Modeling DNA Thermodynamics under Torsional Stress

Negatively twisted DNA is essential to many biological functions. Due to torsional stress, duplex DNA can have local, sequence-dependent structural defects. In this work, a thermodynamic model of DNA was built to qualitatively predict the local sequence-dependent mechanical instabilities under torsional stress. The results were compared to both simulation of a coarse-grained model and experiment results. By using the Kirkwood superposition approximation, we built an analytical model to represent the free energy difference ΔW of a hydrogen-bonded basepair between the B-form helical state and the basepair opened (or locally melted) state, within a given sequence under torsional stress. We showed that ΔW can be well approximated by two-body interactions with its nearest-sequence-neighbor basepairs plus a free energy correction due to long-range correlations. This model is capable of rapidly predicting the position and thermodynamics of local defects in a given sequence. The result qualitatively matches with an in vitro experiment for a long DNA sequence (>4000 basepairs). The 12 parameters used in this model can be further quantitatively refined when more experimental data are available.     

Thermodynamics of DNA branching.

Branched DNA molecules arise transiently as intermediates in genetic recombination or on extrusion of cruciforms from covalent circular DNA duplexes that contain palindromic sequences. The free energy of these structures relative to normal DNA duplexes is of interest both physically and biologically. Oligonucleotide complexes that can form stable branched structures, DNA junctions, have made it possible to model normally unstable branched states of DNA such as Holliday recombinational intermediates. We present here an evaluation of the free energy of creating four-arm branch points in duplex DNA, using a system of two complementary junctions and four DNA duplexes formed from different combinations of the same set of eight 16-mer strands. The thermodynamics of formation of each branched structure from the matching pair of intact duplexes have been estimated in two experiments. In the first, labeled strands are allowed to partition between duplexes and junctions in a competition assay on polyacrylamide gels. In the second, the heats of forming branched or linear molecules from the component strands have been determined by titration microcalorimetry at several temperatures. Taken together these measurements allow us to determine the standard thermodynamic parameters for the process of creating a branch in an otherwise normal DNA duplex. The free energy for reacting two 16-mer duplexes to yield a four-arm junction in which the branch site is incapable of migrating is + 1.1 (+/- 0.4) kcal mol-1 (at 18 degrees C, 10 mM-Mg2+). Analysis of the distribution of duplex and tetramer products by electrophoresis confirms that the free energy difference between the four duplexes and two junctions is small at this temperature. The associated enthalpy change at 18 degrees C is +27.1 (+/- 1.3) kcal mol-1, while the entropy is +89 (+/- 30) cal K-1 mol-1. The free energy for branching is temperature dependent, with a large unfavorable enthalpy change compensated by a favorable entropy term. Since forming one four-stranded complex from two duplexes should be an entropically unfavorable process, branch formation is likely to be accompanied by significant changes in hydration and ion binding. A significant apparent delta Cp is also observed for the formation of one mole of junction, +0.97 (+/-0.05) kcal deg-1 mol-1.     

Torsional tension in metaphase chromosome DNA]

The torsional tension in DNA of isolated metaphase chromosomes from murine fibroblasts was measured by the microfluorescent method. The method is based on the ability of a fluorescent dye ethidium bromide to compensate for the negative torsional tension in topologically closed DNA by intercalation between DNA base pairs. The value of the relative twist difference delta Tw/Tw = -0.1 was found in a bulk (about 3/4) of unconstrained chromosomal DNA. In interphase nuclei, the torsionally stressed DNA comprises about 15%, with value of delta Tw/Tw = -0.075. We suppose that the tension in chromosomal DNA was created in the prophase stage of mitosis by condensines, the drivers of chromosomal condensation.     

