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Michael J Schurr - One of the best experts on this subject based on the ideXlab platform.
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effect of bending strain on the Torsion elastic Constant of dna
Journal of Molecular Biology, 1996Co-Authors: Patrick J Heath, James B Clendenning, Bryant S Fujimoto, Michael J SchurrAbstract:Abstract The Torsion Constants of both circular and linear forms of the same 181 bp DNA were investigated by time-resolved fluorescence polarization anisotropy (FPA) of intercalated ethidium. The ratio of intrinsic ethidium binding Constants of the circular and linear species was determined from the relative fluorescence intensities of intercalated and non-intercalated dye in each case. Possible changes in secondary structure were also probed by circular dichroism (CD) spectroscopy. Upon circularization, the Torsion Constant increased by a factor of 1.42, the intrinsic binding Constant for ethidium increased by about fourfold, and the CD spectrum underwent a significant change. These effects are attributed to an altered secondary structure induced by the bending strain. Quantitative agreement between Torsion Constants obtained from the present FPA studies and previous topoisomer distribution measurements on circular DNAs containing 205 to 217 bp removes a long-standing apparent discrepancy between those two methods. After storage at 4°C for eight months, the Torsion Constant of the circular DNA increased by about 1.25-fold, whereas that of the linear DNA remained unchanged. For these aged circles, both the Torsion Constant and intrinsic binding Constant ratio lie close to the corresponding values obtained previously for a 247 bp DNA by analyzing topoisomer distributions created in the presence of various amounts of ethidium. The available evidence strongly implies that Torsion Constants measured for small circular DNAs with less than 250 bp are specific to the altered secondary structure(s) therein, and are not applicable to linear and much larger circular DNAs with lower mean bending strains.
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effect of bending strain on the Torsion Constant of dna
5th International Conference on Laser Applications in Life Sciences, 1995Co-Authors: Patrick J Heath, Stuart A Allison, Michael J SchurrAbstract:The Torsion Constants of both linear and circular forms of the same approximately 200 base- pair DNA were measured by time-resolved fluorescence polarization anisotropy (FPA). Upon circularization, the Torsion Constant undergoes a substantial increase, and the circular dichroism (CD) spectrum indicates a significant perturbation of the secondary structure. The Torsion Constant of the circular form lies in the range of values determined previously for similarly small circular DNAs by static methods. It is suggested that the long-standing discrepancy between Torsion Constants obtained via static methods for small circles and those obtained via the FPA method for linear DNAs may be due to such an alteration of the secondary structure and enhancement of the Torsion Constant by coherent bending strain present in the small circles.© (1995) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.
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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, Lu Song, Bryant S Fujimoto, Doyle W Stewart, Michael J SchurrAbstract:Abstract The value of the twist energy parameter ( E T ) of pBR322 is determined near zero superhelix density from topoisomer distributions created under various conditions. The resulting value, E T = 1155 ± 65, at 37°C is essentially unaffected by adding 10 mM Mg 2+ , or by changing the kind of Topo I from chicken-red-cell to calf-thymus. This value significantly exceeds that ( E T = 950 ± 80) measured for p30δ DNA under indentical conditions by the same method in the preceding paper. Decreasing the temperature from 37 to 21°C yields a slightly larger value, E T = 1340 ± 130, but the statistical significance of the increase is marginal. Attempts to determine reliable E T 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 E T . Moreover, the curves of apparent E T 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 E T values obtained from competitive dialysis experiments are typically rather low ( E T ∼ 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. The observed trend of apparent E T values extrapolates to a still larger value at r ≈ 0.07, σ = 0, which significantly exceeds the value obtained from topoisomer distributions near r = 0, σ = 0. These findings cast considerable doubt on the reliability of apparent E T values obtained for pBR322 by ethidium binding studies. The differences between pBR322 and p30δ are attributed to one or more different secondary conformations that prevails over part of the pBR322 sequence, and to the metastable prevalence of one or more of those as dye is added. In the case of pBR322 DNA, the Torsion Constant, molar ellipticity at 250 nm, and plateau diffusion coefficient from dynamic light scattering at K = 4.5 × 10 5 cm −1 all vary with superhelix density in a similar way, showing dips at σ = −0.010 and jumps at σ = −0.037. This behavior, which is similar to that observed previously for pUC8 dimer DNA, suggests that two successive transitions in secondary structure are induced in pBR322 by increasing negative superhelix density. The possible role of such structural transitions in the contrasting behaviors of pBR322 and p30δ is discussed.
