Energetics of superhelicity and of B-Z transitions in superhelical DNA

The linking difference, alpha, imposed upon a superhelically constrained DNA molecule must be partitioned between twisting and bending deformations. Transitions to alternative secondary structures can occur at susceptible sites, altering the local molecular twist by an amount delta Twtrans. That part of the linking difference not accommodated in this way, the residual linking difference alpha res, must be manifested as smooth torsional and flexural deformations of secondary structure. The competition among the alternative ways of accommodating the imposed linking difference alpha determines a stressed equilibrium state. The superhelical free energy, G(alpha), is the excess free energy of the equilibrium state at linking difference alpha above that of the relaxed state under identical conditions. In this paper a method is described by which the free energies associated both to linking, G(alpha), and to residual linking differences can be determined from data on superhelical conformational transitions. The application of this approach to previously published experimental data on the B-Z transition suggests that the free energy associated with alpha res is about 30% larger at substantial superhelicities than it is near the relaxed state. At the onset of transition the functional form of G(alpha) is shown to change in a manner dependent upon the length of the Z-susceptible site.     

Slight changes in conditions influence the family of non-B-DNA conformations of the herpes simplex virus type 1 DR2 repeats

The segment inversion site of herpes simplex virus type 1 contains a series of tandem repeats with a purine bias on one strand and high G + C content (DR2 repeats) capable of adopting a non-B-DNA structure under a variety of conditions. Plasmids carrying eight contiguous copies of DR2 sequences undergo a series of supercoil-driven conformational transitions resulting in different extents of relaxation at pH 5.0. These transitions depend on the presence of an appropriate concentration of divalent cations (Mg2+ and Ca2+) which seem to interact specifically with the alternate structure(s). The transitions occurred at approximately the same superhelical density for all lengths of inserts studied. However, the onset of the transition can be shifted to lower negative superhelical densities by increasing NaCl concentrations. This leads to a reduction of the cooperativity of the transition, which takes place over a range of linking isomers under these conditions. Extrapolating from these results, we established physiological conditions where the alternate DNA structure is found at negative superhelical densities as low as -0.035. The existence of non-B-DNA conformations and/or the structural transitions of these sequences located in this region of intense biological activity implies their involvement in the life cycle of the virus.     

Energetics of superhelicity and of B-Z transitions in superhelical DNA

The linking difference, α, imposed upon a superhelically constrained DNA molecule must be partitioned between twisting and bending deformations. Transitions to alternative secondary structures can occur at susceptible sites, altering the local molecular twist by an amount ΔTw _trans. That part of the linking difference not accommodated in this way, the residual linking difference α_res, must be manifested as smooth torsional and flexural deformations of secondary structure. The competition among the alternative ways of accommodating the imposed linking difference α determines a stressed equilibrium state. The superhelical free energy,G(α), is the excess free energy of the equilibrium state at linking difference α above that of the relaxed state under identical conditions. In this paper a method is described by which the free energies associated both to linking,G(α), and to residual linking differences can be determined from data on superhelical conformational transitions. The application of this approach to previously published experimental data on the B-Z transition suggests that the free energy associated with α_res is about 30% larger at substantial superhelicities than it is near the relaxed state. At the onset of transition the functional form ofG(α) is shown to change in a manner dependent upon the length of the Z-susceptible site.     

Topoisomerase mutants and physiological conditions control supercoiling and Z-DNA formation in vivo.

The influence of topoisomerase I and gyrase mutations in Escherichia coli on the supercoiled density of recombinant plasmids and the stability of left-handed Z-DNA was investigated. The formation of Z-DNA in vivo by dC-dG sequences of different lengths was used to determine the effective plasmid supercoil densities in the mutant strains. The presence of Z-DNA in the cells was detected by linking number and EcoRI methylase inhibition assays. A change in the unrestrained superhelical tension in vivo directly effects the B- to Z-DNA transition. Alterations in the internal or external environment of the cells, such as the inactivation of gyrase or topoisomerase I, a gyrase temperature-sensitive mutant, or starvation of cells, have a dramatic influence on the topology of plasmids. Also, E. coli has significantly more superhelical strain than Klebsiella, Morganella, or Enterobacter. These studies indicate that linking deficiency and effective supercoil density are mutually independent variables of plasmid tertiary structure. A variety of factors, such as protein-DNA interactions, activity of topoisomerases, and the resulting supercoil density, contribute to the B to Z transition inside living cells.     