Torsional control of double-stranded DNA branch migration

DNA branched junctions are analogues of Holliday junction recombination intermediates. Partially mobile junctions contain a limited amount of homology flanking the branch point. A partially mobile DNA branched junction has been incorporated into a synthetic double-stranded circular DNA molecule. The junction is flanked by four homologous nucleotide pairs, so that there are five possible locations for the branch point. Two opposite arms of the branched junction are joined to form the circular molecule, which contains 262 nucleotides to the base of the junction. This molecule represents a system whereby torque applied to the circular molecule can have an impact on the junction, by relocating its branch point. Ligation of the molecule produces two topoisomers; about 87% of the product is a relaxed molecule, and the rest is a molecule with one positive supercoil. The position of the branch point is assayed by cleaving the molecule with endonuclease VII. We find that the major site of the branch point in the relaxed topoisomer is at the maximally extruded position in the relaxed molecule. Upon the addition of ethidium, the major site of the branch point migrates to the minimally extruded position. © 1998 John Wiley & Sons, Inc. Biopoly 45: 69–83, 1998     

Thermodynamics of cation-induced DNA condensation.

An estimation of the various free energy contributions to DNA collapse into toroidal particles is made, considering DNA bending and segment mobility, electrostatic repulsions between DNA chains, and attractive forces resulting from correlated counterion fluctuations. It is shown that the process of DNA condensation becomes spontaneous in the presence of divalent cations in methanol, and in the presence of tri- or tetravalent cations in water media. This is a consequence of the large decrease in the electrostatic repulsion between charged DNA segments, allowing the attractive force resulting from correlated fluctuations of bound counterions to become dominant. Our calculations indicate that short DNA fragments would condense into multimolecular particles in order to maximize the attractive force due to counterion fluctuations.     

Torsional regulation of hRPA-induced unwinding of double-stranded DNA

All cellular single-stranded (ss) DNA is rapidly bound and stabilized by single stranded DNA-binding proteins (SSBs). Replication protein A, the main eukaryotic SSB, is able to unwind double-stranded (ds) DNA by binding and stabilizing transiently forming bubbles of ssDNA. Here, we study the dynamics of human RPA (hRPA) activity on topologically constrained dsDNA with single-molecule magnetic tweezers. We find that the hRPA unwinding rate is exponentially dependent on torsion present in the DNA. The unwinding reaction is self-limiting, ultimately removing the driving torsional stress. The process can easily be reverted: release of tension or the application of a rewinding torque leads to protein dissociation and helix rewinding. Based on the force and salt dependence of the in vitro kinetics we anticipate that the unwinding reaction occurs frequently in vivo. We propose that the hRPA unwinding reaction serves to protect and stabilize the dsDNA when it is structurally destabilized by mechanical stresses.     

Thermodynamics of polymolecular duplexes between phosphate-methylated DNA and natural DNA

Phosphate-methylated (P.M.) DNA possesses a very high affinity for complementary natural DNA, as a result of the absence of interstrand electrostatic repulsions. In this study, a model system phosphate-methylated d[Cn] with natural d(Gk) (n less than k) is chosen for an investigation of the thermodynamic properties that determine duplex stability. The enthalpy change of a melting transition is shown to be considerably larger than is observed for corresponding natural DNA duplexes. It is found that delta Hn0 of GG/CC nearest neighbor pairwise interaction equals -15.6 kcal/mol, compared to -11.0 kcal/mol for the natural analog. The entropy change is strongly dependent on the length of the natural DNA strand and the number of phosphate-methylated DNA oligomers hybridized. The results are explained by means of a model in which a cooperative effect for subsequent hybridizations of phosphate-methylated DNA oligomers is assumed, thus giving additional stability.     