Jianyu Zhang - One of the best experts on this subject based on the ideXlab platform.
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effect of mean stress on 2a12 t4 aluminum alloy under tension Torsion Constant amplitude loading
Key Engineering Materials, 2016Co-Authors: Jianyu ZhangAbstract:Fatigue tests have been carried out to investigate the effects of mean-stress and phase-difference on the tension-Torsion fatigue failure of 2A12-T4 aluminum alloy. The results show that for fully reversed tension-Torsion loading, the fatigue life increases with the increase of phase angle, but the fatigue life decreases with the increase of phase angle, when mean-stress exists, both for shear mean-stress and normal mean-stress. Fracture appearance shows that the crack initiation is on the direction of maximum shear stress amplitude plane. Critical plane criteria based on the linear combination of the maximum shear stress amplitude and maximum normal stress are studied and further discussion on the drawbacks of this kind of criteria are performed.
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high cycle fatigue and fracture mode analysis of 2a12 t4 aluminum alloy under out of phase axial Torsion Constant amplitude loading
International Journal of Fatigue, 2012Co-Authors: Jianyu ZhangAbstract:Abstract Fatigue experiments with solid cylindrical bar specimens are carried out to investigate the effects of stress amplitude ratios and phase angles on the axial–Torsion high-cycle fatigue failure of 2A12–T4 aluminum alloy. Macro- and micro-analysis of the specimen fracture appearance is conducted in order to obtain the fracture characteristics under combined axial–Torsion loading with different stress amplitude ratios and different phase angles. The soft factor of fatigue loading is introduced to estimate the failure mode under the combined axial–Torsion loading. The result indicates that the estimation about the soft factor of fatigue loading is consistent with the macro- and micro-analysis.
Bryant S Fujimoto - One of the best experts on this subject based on the ideXlab platform.
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effect of bending strain on the Torsion elastic Constant of dna
Journal of Molecular Biology, 1996Co-Authors: Patrick J Heath, James B Clendenning, Bryant S Fujimoto, Michael J SchurrAbstract:Abstract The Torsion Constants of both circular and linear forms of the same 181 bp DNA were investigated by time-resolved fluorescence polarization anisotropy (FPA) of intercalated ethidium. The ratio of intrinsic ethidium binding Constants of the circular and linear species was determined from the relative fluorescence intensities of intercalated and non-intercalated dye in each case. Possible changes in secondary structure were also probed by circular dichroism (CD) spectroscopy. Upon circularization, the Torsion Constant increased by a factor of 1.42, the intrinsic binding Constant for ethidium increased by about fourfold, and the CD spectrum underwent a significant change. These effects are attributed to an altered secondary structure induced by the bending strain. Quantitative agreement between Torsion Constants obtained from the present FPA studies and previous topoisomer distribution measurements on circular DNAs containing 205 to 217 bp removes a long-standing apparent discrepancy between those two methods. After storage at 4°C for eight months, the Torsion Constant of the circular DNA increased by about 1.25-fold, whereas that of the linear DNA remained unchanged. For these aged circles, both the Torsion Constant and intrinsic binding Constant ratio lie close to the corresponding values obtained previously for a 247 bp DNA by analyzing topoisomer distributions created in the presence of various amounts of ethidium. The available evidence strongly implies that Torsion Constants measured for small circular DNAs with less than 250 bp are specific to the altered secondary structure(s) therein, and are not applicable to linear and much larger circular DNAs with lower mean bending strains.
