Base sequence and helix structure variation in B and A DNA

The observed propeller twist in base-pairs of crystalline double-helical DNA oligomers improves the stacking overlap along each individual helix strand. But, as proposed by Calladine, it also leads to clash or steric hindrance between purines at adjacent base-pairs on opposite strands of the helix. This clash can be relieved by: (1) decreasing the local helix twist angle between base-pairs; (2) opening up the roll angle between base-pairs on the side on which the clash occurs; (3) separating purines by sliding base-pairs along their long axes so that the purines are partially pulled out of the stack (leading to equal but opposite alterations in main-chain torsion angle delta at the two ends of the base-pair); and (4) flattening the propeller twist of the offending base-pairs. Simple sum functions, sigma 1 through sigma 4, are defined, by which the expected local variation in helix twist, base roll angle, torsion angle delta and propeller twist may be calculated from base sequence. All four functions are quite successful in predicting the behavior of B DNA. Only the helix twist and base roll functions are applicable to A DNA, and the helix twist function begins to fail for an A helical RNA/DNA hybrid. Within these limits, the sequence-derived sum functions match the observed helix parameter variation quite closely, with correlation coefficients greater than 0.900 in nearly all cases. Implications of this sequence-derived helix parameter variation for repressor-operator interactions are considered.     

Amide protein exchange and surface conformation of the basic pancreatic trypsin inhibitor in solution. Studies with two-dimensional nuclear magnetic resonance

Dickerson and his colleagues have described the structure of the DNA dodecamer C-G-C-G-A-A-T-T-C-G-C-G in the B form at a level that shows clearly several aspects of some base sequence-dependent departures from the ideal, regular helical structure of B-DNA. I argue that the detailed conformation is a consequence of simple steric repulsive forces between purine bases in consecutive base-pairs but on opposite backbones. These repulsions are a consequence of the “propeller twist” of the base-pairs, together with the larger size of the purine bases, and they may occur in either the major or the minor groove. The argument is conducted in terms of the structural mechanics of a deformable elastic system. These repulsive forces between the base-pairs are resisted by stresses in the helical backbones, which may be studied quantitatively via the variation in torsion angles δ along the backbones, at the points where the sugar rings are connected. There is also a correlation between the cross-chain purine repulsions and the perturbations in helical twist angle between successive base-pairs. The work suggests some comments on the proposed “alternating B” form, the Z form and the A form of DNA.     

Determining local conformational variations in DNA. Nuclear magnetic resonance structures of the DNA duplexes d(CGCCTAATCG) and d(CGTCACGCGC) generated using back-calculation of the nuclear Overhauser effect spectra, a distance geometry algorithm and constrained molecular dynamics

Two-dimensional nuclear magnetic resonance (n.m.r.) spectroscopy and a variety of computational techniques have been used to generate three-dimensional structures of the two DNA duplexes d(CGCCTAATCG) and d(CGTCACGCGC). The central six base-pairs in these two decamers contain all ten dinucleotide pairs in DNA and thus, represent a model system for investigating how the local structure of DNA varies with base sequence. Resonance assignments were made for the non-exchangeable base protons and most of the C-1'-C-4' sugar protons in both decamers. Three-dimensional structures were generated using a distance geometry algorithm and these initial structures were refined by optimizing the fit of back-calculated spectra against the experimental two-dimensional nuclear Overhauser effect (NOE) spectra. This back-calculation procedure consists of calculating NOE cross relaxation rates for a given structure by solution of the Bloch equations, and directly accounts for spin diffusion effects. Use of this refinement procedure eliminates some assumptions that have been invoked when generating structures of DNA oligomers from n.m.r. data. Constrained energy minimization and constrained quenched molecular dynamics calculation were also performed on both decamers to help generate energetically favorable structures consistent with the experimental data. Analysis of the local conformational parameters of helical twist, helical rise, propeller twist, displacement and the alpha, beta, gamma, epison and zeta backbone torsion angles in these structures shows that these parameters span a large range of values relative to the X-ray data of nucleic acids. However, the glycosidic and pseudorotation angles are quite well defined in these structures. The implications that these results have for determination of local structural variations of DNA in solution, such as those predicted by Callidine's rules, are discussed. Our results differ significantly from some previous studies on determining local conformations of nucleic acids and comparisons with these studies are made.     

