DNA bending: the prevalence of kinkiness and the virtues of normality.

DNA bending in 86 complexes with sequence-specific proteins has been examined using normal vector plots, matrices of normal vector angles between all base pairs in the helix, and one-digit roll/slide/twist tables. FREEHELIX, a new program especially designed to analyze severely bent and kinked duplexes, generates the foregoing quantities plus local roll, tilt, twist, slide, shift and rise parameters that are completely free of any assumptions about an overall helix axis. In nearly every case, bending results from positive roll at pyrimidine-purine base pair steps: C-A (= T-G), T-A, or less frequently C-G, in a direction that compresses the major groove. Normal vector plots reveal three well-defined types of bending among the 86 examples: (i) localized kinks produced by positive roll at one or two discrete base pairs steps, (ii) three-dimensional writhe resulting from positive roll at a series of adjacent base pairs steps, or (iii) continuous curvature produced by alternations of positive and negative roll every 5 bp, with side-to-side zig-zag roll at intermediate position. In no case is tilt a significant component of the bending process. In sequences with two localized kinks, such as CAP and IHF, the dihedral angle formed by the three helix segments is a linear function of the number of base pair steps between kinks: dihedral angle = 36 degrees x kink separation. Twenty-eight of the 86 examples can be described as major bends, and significant elements in the recognition of a given base sequence by protein. But even the minor bends play a role in fine-tuning protein/DNA interactions. Sequence-dependent helix deformability is an important component of protein/DNA recognition, alongside the more generally recognized patterns of hydrogen bonding. The combination of FREEHELIX, normal vector plots, full vector angle matrices, and one-digit roll/slide/twist tables affords a rapid and convenient method for assessing bending in DNA.     

A Stochastic Model for Helix Bending in B-DNA

Abstract

Bending in double-helical B-DNA apparently occurs only by rolling adjacent base pairs over one another along their long axes. The lifting apart of ends that would be required by tilt or wedge angle contributions is too costly in free energy and does not occur. Roll angles at base steps can be positive (compression of major groove) or negative (compression of minor groove); with the former somewhat easier.

Individual steps may advance or oppose the overall direction of bend, or make lateral excursions, but the result of this series of “random roll” steps is the production of a net bending in the helix axis. Because the natural roll points for bending in a given plane occur every 5 base pairs, one would expect that double-helical DNA wrapped around a nucleosome core would exhibit bends with the same periodicity. Alternate bends might be particularly acute where the major groove faced the nucleosome core and was compressed against it.

The “annealed kinking” model proposed by Fratini et al. (J. Biol. Chem. 257, 14686 (1982) was suggested from the observation that a major bend at a natural roll point is flanked by decreasing roll angles at the steps to either side, as though local strain was being minimized by somewhat blurring the bend out rather than keeping it localized. The random walk model suggested in this paper would describe this as a decreased roll angle as the helix step rotates toward a direction perpendicular to the overall bend. Bending of DNA is seen to be a more stochastic process than had been suspected. Detailed analysis of every helix step reveals both side excursions and backward or retrograde motion, as in any random walk situation. Yet these isolated steps counteract one another, to leave behind a residuum of overall bending in a specific direction.     

Index to Authors,Volume 3

Abstract

Results of calculations using various empirical potentials suggest that base pair buckling, which commonly occurs in DNA crystal structures, is sufficient to eliminate the steric clash at CpG steps in B-DNA, originating from the base pair propeller twisting. The buckling is formed by an inclination of cytosines while deviations of guanines from a plane perpendicular to the double helix axis are unfavorable. The buckling is accompanied by an increased vertical separation of the base pair centers but the buckled arrangement of base pairs is at least as stable as when the vertical separation is normal and buckle zero. In addition, room is created by the increased vertical separation for the bases to propeller twist as is observed in DNA crystal structures. Further stabilization of base stacking is introduced into the buckled base pair arrangement by roll opening the base pairs into the double helix minor groove. The roll may lead to the double helix bending and liberation of guanines from the strictly perpendicular orientation to the double helix axis. The liberated guanines further contribute to the base pair buckling and stacking improvement. This work also suggests a characteristic very stable DNA structure promoted by nucleotide sequences in which runs of purines follow runs of pyrimidine bases.     