Thermodynamics of single strand DNA base stacking

The thermodynamics of the stacking to unstacking transitions of 24 single-stranded DNA sequences (ssDNA), 10-12 bases in length, in sodium phosphate buffer were determined from 10 to 95 degrees C, using differential scanning calorimetry (DSC). An additional 22 ssDNA sequences did not exhibit an S<-->U transition in this temperature range. The transition properties of the ssDNA sequences with 相似文献   

Thermodynamics of Forming a Parallel DNA Crossover

The process of genetic recombination involves the formation of branched four-stranded DNA structures known as Holliday junctions. The Holliday junction is known to have an antiparallel orientation of its helices, i.e., the crossover occurs between strands of opposite polarity. Some intermediates in this process are known to involve two crossover sites, and these may involve crossovers between strands of identical polarity. Surprisingly, if a crossover occurs at every possible juxtaposition of backbones between parallel DNA double helices, the molecules form a paranemic structure with two helical domains, known as PX-DNA. Model PX-DNA molecules can be constructed from a variety of DNA molecules with five nucleotide pairs in the minor groove and six, seven or eight nucleotide pairs in the major groove. A topoisomer of the PX motif is the juxtaposed JX₁ molecule, wherein one crossover is missing between the two helical domains. The JX₁ molecule offers an outstanding baseline molecule with which to compare the PX molecule, so as to measure the thermodynamic cost of forming a crossover in a parallel molecule. We have made these measurements using calorimetric and ultraviolet hypochromicity methods, as well as denaturing gradient gel electrophoretic methods. The results suggest that in relaxed conditions, a system that meets the pairing requirements for PX-DNA would prefer to form the PX motif relative to juxtaposed molecules, particularly for the 6:5 structure.     

Thermodynamics of Polyethylenimine-DNA Binding and DNA Condensation

In this study, polyethylenimine (PEI) binding to DNA was examined by isothermal titration calorimetry. Two types of binding modes were found to describe the interactions between these polyelectrolytes in buffers and in water. One type of binding involves PEI binding to the DNA groove because the enthalpy change of this binding mode is positive, and PEI is deprotonated to bind to DNA. Another likely binding mode involves external binding of PEI to the DNA phosphate backbone, accompanied with DNA condensation. The enthalpy change is negative and PEI is protonated when it binds to DNA in this mode. The intrinsic enthalpy change of first binding mode is 1.1 kJ/mol and −0.88 kJ/mol for the second binding mode. This result implies that the PEI is rearranged from the groove to the phosphate backbone of DNA when DNA is condensed. The mechanism of DNA condensation caused by PEI is discussed in this study.     

Thermodynamics of B-Z transition in supercoiled DNA.

The nature of the possible supercoil-induced B-Z transition has been analyzed from the thermodynamical point of view, by taking into account the effects of the twisting as well as the writhing components of the supercoiling free energy. The cooperative aspects of the transition, as predicted by theory, agrees well with the corresponding experimental data.     

Thermodynamics of sequence-specific binding of PNA to DNA

For further characterization of the hybridization properties of peptide nucleic acids (PNAs), the thermodynamics of hybridization of mixed sequence PNA-DNA duplexes have been studied. We have characterized the binding of PNA to DNA in terms of binding affinity (perfectly matched duplexes) and sequence specificity of binding (singly mismatched duplexes) using mainly absorption hypochromicity melting curves and isothermal titration calorimetry. For perfectly sequence-matched duplexes of varying lengths (6-20 bp), the average free energy of binding (DeltaG degrees ) was determined to be -6.5+/-0.3 kJ mol(-1) bp(-1), corresponding to a microscopic binding constant of about 14 M(-1) bp(-1). A variety of single mismatches were introduced in 9- and 12-mer PNA-DNA duplexes. Melting temperatures (T(m)) of 9- and 12-mer PNA-DNA duplexes with a single mismatch dropped typically 15-20 degrees C relative to that of the perfectly matched sequence with a corresponding free energy penalty of about 15 kJ mol(-1) bp(-1). The average cost of a single mismatch is therefore estimated to be on the order of or larger than the gain of two matched base pairs, resulting in an apparent binding constant of only 0.02 M(-1) per mismatch. The impact of a mismatch was found to be dependent on the neighboring base pairs. To a first approximation, increasing the stability of the surrounding region, i.e., the distribution of A.T and G.C base pairs, decreases the effect of the introduced mismatch.     