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.     

Interaction of paired homologous series of diacridines and triacridines with deoxyribonucleic acid

Viscometric measurements using covalently closed circular DNA and sonicated rod-like DNA fragments were performed to investigate unwinding and extension of the DNA helix associated with binding of paired homologous series of diacridines and triacridines. The maximum interchromophore distance for members of the diacridine series spans from 15.1 to 27.5 A, permitting the largest of these ligands to cover up to 4 or 5 base-pairs, allowing for helical twist and local unwinding in a bisintercalated complex lacking severe bending or kinking of the DNA backbone. Helix unwinding angles and increments in DNA contour length are characteristic of bifunctional reaction for all the diacridines studied, the DNA lattice appearing to saturate with one ligand molecule bound per 4 base-pairs. The triacridines, whose maximum end-to-end interchromophore distances are the same as those of their paired diacridines, have maximum central-to-terminal interchromophore distances covering the range 7.5-13.8 A and thus have the potential to form trisintercalated complexes with one or two base-pairs sandwiched between each chromophore. However, helix extension and unwinding parameters for the triacridines are similar to those of the diacridines, and we find no evidence of a transition from bifunctional to trifunctional reaction as the homologous series is ascended. In general, the binding site size appears to be 5 base-pairs for the triacridines. The stereochemical requirements for trisintercalation of triacridines are discussed with reference to the present findings and to the work of others.     

Modeling high-resolution hydration patterns in correlation with DNA sequence and conformation

Hydration around the DNA fragment d(C5T5).(A5G5) is presented from two molecular dynamics simulations of 10 and 12 ns total simulation time. The DNA has been simulated as a flexible molecule with both the CHARMM and AMBER force fields in explicit solvent including counterions and 0.8 M additional NaCl salt. From the previous analysis of the DNA structure B-DNA conformations were found with the AMBER force-field and A-DNA conformations with CHARMM parameters. High-resolution hydration patterns are compared between the two conformations and between C.G and T.A base-pairs from the homopolymeric parts of the simulated sequence. Crystallographic results from a statistical analysis of hydration sites around DNA crystal structures compare very well with the simulation results. Differences between the crystal sites and our data are explained by variations in conformation, sequence, and limitations in the resolution of water sites by crystal diffraction. Hydration layers are defined from radial distribution functions and compared with experimental results. Excellent agreement is found when the measured experimental quantities are compared with the equivalent distribution of water molecules in the first hydration shell. The number of water molecules bound to DNA was found smaller around T.A base-pairs and around A-DNA as compared to B-DNA. This is partially offset by a larger number of water molecules in hydrophobic contact with DNA around T.A base-pairs and around A-DNA. The numbers of water molecules in minor and major grooves have been correlated with helical roll, twist, and inclination angles. The data more fully explain the observed B-->A transition at low humidity.     

Solution structure of a DNA double helix incorporating four consecutive non-Watson-Crick base-pairs

A series of DNA 21-mers containing a variety of the 4 x 4 internal loop sequence 5'-CAAG-3'/3'-ACGT-5' were studied using nuclear magnetic resonance (NMR) methodology and distance geometry (DG)/molecular dynamics (MD) approaches. Such oligomers exhibit excellent resolution in the NMR spectra and reveal many unusual NOEs (nuclear Overhauser effect) that allow for the detailed characterization of a DNA hairpin incorporating a track of four different non-Watson-Crick base-pairs in the stem. These include a wobble C.A base-pair, a sheared A.C base-pair, a sheared A.G base-pair, and a wobble G.T base-pair. Significantly different twisting angles were observed between the base-pairs in internal loop that results with excellent intra-strand and inter-strand base stacking within the four consecutive mismatches and the surrounding canonical base-pairs. This explains why it melts at 52 degrees C even though five out of ten base-pairs in the stem adopt non-Watson-Crick pairs. However, the 4 x 4 internal loop still fits into a B-DNA double helix very well without significant change in the backbone torsion angles; only zeta torsion angles between the tandem sheared base-pairs are changed to a great extent from the gauche(-) domain to the trans domain to accommodate the cross-strand base stacking in the internal loop. The observation that several consecutive non-canonical base-pairs can stably co-exist with Watson-Crick base-pairs greatly increases the limited repertoire of irregular DNA folds and reveals the possibility for unusual structural formation in the functionally important genomic regions that have potential to become single-stranded.     

