Authors: K V Klenin, Jörg LangowskiAbstract:We considered an irreversible biochemical intrachain reaction of supercoiled DNA as a random event that occurs, with some probability, at the instant of collision between two reactive groups attached to distant sites of the DNA molecule. For sufficiently small intrinsic rate constant kI, the dominant process contributing to the productive collisions is the quasione-dimensional reptation of the strands forming the Superhelix. The mean reaction time is then given by τF+1/kIcL, where τF is the mean time of the first collision caused by reptation, and cL is the local concentration of one reactive group around the other. The internal reptation of DNA strands was simulated by the repton model, in which a Superhelix branch is approximated by a string of beads placed in a row of cells. This simple model allows semiquantitative estimation of τF and cL (in some arbitrary units) for a large range of the DNA lengths L. The repton chain was calibrated with the help of the data available for small supercoiled plasmids ...
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The diameter of the DNA Superhelix decreases with salt concentration: SANS measurements and Monte Carlo simulations
Journal of Applied Crystallography, 2000Co-Authors: Markus Hammermann, K V Klenin, Nathalie Brun, R P May, Katalin Tóth, Jörg LangowskiAbstract:We have measured the static form factor of superhelical DNA by SANS in dilute aqueous solution as a function of salt concentration. Theoretical static form factors were calculated by Monte Carlo simulations. Simulated and measured form factors are in very good agreement and can be interpreted in terms of the Superhelix diameter, which decreases from (16.0±0.9) nm at 10 mM to (9.0±0.7) nm at 100 mM Na + concentration. This result contradicts a lateral collapse of the DNA Superhelix in physiological salt concentrations as recently found by cryo-electron microscopy.
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Superhelix organization by DNA curvature as measured through site-specific labeling
Journal of molecular biology, 1998Co-Authors: Claudia Pfannschmidt, Jörg LangowskiAbstract:Abstract For determining the position of a defined site in a superhelical DNA we have developed a method for introducing a covalent biotin label at a specific sequence while preserving the superhelicity. This is done by first introducing a specific nick, labeling the DNA by limited nick translation and sealing the nick with ligase. The superhelicity is controlled by including ethidium in the ligation reaction. Using scanning force of microscopy on DNAs labeled by this method, we have then compared the position of streptavidin markers at a specific site relative to the end loop of the Superhelix. We found that in DNAs with permanently curved inserts the label is located preferentially at a defined distance from the end loop, while in controls without curved inserts the label position was random. This indicates that curves are located in or near the end loops in a Superhelix.
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Salt-Dependent DNA Superhelix Diameter Studied by Small Angle Neutron Scattering Measurements and Monte Carlo Simulations
Biophysical journal, 1998Co-Authors: Markus Hammermann, K V Klenin, Nathalie Brun, R P May, Katalin Tóth, Jörg LangowskiAbstract:Using small angle neutron scattering we have measured the static form factor of two different superhelical DNAs, p1868 (1868 bp) and pUC18 (2686 bp), in dilute aqueous solution at salt concentrations between 0 and 1.5 M Na+ in 10 mM Tris at 0% and 100% D2O. For both DNA molecules, the theoretical static form factor was also calculated from an ensemble of Monte Carlo configurations generated by a previously described model. Simulated and measured form factors of both DNAs showed the same behavior between 10 and 100 mM salt concentration: An undulation in the scattering curve at a momentum transfer q = 0.5 nm-1 present at lower concentration disappears above 100 mM. The position of the undulation corresponds to a distance of approximately 10-20 nm. This indicated a change in the DNA Superhelix diameter, as the undulation is not present in the scattering curve of the relaxed DNA. From the measured scattering curves of superhelical DNA we estimated the Superhelix diameter as a function of Na+ concentration by a quantitative comparison with the scattering curve of relaxed DNA. The ratio of the scattering curves of superhelical and relaxed DNA is very similar to the form factor of a pair of point scatterers. We concluded that the distance of this pair corresponds to the interstrand separation in the Superhelix. The computed Superhelix diameter of 16.0 +/- 0.9 nm at 10 mM decreased to 9.0 +/- 0.7 nm at 100 mM salt concentration. Measured and simulated scattering curves agreed almost quantitatively, therefore we also calculated the Superhelix diameter from the simulated conformations. It decreased from 18.0 +/- 1.5 nm at 10 mM to 9.4 +/- 1.5 nm at 100 mM salt concentration. This value did not significantly change to lower values at higher Na+ concentration, in agreement with results obtained by electron microscopy, scanning force microscopy imaging in aqueous solution, and recent MC simulations, but in contrast to the observation of a lateral collapse of the DNA Superhelix as indicated by cryo-electron microscopy studies.