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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, Lu Song, Bryant S Fujimoto, Doyle W Stewart, Michael J SchurrAbstract:Abstract The value of the twist energy parameter ( E T ) of pBR322 is determined near zero superhelix density from topoisomer distributions created under various conditions. The resulting value, E T = 1155 ± 65, at 37°C is essentially unaffected by adding 10 mM Mg 2+ , or by changing the kind of Topo I from chicken-red-cell to calf-thymus. This value significantly exceeds that ( E T = 950 ± 80) measured for p30δ DNA under indentical conditions by the same method in the preceding paper. Decreasing the temperature from 37 to 21°C yields a slightly larger value, E T = 1340 ± 130, but the statistical significance of the increase is marginal. Attempts to determine reliable E T 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 E T . Moreover, the curves of apparent E T 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 E T values obtained from competitive dialysis experiments are typically rather low ( E T ∼ 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. The observed trend of apparent E T values extrapolates to a still larger value at r ≈ 0.07, σ = 0, which significantly exceeds the value obtained from topoisomer distributions near r = 0, σ = 0. These findings cast considerable doubt on the reliability of apparent E T values obtained for pBR322 by ethidium binding studies. The differences between pBR322 and p30δ are attributed to one or more different secondary conformations that prevails over part of the pBR322 sequence, and to the metastable prevalence of one or more of those as dye is added. In the case of pBR322 DNA, the Torsion Constant, molar ellipticity at 250 nm, and plateau diffusion coefficient from dynamic light scattering at K = 4.5 × 10 5 cm −1 all vary with superhelix density in a similar way, showing dips at σ = −0.010 and jumps at σ = −0.037. This behavior, which is similar to that observed previously for pUC8 dimer DNA, suggests that two successive transitions in secondary structure are induced in pBR322 by increasing negative superhelix density. The possible role of such structural transitions in the contrasting behaviors of pBR322 and p30δ is discussed.
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Effect of ethidium on the Torsion Constants of linear and supercoiled DNAs
Biophysical Chemistry, 1991Co-Authors: Pengguang Wu, Lu Song, Bryant S Fujimoto, J. Michael SchurrAbstract:Abstract The Torsion elastic Constants (α) of linear pBR322 (4363 bp) and pUC8 (2717 bp) DNAs and supercoiled pBR322 and pJMSII (4375 bp) DNAs are measured in 0.1 M NaCl as a function of added ethidium/base-pair (EB/BP) ratio by studying the fluorescence polarization anisotropy (FPA) of the intercalated ethidium. The time-resolved FPA is measured by using a picosecond dye laser for excitation and time-correlated single photon counting detection. Previously developed theory for the emission anisotropy is generalized to incorporate rotations of the transition dipole due to excitation transfer. The excitation transfers are simulated by a Monte Carlo procedure (Genest et al., Biophys. Chem. 1 (1974) 266–278) and the consequent rotations of the transition dipole are superposed on the Brownian rotations. After accounting for excitation transfer, the Torsion Constants of the linear DNAs are found to be essentially independent of intercalated ethidium up to a binding ratio r = 0.10 dye/bp. Dynamic light scattering measurements on linear pUC8 DNA confirm that the Torsion Constant is independent of binding ratio up to r = 0.20 dye/bp. If α d denotes the Torsion Constant between ethidium and a base-pair, and α 0 that between two base-pairs, then our data imply that α d /α 0 lies in the range 0.65 to 1.64 with a most probable value of 1.0. The Torsion Constants of supercoiled DNAs decrease substantially with increasing binding ratio even after accounting for excitation transfer. At the binding ratio r * = 0.064, where the superhelix density vanishes and superhelical strain is completely relaxed, the Torsion Constant of the supercoiled pBR322 DNA/dye complex lies below that of the corresponding linear DNA/dye complex by about 30%. This contradicts the conventional view according to which linear, nicked circular, and supercoiled DNA/dye complexes with r = r * should coexist with the same concentration of free dye, display the same distribution of bound dye, and exhibit identical secondary structures, twisting and bending rigidities, and FPA dynamics. These and other observations imply the existence of metastable secondary structure in freshly relaxed supercoiled DNAs. A tentative explanation is presented for these and other unexpected observations on supercoiled DNAS.