Helix geometry and hydration in an A-DNA tetramer: IC-C-G-G

The DNA oligomer of sequence IC-C-G-G has been synthesized, and its X-ray crystal structure solved at a resolution of 2.0 A, using anomalous scattering from iodines in phase analysis: 48 cycles of Jack-Levitt restrained least-squares refinement resulted in a residual error of 19.9% over all data, or 16.5% for two-sigma data. Two double-helical tetramers stack in the crystal to form a continuous octamer, except for the two missing phosphate connections across the center. The octamer has a mean helix rotation of 33.7 degrees (10.7 base-pairs per turn), rise of 2.87 A, mean inclination angle of base-pairs of 14 degrees, and mean base-pair propeller twist of +16.3 degrees. Local variations in both helix rotation and base plane roll angles, including those across the center of the octamer, are as predicted from base sequence by sum functions sigma 1 and sigma 2. The three known DNA octamers: IC-C-G-G/IC-C-G-G, G-G-T-A-T-A-C-C and G-G-C-C-G-G-C-C, make up a graded series in this order, with monotonically changing structural parameters. An exhaustive comparison of torsion angle correlations among the known A helices confirms some structural expectations and reveals some new features. 86 water molecules have been located per double-helical IC-C-G-G tetramer (the asymmetric unit), of which 451/2 per tetramer lie within a first hydrogen-bonded shell of hydration. No ordered water structure is observed comparable to the minor groove spine of hydration in B-DNA.     

Effect of spontaneous twist on DNA minicircles

Monte Carlo simulations are used to study the effect of spontaneous (intrinsic) twist on the conformation of topologically equilibrated minicircles of dsDNA. The twist, writhe, and radius of gyration distributions and their moments are calculated for different spontaneous twist angles and DNA lengths. The average writhe and twist deviate in an oscillatory fashion (with the period of the double helix) from their spontaneous values, as one spans the range between two neighboring integer values of intrinsic twist. Such deviations vanish in the limit of long DNA plasmids.     

The solution structure of a B-DNA undecamer comprising a portion of the specific target site for the cAMP receptor protein in the gal operon. Refinement on the basis of interproton distance data.

A restrained least squares refinement of the solution structure of the double-stranded DNA undecamer 5'd(AAGTGT-GACAT).5'd(ATGTCACACTT) comprising a portion of the specific target site of the cAMP receptor protein in the gal operon is presented. The structure is refined on the basis of both distance and planarity restraints, 2331 in all. The distance restraints comprise 150 interproton distances determined from pre-steady state nuclear Overhauser enhancement measurements and 2159 other interatomic distances derived from idealized geometry (i.e., distances between covalently bonded atoms, between atoms defining fixed bond angles, and between atoms defining hydrogen bonding in AT and GC base pairs). Two refinements were carried out and in both cases the final RMS difference between the experimental and calculated interproton distances was 0.2 A. The difference between the two refined structures is small (overall RMS difference of 0.23 A) and represents the error in the refined coordinates. Although the refined structures have an overall B-type conformation there are large variations in many of the local conformational parameters including backbone and glycosidic bond torsion angles, helical twist and propellor twist, base roll and base tilt angles.     