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Superhelix dimensions of a 1868 base pair plasmid determined by scanning force microscopy in air and in aqueous solution
Nucleic acids research, 1997Co-Authors: Karsten Rippe, Norbert Mücke, Jörg LangowskiAbstract:We have used scanning force microscopy (SFM) to study the conformation of a 1868 base pair plasmid (p1868) in its open circular form and at a superhelical density of σ = ‐0.034. The samples were deposited on a mica surface in the presence of MgCl2. DNA images were obtained both in air and in aqueous solutions, and the dimensions of the DNA Superhelix were analysed. Evaluation of the whole plasmid yielded average Superhelix dimensions of 27 ± 9 nm (outer Superhelix diameter D), 107 ± 51 nm (Superhelix pitch P), and 54 ± 8 (Superhelix pitch angle α). We also analysed compact superhelical regions within the plasmid separately, and determined values of D = 9.2 ± 3.3 nm, P = 42 ± 13 nm and α = 63 ± 20 for samples scanned in air or rehydrated in water. These results indicate relatively large conformation changes between superhelical and more open regions of the plasmid. In addition to the analysis of the DNA Superhelix dimensions, we have followed the deposition process of open circular p1868 to mica in real time. These experiments show that it is possible to image DNA samples by SFM without prior drying, and that the surface bound DNA molecules retain some ability to change their position on the surface.
K V Klenin - One of the best experts on this subject based on the ideXlab platform.
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Kinetics of intrachain reactions of supercoiled DNA: Theory and numerical modeling
The Journal of Chemical Physics, 2001Co-Authors: K V Klenin, Jörg LangowskiAbstract:We considered an irreversible biochemical intrachain reaction of supercoiled DNA as a random event that occurs, with some probability, at the instant of collision between two reactive groups attached to distant sites of the DNA molecule. For sufficiently small intrinsic rate constant kI, the dominant process contributing to the productive collisions is the quasione-dimensional reptation of the strands forming the Superhelix. The mean reaction time is then given by τF+1/kIcL, where τF is the mean time of the first collision caused by reptation, and cL is the local concentration of one reactive group around the other. The internal reptation of DNA strands was simulated by the repton model, in which a Superhelix branch is approximated by a string of beads placed in a row of cells. This simple model allows semiquantitative estimation of τF and cL (in some arbitrary units) for a large range of the DNA lengths L. The repton chain was calibrated with the help of the data available for small supercoiled plasmids ...
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The diameter of the DNA Superhelix decreases with salt concentration: SANS measurements and Monte Carlo simulations
Journal of Applied Crystallography, 2000Co-Authors: Markus Hammermann, K V Klenin, Nathalie Brun, R P May, Katalin Tóth, Jörg LangowskiAbstract:We have measured the static form factor of superhelical DNA by SANS in dilute aqueous solution as a function of salt concentration. Theoretical static form factors were calculated by Monte Carlo simulations. Simulated and measured form factors are in very good agreement and can be interpreted in terms of the Superhelix diameter, which decreases from (16.0±0.9) nm at 10 mM to (9.0±0.7) nm at 100 mM Na + concentration. This result contradicts a lateral collapse of the DNA Superhelix in physiological salt concentrations as recently found by cryo-electron microscopy.