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Evidence for allosteric transitions in secondary structure induced by superhelical stress
Journal of Molecular Biology, 1990Co-Authors: Lu Song, Bryant S Fujimoto, John H. Shibata, Pengguang Wu, John C. Thomas, J. Michael SchurrAbstract:Previous studies suggest that the global secondary structures of native supercoiled and equilibrium linear DNAs may differ somewhat. Recent evidence also indicates that metastable secondary structure commonly persists following complete relaxation of the superhelical stress by intercalating dyes or by the action of topoisomerase I. In this work, the Torsion Constants (α) of pBR322, pUC8 and M13mp7 (replicative form) DNAs are determined by time-resolved fluorescence polarization anisotropy at various times subsequent to linearization. In all three cases, the Torsion Constants are relatively low immediately after linearization, and evolve for eight to ten weeks before reaching their apparent equilibrium values. It is shown in detail how the persistence of metastable secondary structure, subsequent to relaxation of superhelical stress, necessarily implies that one or more transitions in equilibrium secondary structure are induced as the superhelix density is varied from zero to native, orvice versa. Samples of pUC8 dimer (5434 base-pairs) with different superhelix densities are prepared by the action of topoisomerase I in the presence of various amounts of ethidium. Their median linking number differences are determined by standard band counting methods. The translational diffusion coefficient (D0) and the plateau diffusion coefficient (Dplat) characterizing internal motions over short distances (225A˚) are determined by dynamic light-scattering. The Torsion Constant (α) between base-pairs and the circular dichroism spectrum are also measured for each sample. Curves ofDplat,D0, α and molar ellipticity ([θ]) (at the minimum near 250 nm)versus superhelix density (σ) are constructed. The curve ofD0versus σ is very similar to that for sedimentation coefficientversus σ for simian virus 40 (SV40) and polyoma DNAs. The curves ofDplat,D0, α and [θ]versus θ show that, with increasing negative superhelix density, a structural transition occurs nearσ = −0.020to an intermediate state with low Torsion Constant, and a second structural transition occurs nearσ = −0.035 to a state that exhibits more normal properties by σ = −0.048. These data are consistent with the hypothesis that supercoiling induces two successive allosteric transitions to alternative global secondary structures. The data are much less consistent with the hypothesis that supercoiling induces some radical secondary structure at one or a few sites of small extent atσ = −0.020, and at other sites atσ = −0.035, or with hypotheses based on changes in tertiary structure alone. It is proposed that extremely slow kinetics of the transition atσ = −0.020 results in extended trapping of the intermediate state with low Torsion Constant, when superhelical strain is released. This may account for the observed failure of supercoiled DNAs to convert completely toB-helix upon relaxation of the superhelix density, and also for other contradictions of the standard model of supercoiled DNAs.
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Dependence of the Torsional rigidity of DNA on base composition
Nature, 1990Co-Authors: Bryant S Fujimoto, J. Michael SchurrAbstract:THE Escherichia coli phage 434 represser binds as a dimer to the operator of the DNA helix. Although the centre of the operator is not in contact with protein, the represser binding affinity can be reduced at least 50-fold by changing the sequence there^1: operators with A·T base pairs near their centre bind the represser more strongly than do operators with G·C base pairs at the same positions. To explain these observations, it has been proposed that the base composition at the centre of the operator affects the affinity of the operator for represser by altering the ease with which operator DNA can undergo the Torsional deformation necessary for complex formation ^1,2. In this model, the variation in binding affinity would require the Torsion Constant to have specific values and to change in a sequence-dependent manner^1. We have now measured Torsion Constants for DNAs with widely different base compositions. Our results indicate that the Torsion Constants depend only slightly on the overall composition, and firmly delimit the range of values for each. Even the upper-limit values are much too small to account for the observed changes in affinity of the 434 represser. These results rule out simple models that rely on substantial generic differences in Torsion Constant between A·T-rich sequences and G·C-rich sequences, although they do not rule out the possibility of particular sequences having abnormal Torsion Constants.
