On the toroidal condensed state of closed circular DNA

The influence of double helix torsional elasticity on the compaction and structure of circular DNA compact form is studied theoretically in the case when the compact (globular) form has torus shape. For closed circular DNA the topological invariant, the linking number, yields a strict connection between conformation of the double helix considered as unifilar homopolymer and elastic energy of torsional twisting. The contribution of torsional elasticity to the free energy of the toruslike globule is calculated. This contribution is shown to be proportional to the square of superhelical density. Allowance of the torsional elasticity decreases the equilibrium radius of the toruslike globule formed by circular DNA. Closure of linear DNA into a ring widens the stability range of the relatively short DNA compact form and tightens it for long DNA.     

Toroidal globular state of circular DNA

The influence of torsional elasticity of the double helix on compactization and structure of circular DNA in a compact form is studied in the case when the compact (globular) particle has a torus shape. For closed circular DNA the topological invariant, linking number of two strains, yields strict connection between conformation of double helix, considered as a unifilar homopolymer, and elastic energy of torsional twisting. The contribution of torsional elasticity to free energy of the toruslike globule is calculated. This contribution is shown to be proportional to the square of superturn's density. Torsional elasticity decreases the equilibrium radius of the toruslike globule formed by circular DNA in comparison with the case of linear DNA. Closure of linear DNA into a ring widens the stability range of the relatively short DNA compact form and tightens it for long DNA.     

Variance of Writhe for Wormlike DNA Rings with Excluded Volume

Abstract

We have calculated the variance of the equilibrium distribution of a circular wormlike polymer chain over the writhing number, <((Wr)² )>, with allowance for the excluded volume effects. Within this model the <((Wr)² )> value is a function of the number of Kuhn statistical segments, n, and the chain diameter, d measured in Kuhn statistical lengths, b. Simulated DNA chains varied from 200 to 10,000 base pairs and the d value varied from 0.02 to 0.2. Theory predicts a considerable ionic strength dependence of the DNA superhelix energy as a consequence of the change in the DNA diameter. A comparison with the available experimental data has yielded an estimate of the DNA torsional rigidity, the Kuhn statistical length, and the effective diameter of the double helix under conditions of the complete screening of the DNA electrostatic potential.     

Theoretical Study of Ethidium Intercalation in Triple-Stranded DNA and at Triplex-Duplex Junctions

Abstract

The contribution of different factors in the interaction of ethidium intercalated into various sequences of a triple helix, or in the region of the junction between the double- and triple-stranded DNA has been studied by energy minimization. It is found that in the total energy of the ethidium - triple helix complexes, a particular electrostatic contribution emerges due to the presence of protonated cytosines in the triple helix. This parameter is determinant in the sequence-specificity of ethidium binding to the triple helix. The preferred intercalation sites of ethidium in the triple helix are proposed. The interaction of ethidium at the triplex-duplex junction, and its effects are also discussed. This study is aimed at searching for new drugs specific for the triple helix, or for the triplex-duplex junctions.     

Molecular Modelling Study of the Netropsin Complexation With a Nucleic Acid Triple Helix

Abstract

A detailed molecular mechanical study has been made on the complexes of netropsin with the double stranded oligonucleotide (dA)₁₂.(dT)₁₂ and with the triple helix (dA)₁₂.(dT)₁₂.(dT)₁₂. The complexes were built using computer graphics and energy refined using JUMNA program. In agreement with circular dichroïsm experiments we have shown that 3 netropsins can bind the minor grooves of the triple helix and of the double helix. The groove geometry in the duplex and in the triplex is very similar. However a detailed analysis of the energetic terms shows, in agreement with thermal denaturation studies, that the affinity of netropsin toward the double helices is larger than towards triple helices.     

