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.     

The Energetics of the B-Z Transition in DNA

Abstract

The paper deals with the energetics of the transition to left-handed Z form in DNA with an arbitrary base sequence. There is a brief outline of the statistical-mechanical model of the B-Z transition allowing for three possible states of each base pair. The parameters of the model can be determined by comparing the theory with experimental data for the B-Z transition in inserts with given sequences in circular DNA The model contains six energy parameters, most of which have been determined before. In order to find the remaining parameters of the model and test its adequacy, a number of oligonucleotide sequences were synthesized and inserted into the pUC 19 plasmid. Two-dimensional gel electrophoresis was used to determine the superhelical density at which the inserts adopt the Z form. A statistical-mechanical treatment of these data yielded a complete set of six energy parameters for the B-Z transition. The theoretical assumption that the free energy of Z-form pairs does not depend on the type of adjacent pairs proved to be in agreement with the experimental data.     

Extrusion of an imperfect palindrome to a cruciform in superhelical DNA: complete determination of energetics using a statistical mechanical model

We present a detailed study of the extrusion of an imperfect palindrome, derived from the terminal regions of vaccinia virus DNA and contained in a superhelical plasmid, into a cruciform containing bulged bases. We monitor the course of extrusion by two-dimensional gel electrophoresis experiments as a function of temperature and linking number. We find that extrusion pauses at partially extruded states as negative superhelicity increases. To understand the course of extrusion with changes in linking number, DeltaLk, we present a rigorous semiempirical statistical mechanical analysis that includes complete coupling between DeltaLk, cruciform extrusion, formation of extrahelical bases, and temperature-dependent denaturation. The imperfections in the palindrome are sequentially incorporated into the cruciform arms as hairpin loops, single unpaired bases, and complex local regions containing several unpaired bases. We analyze the results to determine the free energies, enthalpies and entropies of formation of all local structures involved in extrusion. We find that, for each unpaired structure, the DeltaG, DeltaH and DeltaS of formation are all approximately proportional to the number of unpaired bases contained therein. This surprising result holds regardless of the arrangement or composition of unpaired bases within a particular structure. Imperfections have major effects on the overall energetics of cruciform extrusion and on the course of this transition. In particular, the extent of the DeltaLk change necessary to extrude an imperfect palindrome is considerably greater than that required for extrusion of the underlying perfect palindrome. Our analysis also suggests that, at higher temperatures, significant denaturation at the base of an imperfect cruciform can successfully compete with extension of the cruciform arms.     

Changes of hydration during conformational transitions of DNA

Fiber X-ray diffraction and measurement of fiber dimensions yields information about the hydration of DNA in fibers. The results obtained give us the fraction of nucleotides in the B form for the A-B transition or the rate of progression for the B-C transition as functions of the number of water molecules per nucleotide. The present experimental results confirm the importance of cooperativity in the A-B transition and the progressive change of the DNA double helix conformation during the C-B transition. At least twenty additional water molecules per nucleotide are necessary to stabilize the B form for DNA molecules in fibers following the A to B transition whereas only ten are sufficient when the B conformation is obtained starting from the C form. Offprint requests to: S. Premilat     

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.     

Energetics of B-Z junction formation in a sixteen base-pair duplex DNA

We report analysis of the NaCl-induced B-Z transition in a 16 base-pair duplex DNA with sequence designed such that when NaCl is increased the left half of the molecule undergoes the B-Z transition while the right half remains in the B-form. An equilibrium thermodynamic model based on the body of available published experimental data and the recent theoretical work of Soumpasis, which indicate, in the salt range above approximately 0.9 M-NaCl, the transition free-energy of B-Z conversion in DNA is a linear function of the NaCl concentration, is employed. Analysis of the B-Z transition of the junction-containing molecule indicates the B-Z junction formed in this 16 base-pair DNA is composed of approximately three base-pairs and has a free energy of formation of approximately 4.7 kcal/mol junction. These values for the junction are in excellent agreement with published estimates of B-Z junction size and energy derived from much longer DNA pieces.     

Electrostatic effects in short superhelical DNA

We present Monte Carlo simulations of the equilibrium configurations of short closed circular DNA that obeys a combined elastic, hard-sphere, and electrostatic energy potential. We employ a B-spline representation to model chain configuration and simulate the effects of salt on chain folding by varying the Debye screening parameter. We obtain global equilibrium configurations of closed circular DNA, with several imposed linking number differences, at two salt concentrations (specifically at the extremes of no added salt and the high salt regime), and for different chain lengths. Minimization of the composite elastic/long-range potential energy under the constraints of ring closure and fixed chain length is found to produce structures that are consistent with the configurations of short supercoiled DNA observed experimentally. The structures generated under the constraints of an electrostatic potential are less compact than those subjected only to an elastic term and a hard-sphere constraint. For a fixed linking number difference greater than a critical value, the interwound structures obtained under the condition of high salt are more compact than those obtained under the condition of no added salt. In the case of no added salt, the electrostatic energy plays a dominant role over the elastic energy in dictating the shape of the closed circular DNA. The DNA supercoil opens up with increasing chain length at low salt concentration. A branched three-leaf rose structure with a fixed linking number difference is higher in energy than the interwound form at both salt concentrations employed here.     