J. Michael Schurr - One of the best experts on this subject based on the ideXlab platform.
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Effect of anisotropy of the bending rigidity on the supercoiling free energy of small circular DNAs
Biopolymers, 1995Co-Authors: J. Michael Schurr, Hazen P. Babcock, John A. GebeAbstract:In principle, the supercoiling free energy of a small circular DNA will be enhanced by increasing the anisotropy of its bending potential at Constant persistence length. The magnitude of this effect is investigated by Monte Carlo simulation using an extension of a previously proposed algorithm. The supercoiling free energy at 298 K is simulated for circular DNAs containing N = 100 bp with Torsion Constant α = 5.8 × 10−12 dyne cm. persistence lengths P = 500 A and 10,000 A and a range of anisotropies of the bending potential from p = 1.0 to 16.0. The apparent Torsion Constants, reckoned from these supercoiling free energies by assuming an isotropic bending potential, are found to increase by less than 3% as the input anisotropy increases from 1.0 to 16.0. When P = 500 A, the apparent Torsion Constant never rises significantly above the input value over the entire range of input anisotropies. When P = 10,000 A, the apparent Torsion Constant rises only about 3% above the input value for anisotropies ρ = 8.0 and 16.0. Evidently, anisotropy of the bending potential cannot account for the fact that the Torsion Constants reported for small circular DNAs exceed those reported for longlinear DNAs by a factor of 1.6 or more. © 1995 John Wiley & Sons, Inc.
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Effect of ethidium on the Torsion Constants of linear and supercoiled DNAs
Biophysical Chemistry, 1991Co-Authors: Pengguang Wu, Lu Song, Bryant S Fujimoto, J. Michael SchurrAbstract:Abstract The Torsion elastic Constants (α) of linear pBR322 (4363 bp) and pUC8 (2717 bp) DNAs and supercoiled pBR322 and pJMSII (4375 bp) DNAs are measured in 0.1 M NaCl as a function of added ethidium/base-pair (EB/BP) ratio by studying the fluorescence polarization anisotropy (FPA) of the intercalated ethidium. The time-resolved FPA is measured by using a picosecond dye laser for excitation and time-correlated single photon counting detection. Previously developed theory for the emission anisotropy is generalized to incorporate rotations of the transition dipole due to excitation transfer. The excitation transfers are simulated by a Monte Carlo procedure (Genest et al., Biophys. Chem. 1 (1974) 266–278) and the consequent rotations of the transition dipole are superposed on the Brownian rotations. After accounting for excitation transfer, the Torsion Constants of the linear DNAs are found to be essentially independent of intercalated ethidium up to a binding ratio r = 0.10 dye/bp. Dynamic light scattering measurements on linear pUC8 DNA confirm that the Torsion Constant is independent of binding ratio up to r = 0.20 dye/bp. If α d denotes the Torsion Constant between ethidium and a base-pair, and α 0 that between two base-pairs, then our data imply that α d /α 0 lies in the range 0.65 to 1.64 with a most probable value of 1.0. The Torsion Constants of supercoiled DNAs decrease substantially with increasing binding ratio even after accounting for excitation transfer. At the binding ratio r * = 0.064, where the superhelix density vanishes and superhelical strain is completely relaxed, the Torsion Constant of the supercoiled pBR322 DNA/dye complex lies below that of the corresponding linear DNA/dye complex by about 30%. This contradicts the conventional view according to which linear, nicked circular, and supercoiled DNA/dye complexes with r = r * should coexist with the same concentration of free dye, display the same distribution of bound dye, and exhibit identical secondary structures, twisting and bending rigidities, and FPA dynamics. These and other observations imply the existence of metastable secondary structure in freshly relaxed supercoiled DNAs. A tentative explanation is presented for these and other unexpected observations on supercoiled DNAS.
