Applying the stochastic difference equation to DNA conformational transitions: a study of B-Z and B-A DNA transitions

Despite the existence of numerous models to account for the B-Z DNA transition, experimenters have not yet arrived at a conclusive answer to the structural and dynamical features of the B-Z transition. By applying the stochastic difference equation to simulate the B-Z DNA transition, we have shown that the stretched intermediate model of the B-Z transition is more probable than other B-Z transition models such as the Harvey model. This is accomplished by comparing potential energy profiles of various B-Z DNA transition models and calculating relative probabilities based on the stochastic difference equation with respect to length (SDEL) formalism. The results garnered in this article allow for new approaches in determining the structural transition of B-DNA to Z-DNA experimentally. We have also simulated the B-A DNA transition using the stochastic difference equation. Unlike the B-Z DNA transition, the mechanism for the B-A DNA transition is well established. The variation in the pseudorotation angle during the transition is in good agreement with experimental results. Qualitative features of the simulated B-A transition also agree well with experimental data. The SDEL approach is thus a suitable numerical technique to compute long-time molecular dynamics trajectory for DNA molecules.     

Influence of a Mechanical Tension on the B-C and B-C Conformational Transitions in DNA Fibres

Abstract

In the present fibre X-ray study we attempt to quantify the effect of a mechanical tension on the conformations, and transitions between the structural forms of DNA A simple experimental device has been realized in order to apply precise mechanical forces on DNA fibres during X-ray exposure. It is shown that, as the applied tension is increased, the B→A transition can be prevented as well as with a decrease of the sodium salt content A kind of distorted B form is then observed the helical parameters of which change with the relative humidity. On the contrary, the mechanical tension does not prevent the B→C transition; it only slows down the form change and improves the X-ray patterns up to a relative humidity of 0%.     

Effect of a mechanical tension on the hydration of DNA in fibres.

Fiber X-ray diffraction and measurement of fibre dimensions yield information about the effects of a mechanical tension on hydration of DNA in fibres. At a given relative humidity, the mechanical tension changes the DNA conformation but does not modify the number of water molecules associated to a nucleotide. The number of water molecules per nucleotide necessary to maintain B form decreases for increasing tensions applied to the DNA fibre. Form transitions can be opposed by mechanical tensions; an energy of 1 Kcal per mole of nucleotide pairs is sufficient to prevent the B to A transition.     

Mapping the B-A conformational transition along plasmid DNA

A simple method is presented to monitor conformational isomerizations along genomic DNA. We illustrate properties of the method with the B-A conformational transition induced by ethanol in linearized pUC19 plasmid DNA. At various ethanol concentrations, the DNA was irradiated with ultraviolet light, transferred to a restriction endonuclease buffer and the irradiated DNA was cleaved by 17 restriction endonucleases. The irradiation damaged DNA and the damage blocked the restrictase cleavage. The amount of uncleaved, i.e. damaged, DNA depended on the concentration of ethanol in a characteristic S-shape way typical of the cooperative B-A transition. The transition beginning and midpoint were determined for each restriction endonuclease. These data map the B-A transition along the whole polylinker of pUC19 DNA and six evenly distributed recognition sequences within the rest of the plasmid. The transition midpoints fell within the B-A transition region of the plasmid simultaneously determined by CD spectroscopy. The present method complements the previous methods used to study the B-A transition. It can be employed to analyze multikilobase regions of genomic DNA whose restriction endonuclease cleavage fragments can be separated and quantified on agarose gels.     

Sequence dependence of the B-A conformational transition of DNA

We have studied, by conformational analysis, the sequence dependence of DNA conformational transition between B- and A-forms. We have considered intramolecular interactions between base pairs, without backbone, to examine their role in the conformational transition between B- and A-forms, and found that base pairs themselves usually have intrinsic conformational preferences for the B- or A-form. Calculation of all ten possible base steps shows that the base combinations, CC (or GG), GC, AT, and TA, have tendencies to assume the A-conformation. Results show that it is particularly easy to slide along the long axis of the base pair for these steps, with AT and CC showing especially flat energies. These calculations show that a preference for the B- or A-conformation depends on the electrostatic energy parameters, in particular, on dielectric and shielding constants; the A-conformation is preferred for low dielectric constant or low shielding. Both the A- and B-conformations are mainly stabilized by electrostatic interactions between favorably juxtaposed atomic charges on base pairs; however, the B-conformation generally has more favorable van der Waals interactions than the A-form. These sequence-dependent conformational preference and environmental effects agree roughly with experimental observations, suggesting that the origin of the conformational polymorphism is attributable to the intrinsic conformational preference of base pairs.     