A 2H-NMR study of the A-DNA conformation in films of oriented Na-DNA: evidence of a disordered B-DNA contribution

Solid-state ²H-nmr spectra have been obtained from folded films of oriented Li- and Na-DNA molecules with the purine bases selectively deuterium labeled at the 8 position. From line shape simulations, we find that the Na-DNA sample at 75% relative humidity (rh) contains both A-DNA and surprisingly large amounts of B-DNA

1 Here, B-DNA refers to “B-DNA family” (i.e. B- or C-DNA).

(57%). For the A-DNA component the average base tilt is 23°, and the total distribution width of tilt angles and helix axis orientations is ～ 4° (standard deviation). In the B-DNA component the base tilt is ～ 0° and the total distribution width is ～ 20°. In contrast, films of Li-DNA only exhibit the B-form line shape, consistent with a base tilt of ～ 0° and a total distribution width of base tilt angles and helix axis orientations of 9°. The nmr results that demonstrate the presence of large amounts of B-DNA in the Na-DNA sample contrast with the x-ray diffraction measurements that indicated mainly A-form. The nmr spectra are used to monitor the B-DNA content in the Na-films and to evaluate procedures for increasing the A-DNA fraction.     

Mediation of the A/B-DNA helix transition by G-tracts in the crystal structure of duplex CATGGGCCCATG

The crystal structure of the DNA dodecamer duplex CATGGGCCCATG lies on a structural continuum along the transition between A- and B-DNA. The dodecamer possesses the normal vector plot and inclination values typical of B-DNA, but has the crystal packing, helical twist, groove width, sugar pucker, slide and x-displacement values typical of A-DNA. The structure shows highly ordered water structures, such as a double spine of water molecules against each side of the major groove, stabilizing the GC base pairs in an A-like conformation. The different hydration of GC and AT base pairs provides a physical basis for solvent-dependent facilitation of the A↔B helix transition by GC base pairs. Crystal structures of CATGGGCCCATG and other A/B-DNA intermediates support a ‘slide first, roll later’ mechanism for the B→A helix transition. In the distribution of helical parameters in protein–DNA crystal structures, GpG base steps show A-like properties, reflecting their innate predisposition for the A conformation.     

Base pair buckling can eliminate the interstrand purine clash at the CpG steps in B-DNA caused by the base pair propeller twisting

Results of calculations using various empirical potentials suggest that base pair buckling, which commonly occurs in DNA crystal structures, is sufficient to eliminate the steric clash at CpG steps in B-DNA, originating from the base pair propeller twisting. The buckling is formed by an inclination of cytosines while deviations of guanines from a plane perpendicular to the double helix axis are unfavorable. The buckling is accompanied by an increased vertical separation of the base pair centers but the buckled arrangement of base pairs is at least as stable as when the vertical separation is normal and buckle zero. In addition, room is created by the increased vertical separation for the bases to propeller twist as is observed in DNA crystal structures. Further stabilization of base stacking is introduced into the buckled base pair arrangement by roll opening the base pairs into the double helix minor groove. The roll may lead to the double helix bending and liberation of guanines from the strictly perpendicular orientation to the double helix axis. The liberated guanines further contribute to the base pair buckling and stacking improvement. This work also suggests a characteristic very stable DNA structure promoted by nucleotide sequences in which runs of purines follow runs of pyrimidine bases.     