Cruciform extrusion in plasmids bearing the replicative intermediate configuration of a poxvirus telomere

The transition from lineform DNA to cruciform DNA (cruciformation) within the cloned telomere sequences of the Leporipoxvirus Shope fibroma virus (SFV) has been studied. The viral telomere sequences have been cloned in recombination-deficient Escherichia coli as a 322 base-pair, imperfect palindromic insert in pUC13. The inverted repeat configuration is equivalent to the arrangement of the telomere structures observed within viral DNA replicative intermediates. A major cruciform structure in the purified recombinant plasmid has been identified and mapped using, as probes, the enzymes AflII, nuclease S1 and bacteriophage T7 endonuclease I. It was extruded from the central axis of the cloned viral inverted repeat and, by unrestricted branch migration, attained a size commensurate with the superhelical density of the plasmid molecule at native superhelical densities. This major cruciform extrusion event was the only detectable duplex DNA perturbation, induced by negative superhelical torsion, in the insert viral sequences. No significant steady-state pool of extruded cruciform was identified in E. coli. However, the identification of a major deletion variant generated even in the recombination-deficient E. coli strain DB1256 (recA recBC sbcB) suggested that the cruciform may be extruded transiently in vivo. The lineform to cruciform transition has been further characterized in vitro using two-dimensional agarose gel electrophoresis. The transition was marked by a high energy of formation (delta Gf = 44 kcal/mol), and an apparently low activation energy that enabled facile transitions at physiological temperatures provided there was sufficient torsional energy. By comparing cruciformation in a series of related bidirectional central axis deletions of the telomeric insert, it has been concluded that the presence of extrahelical bases in the terminal hairpin structures contributes substantially to the high delta Gf value. Also, viral sequences flanking the extruded cruciform were shown to influence the measured delta Gf value. Several general features of poxvirus telomere structure that would be expected to influence the facility of cruciform extrusion are discussed along with the implications of the observed cruciform transition event on the replicative process of poxviruses in vivo.     

Conformational variation in superhelical deoxyribonucleic acid.

Sedimentation experiments have shown that superhelical DNA undergoes a sharp structural transition at low ionic strength. Light-scattering experiments show that this is due to a change in conformation of the DNA rather than to a change in interactions among DNA molecules. The results show that two possible conformations can occur for superhelical DNA under routine experimental conditions and may explain the discrepancies in the number of early unwinding sites exposed by different techniques.     

Evidence for a novel conformational transition in superhelical DNA

On the assumption that the dependence of the electrophoretic mobility of superhelical DNA upon the number of tertiary turns (Wr) is a monotonously increasing function devoid of points of inflection, it is concluded that the inflection (change of sign of the first derivative) observed on the curve gives evidence for a conformational transition in DNA secondary structure that begins long before the BZ or B cruciform transitions. The transition consumes 60% of the topological turns at native levels of supercoiling. It is proposed that the conformation produced belongs to the A-family. Provided that this transition indeed yields the A form (11 base pairs per turn), the energy of the BA conformational transition is estimatd to be 5.8–10.3 cal per base pair for different nucleotide sequences at physiological ionic strength. The energies of BZ and B cruciform transitions in superhelical DNA estimated from electrophoretic mobilities by the present method coincide perfectly with the values obtained by other authors using other methods. In addition, on the basis of the data of Brady et al. (1983) on the number of tertiary turns in superhelical DNA determined by X-ray scattering, it is concluded that the initial assumption is justified and the ratio of bending to twisting stiffnesses of superhelical DNA is estimated as 71 (in the fully supercoiled molecule containing 50% of the supposed A-conformation).     

Energetics of the strand separation transition in superhelical DNA.

In this paper the values of three free energy parameters governing the superhelical strand separation transition are determined by analysis of available experimental data. These are the free energy, a, needed to initiate a run of separation, the torsional stiffness, C, associated with interstrand winding of the two single strands comprising a separated site and the coefficient, K, of the quadratic free energy associated to residual linking. The experimental data used in this analysis are the locations and relative amounts of strand separation occurring in the pBR322 DNA molecule and the measured residual linking, both evaluated over a range of negative linking differences. The analytic method used treats strand separation as a heteropolymeric, co-operative, two-state transition to a torsionally deformable alternative conformation, which takes place in a circular DNA molecule constrained by the constancy of its linking number. The values determined for these parameters under the experimental conditions (T = 310 K, pH = 7.0, monovalent cation concentration = 0.01 M) are a = 10.84(+/- 0.2) kcal/mol, C = 2.5(+/- 0.3) x 10(-13) erg/rad2 and K = 2350(+/- 80) RT/N, where N is the molecular length in base-pairs. In order to assess the accuracy of the author's theoretical methods, these free energy parameters are incorporated into the analysis of superhelical strand separation in different molecules and under other conditions than those used in their evaluation. First, the temperature dependence of transition is treated, then superhelical strand separation is analyzed in a series of DNA molecules having systematic sequence modifications, and the results of these theoretical analyses are compared with those from experiments. In all molecules, transition is predicted in the range of linking differences where it is seen experimentally. Moreover, it occurs at the specific sequence locations that the analysis predicts, and with approximately the predicted relative amounts of transition at each location. The known sensitivities of this transition to changes of temperature and to small sequence modifications are predicted in a quantitatively precise manner by the theoretical results. The demonstrated high-level precision of these theoretical methods provides a tool for the screening of DNA sequences for sites susceptible to superhelical strand separation, some of which may have regulatory or other biological significance.     