The thermodynamic parameters of the conformational transitions in Mycoplasma DNA

DNA preparations have been isolated from 10 strains of phytopathogenic mycoplasms and collection culture Achole plasma laidlawii PG-8. Thermodynamic parameters of denaturation changes in the secondary structure (transconformation) of DNA of these mycoplasms have been determined. It is shown that denaturation temperature is 82.3-83.1 degree C and enthalpy of conformational DNA transitions calculated per 1 g of dry substance is 56.2-61.9 J/g. Changes in the enthalpy (delta H) and entropy (delta S) calculated per 1 mol of nucleotide pairs varied in the range of 35.6-39.0 J/m.p. and 995-109.6 J degree m.p. respectively. No linear dependence of transconformational thermal adsorption on the temperature of DNA denaturation of the studied strains of mycoplasms are revealed, which is probably connected with structural peculiarities of DNA of these microorganisms.     

Experimental method for studying conformational transitions in DNA

An experimental method combining fiber X-ray and direct fiber dimension measurements is proposed for the study of DNA conformational transitions. Curves corresponding to the A-B and B-C transitions are obtained by using the proportionality which exists between the fiber length and the axial rise per nucleotide in the DNA helix. The A-B transition is shown to be cooperative while the B-C one is a progressive change of helical conformation.     

Influence of lipid membranes on the conformational transitions of nucleic acids

The conformational transitions of nucleic acids which were enclosed in reverse phase evaporation vesicles (REV) were studied by thermal denaturation with optical recording. Cloned fragments of double-stranded DNA containing 179 base pairs and 187 base pairs, respectively, and polyA.polyU were enclosed in REV with a yield up to every vesicle containing 50 nucleic acid molecules. With the 179 base pairs DNA enclosed in the vesicle from egg lecithin two well resolved helix-coil transitions could be measured; one is very similar in the midpoint-temperature Tm and halfwidth delta T1/2 to the transition of the free nucleic acid, and the other transition occurs stabilized at a 3.5 degrees C higher Tm-value and with a broader delta T1/2, 2.7 degrees C instead of 0.6 degree C. Both transitions are from nucleic acids inside the vesicles. Varying the surface charge of the lipid membrane by adding the negatively charged phosphatidylserine or phosphatidylglycerol, an optimum in the yield of enclosure and a maximum in the increase in Tm (4.5 degrees C) and delta T1/2 (5.5 degrees C instead of 1.0 degrees C) was obtained at 20% phosphatidylserine or phosphatidylglycerol. In vesicles from pure negatively charged lipids no second population of nucleic acids was observed. Qualitatively, similar effects were observed with polyA.polyU. Stabilization and broadening of the second transition is higher for nucleic acids inside vesicles from lipids with unsaturated fatty acids, as dioleoyl-phosphatidylcholine, than with saturated fatty acids, dipalmitoyl-phosphatidylcholine. Stabilization and broadening decrease with increasing ionic strength, whereas the relative contributions of both transitions to the total hypochromicity remain unchanged; the second transition coincides with the first at 90 mM Na+. From the experimental results it was concluded that the interaction of nucleic acids and lipid membranes is mainly of electrostatic nature. The nucleic acids exist inside the vesicles in two populations, one behaving like nucleic acid free in solution and one influenced by the contact with the membrane. All results are in accordance with a model in which the interaction between the nucleic acid and the membrane is in competition with the dipole-dipole interaction inside the membrane surface.     