Analysis of local helix geometry in three B-DNA decamers and eight dodecamers

Local variations in B-DNA helix structure are compared among three decamers and eight dodecamers, which contain examples of all ten base-pair step types. All pairwise combinations of helix parameters are compared by linear regression analysis, in a search for internal relationships as well as correlations with base sequence. The primary conclusions are: (1) Three-center hydrogen bonds between base-pairs occur frequently in the major groove at C-C, C-A, A-A and A-C steps, but are less convincing at C-C and C-T steps in the minor groove. The requirements for large base-pair propeller are (1) that the base-pair should be A.T rather than G.C, and (2) that it be involved in a major groove three-center hydrogen bond with the following base-pair. Either condition alone is insufficient. Hence, a large propeller is expected at the leading base-pair of A-A and A-C steps, but not at A-T, T-A, C-A or C-C steps. (2) A systematic and quantitative linkage exists between helix variables twist, rise, cup and roll, of such strength that the rise between base-pairs can hardly be described as an independent variable at all. Two typical patterns of behavior are observed at steps from one base-pair to the next: high twist profile (HTP), characterized by high twist, low rise, positive cup and negative roll, and low twist profile (LTP), marked by low twist, high rise; negative cup and positive roll. Examples of HTP are steps G-C, G-A and Y-C-A-R, where Y is pyrimidine and R is purine. Examples of LTP steps are C-G, G-G, A-G and C-A steps other than Y-C-A-R. (3) The minor groove is especially narrow across the two base-pairs of the following steps: A-T, T-A, A-A and G-A. (4) In general, base step geometry cannot be correlated solely with the bases that define the step in question; the two flanking steps also must be taken into account. Hence, local helix structure must be studied in the context, not of two base-pairs: A-B, but of four: x-A-B-y. Calladine's rules, although too simple in detail, were correct in defining the length of sequence over which a given perturbation is expressed. Whereas ten different two-base steps are possible, allowing for the identity of complementary sequences, there are 136 different four-base steps. Only 33 of these 136 four-base steps are represented in the decamer and dodecamer structures solved to date, and hence it is premature to try to set up detailed structural algorithms. (5) The sugar-phosphate backbone chains of B-DNA place strong limits on sequence-induced structural variation, damping down most variables within four or five base-pairs, and preventing purine-purine anti-anti mismatches from causing bulges in the double helix. Hence, although short-range sequence-induced deformations (or deformability) are observed, long-range deformations propagated down the helix are not to be expected.     

Base Pair Opening Pathways in B-DNA

Abstract

Molecular modeling is used to study the opening pathways of bases within a B-DNA oligomer. It is demonstrated that many open states are possible for a single base pair, although a preference for opening towards the major groove of the double helix is found. In addition we show that opening is strongly influenced by the nature of the base involved and is also coupled in many cases to DNA bending.     

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.     

Anisotropic flexibility of DNA depends on the base sequence. Conformation calculations of double-stranded tetranucleotides AAAA:TTTT, (AATT)2, (TTAA)2, GGGG:CCCC, (GGCC)2, (CCGG)2

The bending flexibility of six tetramers was studied in an assumption that they were extended in the both directions by regular double helices. The bends of B-DNA in different directions were considered. The stiffness of the B-DNA double helix when bent into the both grooves proved to be less pronounced than in the perpendicular direction by the order of magnitude. Such an anisotropy is a feature of the sugar-phosphate backbone structure. The calculated fluctuations of the DNA bending along the dyad axis, 5-7 degrees, are in agreement with the experimental value of DNA persistence length. Anisotropy of the double helix is sequence-dependent: most easily bent into the minor groove are the tetramers with purine-pyrimidine dimer (RY) in the middle. In contrast, YR dinucleotides prefer bending into the major groove, moreover, they have an equilibrium bend of 6-12 degrees into this groove. The above inequality is caused by the stacking interaction of the bases. The bend in the central dimers is distributed to some extent between the adjacent links, though the main fraction of the bend remains within the central link. Variation of the sugar-phosphate geometry in the bent helix is unessential, so that DNA remains within the limits of the B-family of forms: namely, when the helical axis is bent by 20 degrees the backbone dihedral angles vary by no more than 15 degrees. The obtained results are in accord with the X-ray structure of B-DNA dodecamer; they further substantiate our earlier model of DNA wrapping in the nucleosome by means of "mini-kinks" separated by a half-pitch of the double helix, i.e. by 5-6 b. p. Sequence-dependent anisotropy of DNA presumably dictates the three-dimensional structure of DNA in solution as well. We have found that nonrandom allocation of YR dimers leads to the systematic bends in the equilibrium structure of certain DNA fragments. To the four "Calladine rules" two more can be added: the minor-groove steric clash of purines in the YR sequences are avoided by: (1) bending of the helix into the major groove; (2) increasing the distance between the base pairs (stretching the double helix).     