Alternate g−g−. conformation for dinucleoside phosphates in solution

An alternative g^?g^? conformation (conformer I') for dinucleosides in solution has been deduced, based on potential energy calculations and nuclear magnetic resonance spectroscopy. This conformation is characterized by larger glycosidic torsional angles (X=94–111°) than those of conformer 1 (X=8–35°), although the other torsional angles are similar. There are thus four stable confonners (I, I', II and III) for dinucleosides in equilibrium with the open forms. The structure of conformer I' supports that of the ‘vertical’ double helix constructed by Olson (W.K. Olson. Proc. Natl. Acad. Sci. U.S.A. 74, (1977) 1775). Our data may suggest the possibility of interconversion between the vertical double helix and the regular double helix of A-form DNA, RNA or A'-form RNA.     

The torsional state of DNA within the chromosome

Virtually all processes of the genome biology affect or are affected by the torsional state of DNA. Torsional energy associated with an altered twist facilitates or hinders the melting of the double helix, its molecular interactions, and its spatial folding in the form of supercoils. Yet, understanding how the torsional state of DNA is modulated remains a challenging task due to the multiplicity of cellular factors involved in the generation, transmission, and dissipation of DNA twisting forces. Here, an overview of the implication of DNA topoisomerases, DNA revolving motors, and other DNA interactions that determine local levels of torsional stress in bacterial and eukaryotic chromosomes is provided. Particular emphasis is made on the experimental approaches being developed to assess the torsional state of intracellular DNA and its organization into topological domains.     

Theoretical Analysis of Disruptions in DNA Minicircles

Under sufficient bending stress, which appears in DNA minicircles and small DNA loops, the double helix experiences local disruptions of its regular structure. We developed a statistical-mechanical treatment of the disruptions in DNA minicircles, studied experimentally by Du et al. The model of disruptions used in our Monte Carlo simulation of minicircle conformations specifies these conformations by three parameters: DNA bend angle at the disruption, θ_d; local DNA unwinding caused by the disruption; and the free energy associated with the disruption in the unstressed double helix, G_d. The model is applicable to any structural type of disruption, kinks or opening of single basepairs. The simulation shows that accounting for both torsional and bending deformation associated with the disruptions is very important for proper analysis. We obtained a relationship between values of G_d and θ_d under which the simulation results are compatible with the experimental data. The relationship suggests that the free energy of basepair opening, which includes flipping out both bases, is significantly higher than the generally accepted value. The model is also applied to the analysis of j-factors of very short DNA fragments.     

How Ala-->Gly mutations in different helices affect the stability of the apomyoglobin molten globule

The apomyoglobin molten globule has a complex, partly folded structure with a folded A[B]GH subdomain; the factors determining its stability are not yet known in detail. Ala-->Gly mutations, made at solvent-exposed positions, are used to probe the role of helix propensity of individual helices in stabilizing the molten globule. Molten globule stability is measured by reversible urea unfolding, monitored both by circular dichroism and by tryptophan fluorescence. Two-state unfolding is tested by superposition of these two unfolding curves, and stability data are reported only for variants which satisfy the superposition test. Results for sites Q8 in the A helix and E109 in the G helix confirm that the helix propensities of the A and G helices both strongly affect molten globule stability, in contrast to results for the G65A/G73A double mutant which show that changing the helix propensity of the E-helix sequence has no significant stabilizing effect. Changing the helix propensity of the B-helix sequence with the G23A/G25A double mutant affects molten globule stability to an intermediate extent, confirming an earlier report that this mutant has increased stability. These results are consistent with the bipartite structure for the molten globule in which the A, G, and H helices are stably folded, while the long E helix is unfolded and the B helix has intermediate stability. Some differences are found in the shapes of the unfolding curves of different mutants even though they satisfy the superposition test for two-state unfolding, and possible explanations are discussed.     

5-Deazaflavins: New Very Efficient DNA Photosensitisers,Synthesis of Oligonucleotide Conjugates

Abstract

In order to study electron transfer processes through the DNA double helix, we have synthesised a series of 5-deazaflavin derivatives 1–4 and demonstrated their ability to induce very efficiently 2′-deoxyguanosine and DNA oxidations by electron transfer from guanine. 5-Deazaflavin-oligonucleotide adducts were also synthesised for the study of electron transfers through double or triple helix formed with their complementary sequence.     