Contribution of the intrinsic curvature to measured DNA persistence length

The persistence length of DNA, a, depends both on the intrinsic curvature of the double helix and on the thermal fluctuations of the angles between adjacent base-pairs. We have evaluated two contributions to the value of a by comparing measured values of a for DNA containing a generic sequence and for an "intrinsically straight" DNA. In each 10 bp segment of the intrinsically straight DNA an initial sequence of five bases is repeated in the sequence of the second five bases, so any bends in the first half of the segment are compensated by bends in the opposite direction in the second half. The value of a for the latter DNA depends, to a good approximation, on thermal fluctuations only; there is no intrinsic curvature. The values of a were obtained from measurements of the cyclization efficiency for short DNA fragments, about 200 bp in length. This method determines the persistence length of DNA with exceptional accuracy, due to the very strong dependence of the cyclization efficiency of short fragments on the value of a. We find that the values of a for the two types of DNA fragment are very close and conclude that the contribution of the intrinsic curvature to a is at least 20 times smaller than the contribution of thermal fluctuations. The relationship between this result and the angles between adjacent base-pairs, which specify the intrinsic curvature, is analyzed.     

Refinement of the solution structure of the B DNA hexamer 5'd(C-G-T-A-C-G)2 on the basis of inter-proton distance data

A restrained least-squares refinement of the solution structure of the self-complementary B DNA hexamer 5'd(C-G-T-A-C-G)2 is presented. The structure is refined on the basis of 190 inter-proton distances determined by pre-steady-state nuclear Overhauser enhancement measurements. Two refinements were carried out starting from two initial B DNA structures differing by an overall root-mean-square (r.m.s.) difference of 0.32 A. In both cases, the final r.m.s. difference between the experimental and calculated inter-proton distances was 0.12 A compared to 0.61 A and 0.58 A for the two initial structures. The difference between the two refined structures is small, with an overall r.m.s. difference of 0.16 A, and represents the error in the refined co-ordinates. The refined structures have a B-type conformation with local structural variations in backbone and glycosidic bond torsion angles, and base-pair propellor twist, base roll, base tilt and local helical twist angles.     

DNA bending and its relation to nucleosome positioning

X-ray and solution studies have shown that the conformation of a DNA double helix depends strongly on its base sequence. Here we show that certain sequence-dependent modulations in structure appear to determine the rotational positioning of DNA about the nucleosome. Three different experiments are described. First, a piece of DNA of defined sequence (169 base-pairs long) is closed into a circle, and its structure examined by digestion with DNAase I: the helix adopts a highly preferred configuration, with short runs of (A, T) facing in and runs of (G, C) facing out. Secondly, the same sequence is reconstituted with a histone octamer: the angular orientation around the histone core remains conserved, apart from a small uniform increase in helix twist. Finally, it is shown that the average sequence content of DNA molecules isolated from chicken nucleosome cores is non-random, as in a reconstituted nucleosome: short runs of (A, T) are preferentially positioned with minor grooves facing in, while runs of (G, C) tend to have their minor grooves facing out. The periodicity of this modulation in sequence content (10.17 base-pairs) corresponds to the helix twist in a local frame of reference (a result that bears on the change in linking number upon nucleosome formation). The determinants of translational positioning have not been identified, but one possibility is that long runs of homopolymer (dA) X (dT) or (dG) X (dC) will be excluded from the central region of the supercoil on account of their resistance to curvature.     

Alternation of DNA and solvent layers in the A form of d(GGCGCC) obtained by ethanol crystallization

We have determined by X-ray crystallography the structure of the hexamer duplex d(GGCGCC)2 in the A-form using ethanol as a precipitant. The same sequence had previously been crystallized in the B-form, but with 2-methyl-2,4-pentanediol as a precipitant. It appears that ethanol precipitation is a useful method to induce the formation of A-form crystals of DNA. Packing of the molecules in the crystal has unique features: the known interaction of A-DNA duplexes between terminal base-pairs and the minor groove of neighbor molecules is combined with a superstructure consisting in an alternation of DNA layers and solvent layers (water/ions). This organization in layers has been observed before, also with hexamers in the A conformation which crystallize in the same space group (C2221). The solvent layer has a precise thickness, although very few ordered water molecules can be detected. Another feature of this crystal is its large unit cell, which gives rise to an asymmetric unit with three hexamer duplexes. One of the three duplexes is quite different from the other two in several aspects: the number of base pairs per turn, the twist pattern, the mean value of the twist angle and the fact that one terminal base-pair is not stacked as part of the duplex and appears to be disordered. So the variability in conformation of this sequence is remarkable.     