Sequence-Dependent Anisotropic Flexibility of B-DNA A Conformational Study

Abstract

Bending flexibility of the six tetrameric duplexes was investigated d(AAAA):d(TTTT), d(AATT)₂, d(TTAA) ₂, d(GGGG):d(CCCC), d(GGCC) ₂ and d(CCGG) ₂. The tetramers were extended in the both directions by regular double helices. The stiffness of the B-DNA double helix when bent into the both grooves proved to be less than that in the perpendicular direction by an order of magnitude. Such an anisotropy is a property of the sugar-phosphate backbone structure. The calculated fluctuations of the DNA bending along the dyad axis, 5–7°, are in agreement with experimental value of the DNA persistence length.

Anisotropy of the double helix is sequence-dependent: most easily bent into the minor groove are the tetramers with purine-pyrimidine dimer (RY) in the middle. In contrast, YR dinucleotides prefer bending into the major groove. Moreover, they have an equilibrium bend of 6–12° into this groove. The above inequality is caused by stacking interaction of the bases.

The bend in the central dimer is distributed to some extent between the adjacent links, though the main fraction of the bend remains within the central link. Variation of the sugar-phosphate geometry in the bent helix is inessential, so that DNA remains within the B-family of forms: namely, when the helical axis is bent by 20°, the backbone dihedral angles vary by no more than 15°.

The obtained results are in accord with x-ray structure of the B-DNA dodecamer; they further substantiate our early model of DNA wrapping in the nucleosome by means of “mini-kinks” separated by a half-pitch of the double helix, i.e. by 5–6 b.p. Sequence-dependent anisotropy of DNA presumably dictates the three-dimentional structure of DNA in solution as well. We have found that nonrandom allocation of YR dimers leads to the systematic bends in equilibrium structure of certain DNA fragments.     

Conformational Characteristics and Correlations in Crystal Structures of Nucleic Acid Oligonucleotides: Evidence for Sub-states

Abstract

Sugar phosphate backbone conformations are a structural element inextricably involved in a complete understanding of specific recognition nucleic acid ligand interactions, from early stage discrimination of the correct target to complexation per se, including any structural adaptation on binding. The collective results of high resolution DNA, RNA and protein/DNA crystal structures provide an opportunity for an improved and enhanced statistical analysis of standard and unusual sugar-phosphate backbone conformations together with corresponding dinucleotide sequence effects as a basis for further exploration of conformational effects on binding. In this study, we have analyzed the conformations of all relevant crystal structures in the nucleic acids data base, determined the frequency distribution of all possible ?, ζ, α, β and γ backbone angle arrangements within four nucleic acid categories (A-RNA and A-DNA, free and bound B-DNA) and explored the relationships between backbone angles, sugar puckers and selected helical parameters. The trends in the correlations are found to be similar regardless of the nucleic acid category. It is interesting that specific structural effects exhibited by the different unusual backbone sub-states are in some cases contravariant. Certain α/γ changes are accompanied by C3′ endo (north) sugars, small twist angles and positive values of base pair roll, and favor a displacement of nucleotide bases towards the minor groove compared to that of canonical B form structures. Unusual ?/ζ combinations occur with C2′ (south) sugars, high twist angles, negative values of base pair roll, and base displacements towards the major groove. Furthermore, any unusual backbone correlates with a reduced dispersion of equilibrium structural parameters of the whole double helix, as evidenced by the reduced standard deviations of almost all con- formational parameters. Finally, a strong sequence effect is displayed in the free oligomers, but reduced somewhat in the ligand bound forms. The most variable steps are GpA and CpA, and, to a lesser extent, their partners TpC and TpG. The results provide a basis for considering if the variable and non-variable steps within a biological active sequence precisely determine morphological structural features as the curvature direction, the groove depth, and the accessibility of base pair for non covalent associations.     