Coarse-grained modeling reveals the impact of supercoiling and loop length in DNA looping kinetics

Measurements of protein-mediated DNA looping reveal that in vivo conditions favor the formation of loops shorter than those that occur in vitro, yet the precise physical mechanisms underlying this shift remain unclear. To understand the extent to which in vivo supercoiling may explain these shifts, we develop a theoretical model based on coarse-grained molecular simulation and analytical transition state theory, enabling us to map out looping energetics and kinetics as a function of two key biophysical parameters: superhelical density and loop length. We show that loops on the scale of a persistence length respond to supercoiling over a much wider range of superhelical densities and to a larger extent than longer loops. This effect arises from a tendency for loops to be centered on the plectonemic end region, which bends progressively more tightly with superhelical density. This trend reveals a mechanism by which supercoiling favors shorter loop lengths. In addition, our model predicts a complex kinetic response to supercoiling for a given loop length, governed by a competition between an enhanced rate of looping due to torsional buckling and a reduction in looping rate due to chain straightening as the plectoneme tightens at higher superhelical densities. Together, these effects lead to a flattening of the kinetic response to supercoiling within the physiological range for all but the shortest loops. Using experimental estimates for in vivo superhelical densities, we discuss our model’s ability to explain available looping data, highlighting both the importance of supercoiling as a regulatory force in genetics and the additional complexities of looping phenomena in vivo.     

Relationship between the molecular structure of aqueous solutions of polyethylene glycol and the compactness of double-stranded DNA molecules

The dependence of viscosity of the water solutions of poly(ethylene glycol) (PEG) on the molecular weight has been studied. It has been shown that there is a "transitional" region in PEG properties which accounts for the formation of fluctuation polymer network of the PEG molecules. It has been shown that the "transitional" region in properties of PEG which appears at a certain concentration of PEG (CtrPEG) is characteristic of the PEG preparations with molecular weights exceeding 600 and dependence of the value of CtrPEG on the molecular weight of PEG was obtained. Compactization of double-stranded DNA molecules in PEG-containing water-salt solutions has been studied and the dependence of the value of CcrPEG, . i.e. the concentration of PEG at which the compact particles of DNA appear in the solution, on the molecular weight of PEG was obtained. The correlation between these two dependences reflecting quite different physico-chemical processes shows that the double-stranded DNA molecules are constrained within the polymer network of the PEG molecules. The influence of ionic strength and ionic composition of the solution on the formation of a compact form was investigated. The transition of the DNA molecules from a linear to a compact state may occur only at a definite value of ionic strength of the solution. This transition may occur at the change of K+ for Na+ cations (at a constant value of CPEG). The extent of compactization of the DNA molecules in PEG-containing water-salt solutions is monitored by the molecular structure and by the ionic strength of the solvent. It is supposed that the peculiarities of compactization of the DNA molecules in PEG-containing water-salt solutions reflect some characteristics of conformational transitions of the DNA molecules which occur in vivo.     

The expression of the Escherichia coli fis gene is strongly dependent on the superhelical density of DNA

The Escherichia coli DNA architectural protein FIS is a pleiotropic regulator, which couples the cellular physiology with transitions in the superhelical density of bacterial DNA. Recently, we have shown that this effect is in part mediated via DNA gyrase, the major cellular topoisomerase responsible for the elevation of negative supercoiling. Here, we demonstrate that, in turn, the expression of the fis gene strongly responds to alterations in the topology of DNA in vivo, being maximal at high levels of negative supercoiling. Any deviations from these optimal levels decrease fis promoter activity. This strict dependence of fis expression on the superhelical density suggests that fis may be involved in 'fine-tuning' the homeostatic control mechanism of DNA supercoiling in E. coli.     