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     

Theoretical analysis of competitive conformational transitions in torsionally stressed DNA

This paper presents a theoretical analysis of the conformation of a torsionally deformed segment of DNA containing two sites susceptible to stress-induced transitions in secondary structure. A mechanical analysis of the ensuing competitive behavior is developed and applied to several phenomena of possible biological relevance. First, a molecular lesion which disrupts base pairing without strand breakage (such as a pyrimidine dimer) is shown to provide an effective nucleation site for further stress-induced denaturation. A competition is established between strand separation at this lesion site and at the A + T-richest portion of the molecule. The relative importance of these two forms of melting is shown to depend upon the A + T-content of the sites involved, segment length, local environmental conditions and the magnitude of the imposed torsional deformation. A possible alternative mode of behavior of a stressed segment of DNA involves transitions from B-form to Z-form. The second application of this theory analyzes the interplay between B → Z transitions and local denaturation in torsionally stressed DNA. Finally, local melting is shown to be energetically preferred over transitions to A-form under physiologically reasonable conditions in vitro, due primarily to the greater degree of unwinding involved in melting.The mechanical theory presented here makes several simplifying assumptions regarding the nature of the transitions and the sequences involved. First, the theory is developed explicitly for the competition between two sites of possible transition, with no further consideration given to conformational degeneracy or sequence effects. These sites are regarded as being uniform in composition. A multistate, heteropolymeric statistical mechanical transition theory is required to account rigorously for degeneracy and the influence of base sequence. A preliminary formulation of such a theory is used to analyze the denaturation of a segment containing a site of disrupted base pairing.     

Effect of divalent metal ions on DNA conformational transitions in water-ethanol solutions

The influence of different MgCl2 and MnCl2 concentrations on DNA conformational transitions in water-ethanol solutions was studied. It was shown that the presence of magnesium ions in solution at a concentration of 5 x 10(-4) M did not influence the decrease in the size of DNA without change in its persistent length at an alcohol concentration of about 17 % v/v. In contrast, manganese ions prevent this change in DNA parameters. At sufficiently high ethanol concentrations, the compaction of DNA followed by its precipitation takes place, which is accompanied by an increase of scattering in solution. As the concentration of Mg2+ and Mn2+ in solution increases, this process is observed at lower ethanol concentrations.     

Competing B-Z and helix-coil conformational transitions in supercoiled plasmid DNA.

The formation of melted regions from A + T-rich sequences and left-handed Z-DNA by alternating purine-pyrimidine sequences will both be facilitated by negative supercoiling, and thus if the sequences are present within the same plasmid molecule they will compete for the free energy of supercoiling. We have studied a series of plasmids that contain either (CG)8 or (TG)12 sequences in either G + C or A + T-rich contexts, by means of two-dimensional gel electrophoresis and chemical modification. We observe both B-Z and helix-coil transitions in all plasmids at elevated temperatures and low ionic strength. The plasmids fall into a number of different classes, in terms of the conformational behavior. As the superhelix density is increased, pCG8/vec ((CG)8 in G + C-rich context) undergoes an initial B-Z transition, followed by melting transitions in sequences remote from the (CG)8 sequence. The two transitions are coupled through the topology of the molecule but are otherwise independent. When the (CG)8 sequence was placed in an A + T-rich context (pCG8/col), the helix-coil transition was perturbed by the presence of the Z-DNA segment. Replacement of the (CG)8 tracts by (TG)12 sequences resulted in a further level of interaction between the transitions. Statistical mechanical modeling of the transitions suggested that at intermediate levels of negative supercoiling the Z-DNA formed by the (TG)12 sequence has a lowered probability due to the helix-coil transition in the A + T-rich sequences. These studies illustrate the complexities of competing conformational equilibria in supercoiled DNA molecules.     

A method for the experimental study of DNA conformational transitions in fibers

The method proposed for the study of DNA conformational transitions is based on the proportionality, experimentally observed, between the length of a DNA fiber and the axial rise per nucleotide characterizing the molecular helix. Precise curves for the A-B and B-C transitions as a function of the relative humidity are obtained by using X-ray fiber data and measurements of fiber dimensions. It is thus shown that the A-B transition is a cooperative process between two different states, whereas the B-C transition can be considered as a progressive change of conformation. The present method is applied on two natural DNAs differing in base composition so that the effect of the nucleotide content on the conformational changes can be estimated.     