Conformational stability of biological polyelectrolytes: Evaluation of enthalpy and entropy changes of conformational transitions

A new method is proposed for the determination of the enthalpy and entropy changes of nonionic origin upon conformational transition of linear biopolyelectrolytes in solution. For all transition midpoints, defined by given temperature and ionic strength, the total free energy change of the system is zero, which means that the nonionic contribution to the free energy change is equal in value and opposite in sign to the polyelectrolytic one. The counterion condensation theory of linear polyelectrolytes provides for the appropriate analytical expression to be used in such calculations. Linear plots of the proper functions of the calculated free energy changes vs the proper functions of temperature allows for the determination of the enthalpic and entropic terms of the nonionic free energy change of transition. The method has been applied to the extensive available data of the ion-induced conformational change of κ-carrageenan, a linear sulfated galactan extracted from seaweeds. The method has proved very successful, with the results showing a remarkable convergency of the enthalpy values for different monovalent counterions. On the other hand, the above approach has made it possible to explain the known effect of counterion specificity on the transition by a small difference in the nonionic entropic contributions. © 1998 John Wiley & Sons, Inc. Biopoly 45: 203–216, 1998     

Modelling DNA stretching for physics and biology

We have used internal coordinate molecular mechanics calculations to study how the DNA double helix deforms upon stretching. Results obtained for polymeric DNA under helical symmetry constraints suggest that two distinct forms, an unwound ribbon and a narrow fibre, can be formed as a function of which ends of the duplex are pulled. Similar results are also obtained with DNA oligomers. These experiments lead to force curves which exhibit a plateau as the conformational transition occurs. This behaviour is confirmed by applying an increasing force to DNA and observing a sudden length increase at a critical force value. It is finally shown some DNA binding proteins can also stretch DNA locally, to conformations related to those created by nanomanipulation.This revised version was published online in October 2005 with corrections to the Cover Date.     

Assembly of DNA onto the histone octamer facilitates the B-to-Z transition

Nucleosomal core particles containing the right- and left-handed conformations of DNA were examined for their ability to support the BZ or ZB transition. Nucleosomes were assembled onto the B- and Z-conformations of poly[d(Gm5C)] and the B-conformation of poly[d(GC)] as previously described (1). Absorbance and circular dichroic spectroscopy indicated that the DNA on all three core particle populations could undergo the conformational BZ transition. Further, the right- to left-handed transition for both poly[d(Gm5C)] and poly[d(GC)] appeared to be facilitated by the DNAs association with the histone octamer. The DNA remained associated with the protein core subsequent to the transition, and electron microscopy and sedimentation velocity analysis indicated that there were no gross changes in nucleosomal structure. However, a change in the sedimentation value of the poly[d(Gm5C)] core particles was detected when the conformation of the DNA was altered from B to Z, resulting in a lower S20,w value for the Z-form particles than for the corresponding B-form particles.     

Purification and characterization of a novel ATP-independent type I DNA topoisomerase from a marine methylotroph

DNA topoisomerase is involved in DNA repair and replication. In this study, a novel ATP-independent 30-kDa type I DNA topoisomerase was purified and characterized from a marine methylotroph, Methylophaga sp. strain 3. The purified enzyme composed of a single polypeptide was active over a broad range of temperature and pH. The enzyme was able to relax only negatively supercoiled DNA. Mg(2+) was required for its relaxation activity, while ATP gave no effect. The enzyme was clearly inhibited by camptothecin, ethidium bromide, and single-stranded DNA, but not by nalidixic acid and etoposide. Interestingly, the purified enzyme showed Mn(2+)-activated endonuclease activity on supercoiled DNA. The N-terminal sequence of the purified enzyme showed no homology with those of other type I enzymes. These results suggest that the purified enzyme is an ATP-independent type I DNA topoisomerase that has, for the first time, been characterized from a marine methylotroph.     

Strand separation in negatively supercoiled DNA

We consider Benham’s model for strand separation in negatively supercoiled circular DNA, and study denaturation as function of the linking difference density κ<0. We propose a statistical version of this model, based on bayesian segmentation methods of current use in bioinformatics; this leads to new algorithms with priors adapted to supercoiled DNA, taking into account the random nature of the free energies needed to denature base pairs.     

Visualization of alkali-denatured supercoiled plasmid DNA by atomic force microscopy

To study the alkali denaturation of supercoiled DNA, plasmid pBR322 was treated with gradient concentrations of NaOH solution. The results of gel electrophoresis showed that the alkali denaturation of the supercoiled DNA occurred in a narrow range of pH value (12.88-12.90). The alkali-denatured supercoiled DNA ran, as a sharp band, faster than the supercoiled DNA. The supercoiled plasmid DNA of pBR322, pACYC184 and pJGX15A were denatured by NaOH, and then visualized by atomic force microscopy. Compared with the supercoiled DNA, the atomic force microscopy images of the alkali-denatured supercoiled DNA showed rough surface with many kinks, bulges on double strands with inhomogeneous diameters. The apparent contour lengths of the denatured DNA were shortened by 16%, 16% and 50% for pBR322, pACYC184 and pJGX15A, respectively. All evidence suggested that the alkali-denatured supercoiled DNA had a stable conformation with unregistered, topologically constrained double strands and intrastrand secondary structure.     