Light-scattering studies on supercoil unwinding

It has been shown previously that supercoiled [unk]X174 bacteriophage intracellular DNA (mol.wt. 3.2x10(6)) with superhelix density, sigma=-0.025 (-12 superhelical turns) at 25 degrees C is best represented as a Y shape. In this work two techniques have been used to unwind the supercoil and study the changes in tertiary structure which result from changes in the secondary structure. The molecular weights from all experiments were in the range 3.2x10(6)+/-0.12x10(6). In experiments involving temperature change little change in the Y shape was observed between sigma=-0.027 (-13 superhelical turns, 14.9 degrees C) and sigma=-0.021 (-10 superhelical turns, 53.4 degrees C) as evidenced by the root-mean-square radius and the particle-scattering factor P(theta). However, at sigma=-0.0176 (-8 superhelical turns, 74.5 degrees C) the root-mean-square radius fell to between 60 and 70nm from 90nm indicating a large structural change, as did alterations in the P(theta) function. In experiments with the intercalating dye proflavine from values of bound proflavine of 0-0.06mol of dye/mol equiv. of nucleotide which correspond to values of sigma from -0.025 to -0.0004 (-12 to 0 superhelical turns) a similar transition was found when the superhelix density was changed by the same amount, and the molecule was shown to go through a further structural change as the unwinding of the duplex proceeded. At sigma=-0.018 (-9 superhelical turns) the structure was compatible with a toroid, and at sigma=-0.0004 it was compatible with a circle but at no point in the sequence of structure transitions was the structure compatible with the conventional straight interwound model normally visualized as the shape of supercoiled DNA.     

Evidence for allosteric transitions in secondary structure induced by superhelical stress

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 (alpha) 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, or vice 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 (Do) and the plateau diffusion coefficient (Dplat) characterizing internal motions over short distances (225 A) are determined by dynamic light-scattering. The torsion constant (alpha) between base-pairs and the circular dichroism spectrum are also measured for each sample. Curves of Dplat, Do, alpha and molar ellipticity ([theta]) (at the minimum near 250 nm) versus superhelix density (sigma) are constructed. The curve of Do versus sigma is very similar to that for sedimentation coefficient versus sigma for simian virus 40 (SV40) and polyoma DNAs. The curves of Dplat, Do, alpha and [theta] versus sigma show that, with increasing negative superhelix density, a structural transition occurs near sigma = -0.020 to an intermediate state with low torsion constant, and a second structural transition occurs near sigma = -0.035 to a state that exhibits more normal properties by sigma = -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 at sigma = -0.020, and at other sites at sigma = -0.035, or with hypotheses based on changes in tertiary structure alone.(ABSTRACT TRUNCATED AT 400 WORDS)     

DNA curvature influences the internal motions of supercoiled DNA.

We present evidence that short curved DNA segments can act as mediators for the ordering of large domains in superhelical DNA. Using a non-invasive solution method (dynamic light scattering), we investigated the effect of permanently curved inserts on the solution structure and on the internal motions of superhelical plasmid DNA. We find that the dynamics of superhelical DNA are strongly influenced by sequence- or protein-induced bending: in superhelical plasmids containing curved inserts the amplitude of the internal motion is lower than that of non-curved controls. Furthermore, the relative arrangement of curved sequences in the plasmids can influence the overall shape of the superhelical DNA. On linearized forms of the plasmids, these effects are not observed.     

Environmental influences on DNA superhelicity. The effect of ionic strength on superhelix conformation in solution

The techniques of small-angle X-ray scattering and analysis that have been developed by the authors are used to investigate the influence of ionic strength on the superhelical conformation of native COP608 plasmid DNA in solution. For salt concentrations below 0.1 M, the superhelicity is partitioned between twisting (Tw) and writhing (Wr) in the ratio delta Tw/Wr = 2. Near the physiological salt concentration, [Na+] = 0.2 M, a co-operative transition is observed in which the pitch angle of the toroidal superhelix is drastically decreased. This results in an almost complete relaxation of writhe. At salt concentrations in excess of the threshold for this transition, the superhelical partitioning occurs in the ratio delta Tw/Wr greater than 25. Energetic considerations support the suggestion that this transition results from co-operative, superhelical B to Z transconformation reactions at susceptible sites. A method is discussed that will enable the direct measurement of this secondary structural transition by means of X-ray scattering.     

Theoretical model for the equilibrium behavior of DNA superhelices

A statistical-mechanical model for superhelical DNA is presented. The partition function for a DNA superhelix is written by using a combinatorial approach in order to allow for the known relation between the number of superhelical twists and the states of the base pairs in the double helix. While the theory allows any factors which might contribute to the free energy of superhelical twisting to be included in the statistical weights of the superhelical twists, only the reduction in configurational entropy is considered in this paper. Similarities between an imperfectly matched DNA double helix and a DNA superhelix are used in the derivation of expressions for the entropy of superhelical DNA. Although the partition function is presented in a general form, permitting many equilibrium properties of DNA superhelices to be treated, the application considered in this paper is the calculation of helix–coil transition curves. Several experimentally observed features of such transitions are predicted. For example, the curves are bimodal, with an early and a late transition relative to that of a nicked molecule. The results are very sensitive to the volume within which two parts of the double helix must meet when forming a superhelical twist. The free energy of superhelix formation is calculated, and the results are compared with those obtained from the data of Bauer and Vinograd for ethidium bromide intercalation. In the present model, the free energy increases less sharply with an increase in the number of superhelical twists than observed experimentally, indicating that factors other than configurational entropy probably make important contributions to the free energy of superhelix formation.