The effect of hydrogen ions on the B-A transition in DNA

The effects of hydrogen ions binding to DNA on its secondary structure and B to A transition were studied by methods of X-ray diffraction and infrared spectroscopy. Helical parameters of DNA molecules with different degrees of protonation were determined. It was shown that H+-ions binding stabilize the B form of DNA in fibers in the wide range of water and inorganic salt content. Only 0.03 H+-ions bound to each nucleotide are sufficient to prevent B to A transition caused by a relative humidity decrease in DNA fibers, containing 4% of NaCl. The effective stabilization of the DNA B form by H+-ions binding is explained by modifications in DNA - solvent molecules interactions, especially in the major groove of double helices.     

Theory of DNA melting in the interval of B-A transition

Theory of DNA melting within the B--A transition range is presented. The phase diagram in coordinates alcohol--temperature is plotted. The temperature shift of DNA helix--coil transition in the B--A transition point is predicted to be delta T = 3 degrees. The temperature rise of DNA melting in the range of B--A transition is caused by the presence of junctions between regions in B and A forms in helical sections.     

Translin binding to DNA: recruitment through DNA ends and consequent conformational transitions

The human translin protein binds a variety of sequences (chromosomal breakpoint consensus sequences, their sequence variants, as well as nonbreakpoint sequences such as simple AT and GC repeats) at nanomolar protein concentration when short single strands ( approximately 20-30mers) are used as DNA targets. The protein, which is known to exist as an octamer in its free state, undergoes a conformational transition upon binding to short single strands leading either to a compaction or to the dissociation of the oligomer. Moreover, the protein oligomers tend to aggregate into complexes that get progressively larger as the length of the single-stranded DNA target increases. The protein loads onto duplexes via the free ends of DNA, generating higher oligomeric complexes as a function of protein concentration. Interestingly, the conformation of DNA targets encased by translin oligomer is significantly altered such that the single strand is rendered hypersensitive to DNase I. Furthermore, the loading of translin oligomers leads to tighter clamping of duplex ends. All of these observations, taken together, suggest that translin is a bona fide binder of DNA ends, thereby subjecting the DNA to a conformation conducive for repair steps during translocation events. We discuss the results in the perspective of translin biology.     

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.     

Fine structure in the B-A transition of native DNA

The B-A transition caused by high ethanol concentrations has been studied by the multi-dimensional spectrophotometer equipped with the computer-controlled microbullet. When ethanol concentration is increased, the CD signal at 270 nm of linearized ColEl DNA exhibits a biphasic transition; the first broad one and the second sharp one. The B-A transition of the ColEl DNA is much broader than that of alternative copolymers with shorter lengths. In addition, each PvuII restriction fragment of ColEl DNA has a different transition curve. Therefore the stability of the B-A transition varies along a long DNA molecule. The second transition is speculated to be caused by aggregation. When ethanol concentration is decreased, on the other hand, only a single transition shifted to lower ethanol concentration is observed. Thus the B-A transition curve has a hysteresis. A slow dissociation rate of the aggregation seems to cause the hysteresis.     

Effects of A:T base pairs on the B-Z conformational transitions of DNA

Effects of A:T base pairs on the propensity of B to Z conformational transitions have been investigated by the CD salt titrations on d(CG)5' d(GC)5' terminal or central A:T replaced decamers, and terminal A:T appended dodecamers. The presence of A:T at the center greatly inhibits the B to Z transition of both G:C decamers. Moderate Z inhibitions are shown by terminal A:T replacements and additions to d(CG)5' with the former exhibiting a stronger effect. In contrast, the addition and replacement with A:T at the terminals of d(GC)5 facilitate the B to Z conversion, with the replacement exhibiting a somewhat more pronounced effect. These results may be rationalized in terms of the number of contigous CG sequences present in an oligomer and the relative inhibitory effects of other dinucleotide sequences. Our results also suggest that some short oligomers with purine at the 5'-end, such as d[A(CG)nT] with n greater than or equal to 2, may likely crystallize as Z conformations.