Thermodynamics of HMGB1 interaction with duplex DNA

The high mobility group protein HMGB1 is a small, highly abundant protein that binds to DNA in a non-sequence-specific manner. HMGB1 consists of 2 DNA binding domains, the HMG boxes A and B, followed by a short basic region and a continuous stretch of 30 glutamate or aspartate residues. Isothermal titration calorimetry was used to characterize the binding of HMGB1 to the double-stranded model DNAs poly(dAdT).(dTdA) and poly(dGdC).(dCdG). To elucidate the contribution of the different structural motifs to DNA binding, calorimetric measurements were performed comparing the single boxes A and B, the two boxes plus or minus the basic sequence stretch (AB(bt) and AB), and the full-length HMGB1 protein. Thermodynamically, binding of HMGB1 and all truncated constructs to duplex DNA was characterized by a positive enthalpy change at 15 degrees C. From the slopes of the temperature dependence of the binding enthalpies, heat capacity changes of -0.129 +/- 0.02 and -0.105 +/- 0.05 kcal mol(-1) K(-1) were determined for box A and full-length HMGB1, respectively. Significant differences in the binding characteristics were observed using full-length HMGB1, suggesting an important role for the acid tail in modulating DNA binding. Moreover, full-length HMGB1 binds differently these two DNA templates: binding to poly(dAdT).(dTdA) was cooperative, had a larger apparent binding site size, and proceeded with a much larger unfavorable binding enthalpy than binding to poly(dGdC).(dCdG).     

Thermodynamics of the interaction of daunomycin with DNA

The association constant for the interaction of daunomycin with DNA was determined as a function of temperature (using [³H] daunomycin in conventional equilibrium dialysis cells) and ionic strength (using a spectrophotometric titration method). The association constant varied between 3.1 × 10⁶ M^?1 (4°C) and 3.9 × 10⁵ M^?1 (65°C). The free energy change was ?8.2 to ?8.8 $\text{kcal}\text{mol}$, the enthalpy change ?5.3 $\text{kcal}\text{mol}$ and the entropy change +10 to +11 eu, all values being consistent with that expected of an intercalation process. The apparent number of intercalation sites detected (0.15 to 0.16 per nucleotide) was independent of temperature. The large positive entropy change accompanying the interaction appeals to be due to extensive release of water from the DNA and daunomycin. The apparent number of binding sites increased dramatically with decrease of ionic strength, although the apparent association constant remained largely unaffected by ionic strength.     

DNA Methylation in Mouse Spermatozoa under Long-Term Modeling the Effects of Microgravity

Russian Journal of Developmental Biology - The mechanisms of the interaction between cells and the gravitational field are still unknown, and there are hardly any data on the effect of the gravity...     

Torsional rigidity of DNA and length dependence of the free energy of DNA supercoiling

By analyzing the Boltzmann populations of DNA topoisomers that differ only in their linking numbers, the dependence of the free energy delta G tau of DNA supercoiling on the linking number alpha has been determined for DNA rings as small as 200 base-pairs (bp) in length. All experimental data can be fitted by the relation delta G tau = K (alpha-alpha)2, where alpha is a constant for a given DNA at a given set of conditions and K is a DNA length-dependent proportionality constant. For DNA rings with length N larger than 2000 bp, K is inversely proportional to N and the product NK is nearly a constant around 1150 RT X bp. For rings smaller than 2000 bp NK increases steadily with decreasing N; for a 200 bp ring NK is 3900 RT X bp. The increase in NK when N decreases can be interpreted as a result of the decrease in the contribution of the fluctuation in the writhing number to the equilibrium distribution in alpha. Assuming that the writhing contribution approaches zero for DNA rings 200 bp in size, the torsional rigidity of the DNA double helix is calculated to be 2.9 X 10(-19) erg cm. In addition, the large value of K for the small circles allows precise calculation of the helical repeat of DNA. For the 210 bp rings, the repeat is measured to be 10.54 bp.