Sequence-Dependent Bending of DNA and Phasing of Nucleosomes

Abstract

Conformational analysis has revealed anisotropic flexibility of the B-DNA double helix: it bends most easily into the grooves, being the most rigid when bent in a perpendicular direction. This result implies that DNA in a nucleosome is curved by means of relatively sharp bends (“mini-kinks”) which are directed into the major and minor grooves alternatively and separated by 5–6 base pairs. The “mini-kink” model proved to be in keeping with the x-ray structure of the B-DNA dodecamer resolved later, which exhibits two “annealed kinks”, also directed into the grooves.

The anisotropy of B DNA is sequence-dependent: the pyrimidine-purine dimers (YR) favor bending into the minor groove, and the purine-pyrimidine dinucleotides (RY), into the minor one. The RR and YY dimers appear to be the most rigid dinucleotides. Thus, a DNA fragment consisting of the interchanging oligopurine and oligopyrmidine blocks 5–6 base pairs long should manifest a spectacular curvature in solution.

Similarly, a nucleotide sequence containing the RY and YR dimers separated by a half-pitch of the double helix is the most suitable for wrapping around the nucleosomal core. Analysis of the numerous examples demonstrating the specific alignment of nucleosomes on DNA confirms this concept. So, the sequence-dependent “mechanical” properties of the double helix influence the spatial arrangement of DNA in chromatin.     

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.     

Mutual interactions of the phosphate groups in locally deformed backbones of various DNA double helices at high salt concentrations

Changes in the free energy of mutual phosphate group interactions are calculated that accompany bending of the A-, B- and Z-DNA backbones in 0.7, 2.1 and 4.2 mol/l NaCl aqueous solutions. The bending is often found to be favoured in the direction of the double helix grooves; B-DNA prefers bending into the major groove while minor groove is the preferred bending direction of A-DNA in the presence of 0.7 mol/l NaCl. Interestingly, the preferences are reversed in 4.2 mol/l NaCl. Further stabilization of A-DNA and B-DNA backbones is achieved in some cases if bending is combined with suitable local double helix twist alterations. Bending tendencies of Z-DNA backbone are generally weaker and they decrease, in contrast to B-DNA and A-DNA, with the increasing ionic strength.     

Conformational characteristics and correlations in crystal structures of nucleic acid oligonucleotides: evidence for sub-states