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Evidence for allosteric transitions in secondary structure induced by superhelical stress
Journal of Molecular Biology, 1990Co-Authors: Lu Song, Bryant S Fujimoto, John H. Shibata, Pengguang Wu, John C. Thomas, J. Michael SchurrAbstract:Previous studies suggest that the global secondary structures of native supercoiled and equilibrium linear DNAs may differ somewhat. Recent evidence also indicates that metastable secondary structure commonly persists following complete relaxation of the superhelical stress by intercalating dyes or by the action of topoisomerase I. In this work, the Torsion Constants (α) of pBR322, pUC8 and M13mp7 (replicative form) DNAs are determined by time-resolved fluorescence polarization anisotropy at various times subsequent to linearization. In all three cases, the Torsion Constants are relatively low immediately after linearization, and evolve for eight to ten weeks before reaching their apparent equilibrium values. It is shown in detail how the persistence of metastable secondary structure, subsequent to relaxation of superhelical stress, necessarily implies that one or more transitions in equilibrium secondary structure are induced as the superhelix density is varied from zero to native, orvice versa. Samples of pUC8 dimer (5434 base-pairs) with different superhelix densities are prepared by the action of topoisomerase I in the presence of various amounts of ethidium. Their median linking number differences are determined by standard band counting methods. The translational diffusion coefficient (D0) and the plateau diffusion coefficient (Dplat) characterizing internal motions over short distances (225A˚) are determined by dynamic light-scattering. The Torsion Constant (α) between base-pairs and the circular dichroism spectrum are also measured for each sample. Curves ofDplat,D0, α and molar ellipticity ([θ]) (at the minimum near 250 nm)versus superhelix density (σ) are constructed. The curve ofD0versus σ is very similar to that for sedimentation coefficientversus σ for simian virus 40 (SV40) and polyoma DNAs. The curves ofDplat,D0, α and [θ]versus θ show that, with increasing negative superhelix density, a structural transition occurs nearσ = −0.020to an intermediate state with low Torsion Constant, and a second structural transition occurs nearσ = −0.035 to a state that exhibits more normal properties by σ = −0.048. These data are consistent with the hypothesis that supercoiling induces two successive allosteric transitions to alternative global secondary structures. The data are much less consistent with the hypothesis that supercoiling induces some radical secondary structure at one or a few sites of small extent atσ = −0.020, and at other sites atσ = −0.035, or with hypotheses based on changes in tertiary structure alone. It is proposed that extremely slow kinetics of the transition atσ = −0.020 results in extended trapping of the intermediate state with low Torsion Constant, when superhelical strain is released. This may account for the observed failure of supercoiled DNAs to convert completely toB-helix upon relaxation of the superhelix density, and also for other contradictions of the standard model of supercoiled DNAs.
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Dependence of the Torsional rigidity of DNA on base composition
Nature, 1990Co-Authors: Bryant S Fujimoto, J. Michael SchurrAbstract:THE Escherichia coli phage 434 represser binds as a dimer to the operator of the DNA helix. Although the centre of the operator is not in contact with protein, the represser binding affinity can be reduced at least 50-fold by changing the sequence there^1: operators with A·T base pairs near their centre bind the represser more strongly than do operators with G·C base pairs at the same positions. To explain these observations, it has been proposed that the base composition at the centre of the operator affects the affinity of the operator for represser by altering the ease with which operator DNA can undergo the Torsional deformation necessary for complex formation ^1,2. In this model, the variation in binding affinity would require the Torsion Constant to have specific values and to change in a sequence-dependent manner^1. We have now measured Torsion Constants for DNAs with widely different base compositions. Our results indicate that the Torsion Constants depend only slightly on the overall composition, and firmly delimit the range of values for each. Even the upper-limit values are much too small to account for the observed changes in affinity of the 434 represser. These results rule out simple models that rely on substantial generic differences in Torsion Constant between A·T-rich sequences and G·C-rich sequences, although they do not rule out the possibility of particular sequences having abnormal Torsion Constants.
Patrick J Heath - One of the best experts on this subject based on the ideXlab platform.