Is DNA really a double helix?

Do the two chains of the DNA molecule coil round one another plectonemically ? If so, what is the approximate value of Lk (the linking number) for any closed, circular DNA molecule? Experiments using gel electrophoresis have shown that supercoiled DNA molecules usually migrate in a series of discrete bands. The only tenable explanation for this quantized behavior is that the molecules in one band all have the same value of Lk and that this value differs by unity from that of the adjacent bands. Various experiments in which circular DNA is unwound by known amounts show that (given this assumption) Lk for relaxed DNA is very roughly equal to $\text{N}\text{10}$ (where N is the number of base-pairs), as expected from the classical double helix.The original model for the double helix was right-handed. The experimental evidence for this feature is suggestive but not yet completely compelling.     

A molecular mechanical model to predict the helix twist angles of B-DNA

We present here a model for the prediction of helix twist angles in B-DNA, a model composed of a collection of torsional springs. Statistically averaged conformational energy calculations show that, for a specified basepair step, the basepair-basepair conformational energy is quadratically dependent on the helix twist angle, so the calculations provide the spring parameters for the basepair-basepair interactions. Torsional springs can also be used to model the effects of the backbone on the helix twist, and the parameters for those springs are derived by fitting the model to experimental data. The model predicts a macroscopic torsional stiffness and a longitudinal compressibility (Young's modulus) which are both in good agreement with experiment. One biological consequence of the model is examined, the sequence specificity of the Eco RI restriction endonuclease, and it is shown that the discriminatory power of the enzyme receives a substantial contribution from the energetic cost of torsional deformations of the DNA when wrong sequences are forced into the enzyme binding site.     

Natural DNA sequences can form left-handed helices in low salt solution under conditions of topological constraint

The conformation of DNA that originates from association of complementary single-stranded circles (form V DNA) is investigated in solution at low salt concentration. It is shown that circular dichroism extended to the far ultraviolet region (down to 165 nm) represents a powerful tool for determination of the handedness of double helical DNAs in solution. The positive intense band at 186 nm followed by a strong negative band around 170 nm is characteristic of all right-handed helical forms (B,A) of DNA, whereas the circular dichroism spectrum of the Z form of poly[d(G-C)] of opposite helical sense represents a quasi inversion of these far ultraviolet bands. Thus, form V DNA is found to represent a co-existence of left-handed Z-type and right-handed B double helical stretches in addition to negative superturns. The Raman spectrum of form V DNA provides further support for the contribution of a left-handed double helical conformation, as shown by comparison to the high resolution Raman spectra of poly[d(G-C)] in the Z and B forms.The analysis of present spectroscopic data and the analysis of occurrence of alternating [d(G-C)] purine-pyrimidine sequences in the form V DNA used strongly suggest that in DNA of natural sequence, topological constraint may generate left-handed double helices, a conformation thought so far to be limited to the alternating [d(G-C)] sequences. Such structure could play a role in recognition and regulation of gene expression.     

Variance of writhe for wormlike DNA rings with excluded volume

We have calculated the variance of the equilibrium distribution of a circular wormlike polymer chain over the writhing number, less than (Wr)2 greater than, with allowance for the excluded volume effects. Within this model the less than (Wr)2 greater than value is a function of the number of Kuhn statistical segments, n, and the chain diameter, d measured in Kuhn statistical lengths, b. Simulated DNA chains varied from 200 to 10,000 base pairs and the d value varied from 0.02 to 0.2. Theory predicts a considerable ionic strength dependence of the DNA superhelix energy as a consequence of the change in the DNA diameter. A comparison with the available experimental data has yielded an estimate of the DNA torsional rigidity, the Kuhn statistical length, and the effective diameter of the double helix under conditions of the complete screening of the DNA electrostatic potential.     