The potentially Z-DNA-forming sequence d(GTGTACAC) crystallizes as A-DNA

(GT)n/(CA)n sequences have stimulated much interest because of their frequent occurrence in eukaryotic DNA and their potential for forming the left-handed Z-DNA structure. We here report the X-ray crystal structure of a self-complementary octadeoxynucleotide, d(GTGTACAC), at 2.5 A resolution. The molecule adopts a right-handed double-helical conformation belonging to the A-DNA family. In this alternating purine-pyrimidine DNA minihelix the roll and twist angles show alternations qualitatively consistent with Calladine's rules. The average tilt angle of 9.3 degrees is between the values found in A-DNA (19 degrees) and B-DNA (-6 degrees) fibers. It is envisaged that such intermediate conformations may render diversity to genomic DNA. The base-pair tilt angles and the base-pair displacements from the helix axis are found to be correlated for the known A-DNA double-helical fragments.     

Structure of a B-DNA dodecamer. II. Influence of base sequence on helix structure

Detailed examination of the structure of the B-DNA dodecamer C-G-C-G-A-A-T-T-C-G-C-G, obtained by single-crystal X-ray analysis (Drew et al., 1981), reveals that the local helix parameters, twist, tilt and roll, are much more strongly influenced by base sequence than by crystal packing or any other external forces. The central EcoRI restriction endonuclease recognition site, G-A-A-T-T-C, is a B helix with an average of 9.8 base-pairs per turn. It is flanked on either side by single-base-pair steps having aspects of an A-like helix character. The dodecamer structure suggests several general principles, whose validity must be tested by other B-DNA analyses. (1) When an external bending moment is applied to a B-DNA double helix, it bends smoothly, without kinks or breaks, and with relatively little effect on local helix parameters. (2) Purine-3′,5′-pyrimidine steps open their base planes towards the major groove, pyrimidine-purine steps open toward the minor groove, and homopolymer (Pur-Pur, Pyr-Pyr) steps resist rolling in either direction. This behavior is related to the preference of pyrimidines for more negative glycosyl torsion angles. (3) CpG steps have smaller helical twist angles than do GpC, as though in compensation for their smaller intrinsic base overlap. Data on A-T steps are insufficient for generalization. (4) G.C base-pairs have smaller propellor twist than A · T, and this arises mainly from interstrand base overlap rather than the presence of the third hydrogen bond. (5) DNAase I cuts preferentially at positions of high helical twist, perhaps because of increased exposure of the backbone to attack. The correlation of the digestion patterns in solution and helical twist in the crystal argues for the essential identity of the helix structure in the two environments. (6) In the two places where the sequence TpCpG occurs, the C slips from under T in order to stack more efficiently over G. At the paired bases of this CpG step, the G and C are tilted so the angle between base planes is splayed out to the outside of the helix. This TpC is the most favored cutting site for DNAase I by a factor of 4.5 (Lomonossoff et al., 1981). (7) The EcoRI restriction endonuclease and methylase both appear to prefer a cutting site of the type purine-purine-A-T-T-pyrimidine, involving two adjacent homopolymer triplets, and this may be a consequence of the relative stiffness of homopolymer base-stacking observed in the dodecamer.     

Superhelical turns of closed circular DNA in solution and in a gel: evidence for a conformational difference

We have compared the number of superhelical turns, tau, in circular covalently closed plasmid pBR beta G DNA obtained by four different methods, each based on one particular distinguishing principle. Three of the methods allow an unequivocal determination of tau under gel electrophoresis conditions, whilst the fourth enables us to determine its value in solution. We were able to detect a significant difference between the two environments, corresponding to an unwinding of the DNA duplex angle by 0.3 degrees when a sample is transferred from solution to gel. The possible existence of such an effect has been generally overlooked by previous investigators. Our result suggests that the previously reported value for the number of base-pairs per helical turn should be adjusted downwards by about 0.10, so that it applies to conditions in solution.