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On the origin of the temperature dependence of the supercoiling free energy
Biophysical Journal, 1997Co-Authors: Jeffrey J. Delrow, Patrick J Heath, J. M. SchurrAbstract:Monte Carlo simulations using temperature-invariant Torsional and bending rigidities fail to predict the rather steep decline of the experimental supercoiling free energy with increasing temperature, and consequently fail to predict the correct sign and magnitude of the supercoiling entropy. To illustrate this problem, values of the twist energy parameter (E(T)), which governs the supercoiling free energy, were simulated using temperature-invariant Torsion and bending potentials and compared to experimental data on pBR322 over a range of temperatures. The slope, -dE(T)/dT, of the simulated values is also compared to the slope derived from previous calorimetric data. The possibility that the discrepancies arise from some hitherto undetected temperature dependence of the Torsional rigidity was investigated. The Torsion elastic Constant of an 1876-bp restriction fragment of pBR322 was measured by time-resolved fluorescence polarization anisotropy of intercalated ethidium over the range 278–323 K, and found to decline substantially over that interval. Simulations of a 4349-bp model DNA were performed using these measured temperature-dependent Torsional rigidities. The slope, -dE(T)/dT, of the simulated data agrees satisfactorily with the slope derived from previous calorimetric measurements, but still lies substantially below that of Duguet's data. Models that involve an equilibrium between different secondary structure states with different intrinsic twists and Torsion Constants provide the most likely explanation for the variation of the Torsion Constant with T and other pertinent observations.
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effect of bending strain on the Torsion elastic Constant of dna
Journal of Molecular Biology, 1996Co-Authors: Patrick J Heath, James B Clendenning, Bryant S Fujimoto, Michael J SchurrAbstract:Abstract The Torsion Constants of both circular and linear forms of the same 181 bp DNA were investigated by time-resolved fluorescence polarization anisotropy (FPA) of intercalated ethidium. The ratio of intrinsic ethidium binding Constants of the circular and linear species was determined from the relative fluorescence intensities of intercalated and non-intercalated dye in each case. Possible changes in secondary structure were also probed by circular dichroism (CD) spectroscopy. Upon circularization, the Torsion Constant increased by a factor of 1.42, the intrinsic binding Constant for ethidium increased by about fourfold, and the CD spectrum underwent a significant change. These effects are attributed to an altered secondary structure induced by the bending strain. Quantitative agreement between Torsion Constants obtained from the present FPA studies and previous topoisomer distribution measurements on circular DNAs containing 205 to 217 bp removes a long-standing apparent discrepancy between those two methods. After storage at 4°C for eight months, the Torsion Constant of the circular DNA increased by about 1.25-fold, whereas that of the linear DNA remained unchanged. For these aged circles, both the Torsion Constant and intrinsic binding Constant ratio lie close to the corresponding values obtained previously for a 247 bp DNA by analyzing topoisomer distributions created in the presence of various amounts of ethidium. The available evidence strongly implies that Torsion Constants measured for small circular DNAs with less than 250 bp are specific to the altered secondary structure(s) therein, and are not applicable to linear and much larger circular DNAs with lower mean bending strains.
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effect of bending strain on the Torsion Constant of dna
5th International Conference on Laser Applications in Life Sciences, 1995Co-Authors: Patrick J Heath, Stuart A Allison, Michael J SchurrAbstract:The Torsion Constants of both linear and circular forms of the same approximately 200 base- pair DNA were measured by time-resolved fluorescence polarization anisotropy (FPA). Upon circularization, the Torsion Constant undergoes a substantial increase, and the circular dichroism (CD) spectrum indicates a significant perturbation of the secondary structure. The Torsion Constant of the circular form lies in the range of values determined previously for similarly small circular DNAs by static methods. It is suggested that the long-standing discrepancy between Torsion Constants obtained via static methods for small circles and those obtained via the FPA method for linear DNAs may be due to such an alteration of the secondary structure and enhancement of the Torsion Constant by coherent bending strain present in the small circles.© (1995) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.