Topological distributions and the torsional rigidity of DNA. A Monte Carlo study of DNA circles

Distributions of the linking number of circular DNA molecules, defined as the sum of twist and the writhing number, are obtained by Monte Carlo simulations of small, randomly closed DNA circles. We estimate the relative contributions of fluctuations in twist and writhe to the linking number distribution, as functions of DNA size. Published experimental data on topoisomer distributions in circular DNA molecules are interpreted to estimate the torsional rigidity of DNA in solution. We show that ignoring the writhe component of the linking number distribution, even for DNA circles as small as 250 base-pairs, leads to an underestimate for the torsional stiffness of the double helix. The value of the torsional modulus obtained from this analysis, C = 3.4 X 10(-19) erg cm, is from 10 to 40% larger than that estimated by others and more than twice as large as the values obtained from fluorescence depolarization or other time-resolved spectroscopic measurements. We also develop further the theoretical treatment of ring closure probabilities for DNA described in the previous article. It is shown that the torsional part of the ring closure probability, phi 0,1 (tau 0) is a periodic function of DNA length that contributes strongly to the ring closure probability for short chains but makes negligible contributions for chains over 1000 base-pairs in length.     

Theory of the toroidal compact form of DNA in a polymer solution

A diagram of the states of single long rigid DNA macromolecule in solution of short flexible polymer was plotted. It has been shown that the compacting effect of the polymer solvent on DNA can be interpreted as hydrostatic pressure of coils gas on impermeable "walls" of the DNA globule. Conditions were found for the existence of compact (globular) states in the form of a sphere (stable for a very long DNA) and tore (for a relatively short one). Possibility of intramolecular liquidcrystalline orderliness of DNA segments in a globule was analyzed. Regions of realization of both regimes were studied: when compression with an added polymer was equalized by the forces of steric repulsion of DNA segments and when it was balanced by the elasticity of the DNA chain bendings.     

Strong bending of the DNA double helix

During the past decade, the issue of strong bending of the double helix has attracted a lot of attention. Here, we overview the major experimental and theoretical developments in the field sorting out reliably established facts from speculations and unsubstantiated claims. Theoretical analysis shows that sharp bends or kinks have to facilitate strong bending of the double helix. It remains to be determined what is the critical curvature of DNA that prompts the appearance of the kinks. Different experimental and computational approaches to the problem are analyzed. We conclude that there is no reliable evidence that any anomalous behavior of the double helix happens when DNA fragments in the range of 100 bp are circularized without torsional stress. The anomaly starts at the fragment length of about 70 bp when sharp bends or kinks emerge in essentially every molecule. Experimental data and theoretical analysis suggest that kinks may represent openings of isolated base pairs, which had been experimentally detected in linear DNA molecules. The calculation suggests that although the probability of these openings in unstressed DNA is close to 10⁻⁵, it increases sharply in small DNA circles reaching 1 open bp per circle of 70 bp.     

Local and global cooperativity in the human alpha-lactalbumin molten globule

NMR spectroscopy has been used to follow the urea-induced unfolding of the low pH molten globule states of a single-disulfide variant of human alpha-lactalbumin ([28-111] alpha-LA) and of two mutants, each with a single proline substitution in a helix. [28-111] alpha-LA forms a molten globule very similar to that formed by the wild-type four-disulfide protein, and this variant has been used as a model for the alpha-lactalbumin (alpha-LA) molten globule in a number of studies. The urea-induced unfolding behavior of [28-111] alpha-LA is similar to that of the four-disulfide form of the protein, except that [28-111] alpha-LA is less stable and has greater cooperativity in the loss of different elements of structure. For one mutant, L11P, the helix containing the mutation is highly destabilized such that it is completely unfolded even in the absence of urea. By contrast, for the other mutant, Q117P, the helix containing the mutation retains its compact structure. Both mutations, however, show significant long-range destabilization of the overall fold showing that the molten globule state has a degree of global cooperativity. The results reveal that different permutations of three of the four major alpha-helices of the protein can form a stable, locally cooperative, compact structural core. Taken together, these findings demonstrate that the molten globule state of alpha-LA is an ensemble of conformations, with different subsets of structures linked by a range of long-range interactions.