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Salt-Dependent DNA Superhelix Diameter Studied by Small Angle Neutron Scattering Measurements and Monte Carlo Simulations
Biophysical journal, 1998Co-Authors: Markus Hammermann, K V Klenin, Nathalie Brun, R P May, Katalin Tóth, Jörg LangowskiAbstract:Using small angle neutron scattering we have measured the static form factor of two different superhelical DNAs, p1868 (1868 bp) and pUC18 (2686 bp), in dilute aqueous solution at salt concentrations between 0 and 1.5 M Na+ in 10 mM Tris at 0% and 100% D2O. For both DNA molecules, the theoretical static form factor was also calculated from an ensemble of Monte Carlo configurations generated by a previously described model. Simulated and measured form factors of both DNAs showed the same behavior between 10 and 100 mM salt concentration: An undulation in the scattering curve at a momentum transfer q = 0.5 nm-1 present at lower concentration disappears above 100 mM. The position of the undulation corresponds to a distance of approximately 10-20 nm. This indicated a change in the DNA Superhelix diameter, as the undulation is not present in the scattering curve of the relaxed DNA. From the measured scattering curves of superhelical DNA we estimated the Superhelix diameter as a function of Na+ concentration by a quantitative comparison with the scattering curve of relaxed DNA. The ratio of the scattering curves of superhelical and relaxed DNA is very similar to the form factor of a pair of point scatterers. We concluded that the distance of this pair corresponds to the interstrand separation in the Superhelix. The computed Superhelix diameter of 16.0 +/- 0.9 nm at 10 mM decreased to 9.0 +/- 0.7 nm at 100 mM salt concentration. Measured and simulated scattering curves agreed almost quantitatively, therefore we also calculated the Superhelix diameter from the simulated conformations. It decreased from 18.0 +/- 1.5 nm at 10 mM to 9.4 +/- 1.5 nm at 100 mM salt concentration. This value did not significantly change to lower values at higher Na+ concentration, in agreement with results obtained by electron microscopy, scanning force microscopy imaging in aqueous solution, and recent MC simulations, but in contrast to the observation of a lateral collapse of the DNA Superhelix as indicated by cryo-electron microscopy studies.
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conformational and thermodynamic properties of supercoiled dna
Journal of Molecular Biology, 1992Co-Authors: Alexander Vologodskii, Stephen D Levene, K V Klenin, Maxim D Frankkamenetskii, Nicholas R. CozzarelliAbstract:Abstract We used Monte Carlo simulations to investigate the conformational and thermodynamic properties of DNA molecules with physiological levels of supercoiling. Three parameters determine the properties of DNA in this model: Kuhn statistical length, torsional rigidity and effective double-helix diameter. The chains in the simulation resemble strongly those observed by electron microscopy and have the conformation of an interwound Superhelix whose axis is often branched. We compared the geometry of simulated chains with that determined experimentally by electron microscopy and by topological methods. We found a very close agreement between the Monte Carlo and experimental values for writhe, Superhelix axis length and the number of superhelical turns. The computed number of Superhelix branches was found to be dependent on Superhelix density, DNA chain length and double-helix diameter. We investigated the thermodynamics of supercoiling and found that at low Superhelix density the entropie contribution to Superhelix free energy is negligible, whereas at high Superhelix density, the entropie and enthalpic contributions are nearly equal. We calculated the effect of supercoiling on the spatial distribution of DNA segments. The probability that a pair of DNA sites separated along the chain contour by at least 50 nm are juxtaposed is about two orders of magnitude greater in supercoiled DNA than in relaxed DNA. This increase in the effective local concentration of DNA is not strongly dependent on the contour separation between the sites. We discuss the implications of this enhancement of site juxtaposition by supercoiling in the context of protein-DNA interactions involving multiple DNA-binding sites.
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Conformational and Thermodynamic Properties of Supercoiled DNA
Journal of molecular biology, 1992Co-Authors: Alexander Vologodskii, Stephen D Levene, K V Klenin, Maxim D. Frank-kamenetskii, Nicholas R. CozzarelliAbstract:We used Monte Carlo simulations to investigate the conformational and thermodynamic properties of DNA molecules with physiological levels of supercoiling. Three parameters determine the properties of DNA in this model: Kuhn statistical length, torsional rigidity and effective double-helix diameter. The chains in the simulation resemble strongly those observed by electron microscopy and have the conformation of an interwound Superhelix whose axis is often branched. We compared the geometry of simulated chains with that determined experimentally by electron microscopy and by topological methods. We found a very close agreement between the Monte Carlo and experimental values for writhe, Superhelix axis length and the number of superhelical turns. The computed number of Superhelix branches was found to be dependent on Superhelix density, DNA chain length and double-helix diameter. We investigated the thermodynamics of supercoiling and found that at low Superhelix density the entropic contribution to Superhelix free energy is negligible, whereas at high Superhelix density, the entropic and enthalpic contributions are nearly equal. We calculated the effect of supercoiling on the spatial distribution of DNA segments. The probability that a pair of DNA sites separated along the chain contour by at least 50 nm are juxtaposed is about two orders of magnitude greater in supercoiled DNA than in relaxed DNA. This increase in the effective local concentration of DNA is not strongly dependent on the contour separation between the sites. We discuss the implications of this enhancement of site juxtaposition by supercoiling in the context of protein-DNA interactions involving multiple DNA-binding sites.