Energetics of kayaking

The metabolic cost of paddling at low speeds (v) was measured from oxygen uptake (VO2) and anaerobic glycolysis in an annular pool or calculated from submaximal VO2 measured at higher speeds when the kayaker was assisted in overcoming water resistance. Also calculated were the total drag (D) and the net mechanical efficiency (e). Each of the above variables was determined in male (n = 17) and female (n = 7) kayakers ranging in experience from beginners to elite. The VO2 increased with v to a peak of approximately 3.4 l.min-1 (80%-100% of peak VO2 during running) in men and of approximately 2.8 l.min-1 in women, while at higher speeds the additional energy was accounted for by anaerobic glycolysis. In all subjects the energy cost to paddle a given distance (C) increased according to a power function with increasing v. The C was lower for the elite male paddlers than for the unskilled group, while that for elite women was slightly less than that for the elite men. Also the rates of increase of C appeared to be inversely proportional to the subjects' skill. Total D for elite men increased from approximately 15 to 60 N over a range of speeds from 1 to 2.2 m.s-1 while those of unskilled men and skilled women for the same speed range were 10-20 N greater and slightly less, respectively. The e increased linearly, but at a different rate, with increases in v for the unskilled and the elite kayakers (males and females) being 4.2% and 6%, respectively, at v = 1.2 m.s-1.     

DNA plasmid production in different host strains of <Emphasis Type="Italic">Escherichia coli</Emphasis>

We compared plasmid DNA production in 13 strains of Escherichia coli in shake flasks using media containing glucose or glycerol. DNA yield from either carbon source showed small correlation with maximum growth rate. Three strains, SCS1-L, BL21 and MC4100, were selected for a controlled exponential fed-batch process at a growth rate of 0.14 h⁻¹ to an optical density of about 70, followed by a four-hour heat treatment. Prior to heat treatment, SCS1-L generated 15.4 mg DNA/g, BL21 generated 11.0 mg DNA/g and MC4100 generated 7.9 mg DNA/g, while after heat treatment the strains attained DNA yields, respectively, of 18.0, 15.0 and 6.8 mg/g. The strains also varied in their percentage of supercoiled DNA after heat treatment, with SCS1-L averaging 66% supercoiled, BL21 17% and MC4100 40%. We further investigated the two strains that yielded the highest percentage of supercoiled DNA (SCS1-L and MC4100) at a higher growth rate of 0.28 h⁻¹. At this condition, a slightly lower DNA yield was generated faster, and the percentage of supercoiled DNA increased. Heat treatment improved DNA yield, and surprisingly did so to a greater extent at the higher growth rate. As a consequence of these factors, higher growth rates might be advantageous for DNA production.     

Allosteric serine hydroxymethyltransferase from monkey liver: Temperature induced conformational transitions

The homogeneous serine hydroxymethyltransferase from monkey liver was optimally activate at 60°C and the Arrhenius plot for the enzyme was nonlinear with a break at 15°C. The monkey liver enzyme showed high thermal stability of 62°C, as monitored by circular dichroism at 222 nm, absorbance at 280 nm and enzyme activity. The enzyme exhibited a sharp co-operative thermal transition in the range of 50°–70°(T _m= 65°C), as monitored by circular dichroism. L-Serine protected the enzyme against both thermal inactivation and thermal disruption of the secondary structure. The homotropic interactions of tetrahydrofolate with the enzyme was abolished at high temperatures (at 70°C, the Hill coefficient value was 1.0). A plot ofh values vs. assay temperature of tetrahydrofolate saturation experiments, showed the presence of an intermediate conformer with anh value of 1.7 in the temperature range of 45°–60°C. Inclusion of a heat denaturation step in the scheme employed for the purification of serine hydroxymethyltransferase resulted in the loss of cooperative interactions with tetrahydrofolate. The temperature effects on the serine hydroxylmethyltransferase, reported for the first time, lead to a better understanding of the heat induced alterations in conformation and activity for this oligomeric protein.     

The influence of tertiary structural restraints on conformational transitions in superhelical DNA.

This paper examines theoretically the effects that restraints on the tertiary structure of a superhelical DNA domain exert on the energetics of linking and the onset of conformational transitions. The most important tertiary constraint arises from the nucleosomal winding of genomic DNA in vivo. Conformational transitions are shown to occur at equilibrium at less extreme superhelicities in DNA whose tertiary structure is restrained than in unrestrained molecules where the residual linking difference alpha res (that part of the superhelical deformation which is not absorbed by transitions) may be freely partitioned between twisting and bending. In the extreme case of a rigidly held tertiary structure, this analysis predicts that the B-Z transition will occur at roughly half the superhelix density needed to drive the same transition in solution, other factors remaining fixed. This suggests that superhelical transitions may occur at more moderate superhelical deformations in vivo than in solution. The influence on transition behavior of the tertiary structural restraints imposed by gel conditions also are discussed.