J. Michael Schurr - One of the best experts on this subject based on the ideXlab platform.
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Effect of ethidium binding and Superhelix density on the supercoiling free energy and torsion and bending constants of p30δ DNA
Biophysical chemistry, 1994Co-Authors: James B Clendenning, Alexei N Naimushin, Bryant S Fujimoto, Doyle W Stewart, J. Michael SchurrAbstract:Topoisomer distributions created by the action of topoisomerase I on p30 delta DNA in the presence of various concentrations of ethidium are measured and analyzed using recently developed theory to obtain the twist energy parameter (ET) that governs the free energy of supercoiling in each case. Competitive dialysis experiments to investigate the relative affinity of ethidium for linear and supercoiled DNAs at different binding ratios are assayed fluorometrically and the results are analyzed using related theory. The topoisomer distributions and fluorescence intensity ratios agree well with the theory, which is based on the assumption that the supercoiling free energy varies quadratically with the effective linking difference, regardless of ethidium binding or Superhelix density. The topoisomer distribution experiments alone yield an average best-fit value, ET = 950 +/- 80, independent of ethidium binding ratio from r = 0 to 0.082, while the combined topoisomer distribution and ethidium binding experiments yield an average best-fit value, ET = 1030 +/- 90, which is essentially independent of ethidium binding ratio from r = 0 to 0.082 and Superhelix density from sigma = 0 to (-)0.053. One may conclude that the supercoiling free-energy-varies quadratically with effective linking difference over the entire range of observed ethidium binding ratios and Superhelix densities. The independently measured torsion constant (alpha) of p30 delta DNA is likewise essentially independent of Superhelix density and ethidium binding ratio. The observed invariance of ET and alpha implies that the bending constant kappa beta is similarly invariant to Superhelix density and ethidium binding ratio. The apparently ideal behavior displayed by p30 delta DNA is not exhibited by pBR322 DNA, which is discussed in the following companion paper.
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Effect of ethidium binding and Superhelix density on the apparent supercoiling free energy and torsion constant of pBR322 DNA.
Biophysical chemistry, 1994Co-Authors: Alexei N Naimushin, James B Clendenning, Bryant S Fujimoto, Doyle W Stewart, Lu Song, Ug-sung Kim, J. Michael SchurrAbstract:The value of the twist energy parameter (ET) of pBR322 is determined near zero Superhelix density from topoisomer distributions created under various conditions. The resulting value, ET = 1155 +/- 65, at 37 degrees C is essentially unaffected by adding 10 mM Mg2+, or by changing the kind of Topo I from chicken-red-cell to calf-thymus. This value significantly exceeds that (ET = 950 +/- 80) measured for p30 delta DNA under identical conditions by the same method in the preceding paper. Decreasing the temperature from 37 to 21 degrees C yields a slightly larger value, ET = 1340 +/- 130, but the statistical significance of the increase is marginal. Attempts to determine reliable ET values for pBR322 at higher Superhelix densities by ethidium binding were frustrated by the fact that good fits of the equilibrium dialysis results could not be achieved using a single value of ET. Moreover, the curves of apparent ET versus binding ratio r vary considerably from one preparation to another, and for a given preparation vary with time after cell lysis up to about seven weeks, after which they settle in to nearly reproducible behavior. The apparent ET values obtained from competitive dialysis experiments are typically rather low (ET approximately 700) for small r and nearly native Superhelix density, and rise up to 1300 to 1500 with increasing binding ratio (up to r = 0.055) and decreasing negative Superhelix density.(ABSTRACT TRUNCATED AT 250 WORDS)