Sugar phosphate backbone conformations are a structural element inextricably involved in a complete understanding of specific recognition nucleic acid ligand interactions, from early stage discrimination of the correct target to complexation per se, including any structural adaptation on binding. The collective results of high resolution DNA, RNA and protein/DNA crystal structures provide an opportunity for an improved and enhanced statistical analysis of standard and unusual sugar-phosphate backbone conformations together with corresponding dinucleotide sequence effects as a basis for further exploration of conformational effects on binding. In this study, we have analyzed the conformations of all relevant crystal structures in the nucleic acids data base, determined the frequency distribution of all possible epsilon, zeta, alpha, beta and gamma backbone angle arrangements within four nucleic acid categories (A-RNA and A-DNA, free and bound B-DNA) and explored the relationships between backbone angles, sugar puckers and selected helical parameters. The trends in the correlations are found to be similar regardless of the nucleic acid category. It is interesting that specific structural effects exhibited by the different unusual backbone sub-states are in some cases contravariant. Certain alpha/gamma changes are accompanied by C3' endo (north) sugars, small twist angles and positive values of base pair roll, and favor a displacement of nucleotide bases towards the minor groove compared to that of canonical B form structures. Unusual epsilon/zeta combinations occur with C2' (south) sugars, high twist angles, negative values of base pair roll, and base displacements towards the major groove. Furthermore, any unusual backbone correlates with a reduced dispersion of equilibrium structural parameters of the whole double helix, as evidenced by the reduced standard deviations of almost all conformational parameters. Finally, a strong sequence effect is displayed in the free oligomers, but reduced somewhat in the ligand bound forms. The most variable steps are GpA and CpA, and, to a lesser extent, their partners TpC and TpG. The results provide a basis for considering if the variable and non-variable steps within a biological active sequence precisely determine morphological structural features as the curvature direction, the groove depth, and the accessibility of base pair for non covalent associations.     

Groove Width and Depth of B-DNA Structures Depend on Local Variation in Slide

Abstract

The groove widths of DNA helix, especially minor groove width, are generally believed to be important for recognition of DNA by various types of ligands. It has been postulated earlier that large negative propeller twist, in the AT rich regions compresses the minor groove of duplex DNA A systematic study has now been carried out by generating models with different values of local doublet and intra-basepair parameters and calculating their minor groove widths. It is found that several local doublet parameters affect the minor groove width but it depends most strongly on the local step parameters roll and slide when each parameter is considered individually. However, a detailed analysis of the various local parameters within the B-DNA family of crystal structures indicates that propeller twist and slide are most strongly correlated with the observed values of minor groove width. The groove depth is also strongly correlated with slide. Thus the local base sequence dependent variations in slide can modify both the groove width and depth and consequently determine the ligand binding properties of DNA.     

A molecular dynamics simulation of the (dG)6 . (dC)6 minihelix including counterions and water

The results of a 60 ps molecular dynamics (MD) simulation of (dG)6.(dC)6 including 10 Na+ counterions and 292 water molecules are presented. All backbone angles and helix parameters for the hexamer are reported in this paper along with trajectory plots of selected angles. Hydrogen bonding between the bases along the helical axis was observed to fluctuate with time, showing the dynamic nature of the base-pairing interaction. These fluctuations gave rise to unusual hydrogen-bonding patterns. Good intrastrand base stacking and no interstrand base stacking were also observed. The hexamer minihelix retains an essentially B-DNA conformation throughout the entire simulation even though some helix parameters and backbone angles do not have strict B-DNA values. The most striking feature obtained from the simulation was a high propeller twist, which resulted in a narrow minor groove for the minihelix. It is proposed that (dG)n.(dC)n sequences are resistant to DNAase I because of this narrow minor groove in dilute aqueous solution.     

Proton and phosphorus NMR studies of d-CpG(pCpG)n duplexes in solution. Helix-coil transition and complex formation with actinomycin-D.

The Watson–Crick imino and amino exchangeable protons, the nonexchangeable base and sugar protons, and the backbone phosphates for d-CpG(pCpG)_n, n = 1 and 2, have been monitored by high-resolution nmr spectroscopy in aqueous solution over the temperature range 0°–90°C. The temperature dependence of the chemical shifts of the tetramer and hexamer resonances is consistent with the formation of stable duplexes at low temperature in solution. Comparison of the spectral characteristics of the tetranucleotide with those of the hexanucleotide with temperature permits the differentiation and assignment of the cytosine proton resonances on base pairs located at the end of the helix from those in an interior position. There is fraying at the terminal base pairs in the tetranucleotide and hexanucleotide duplexes. The Watson–Crick ring imino protons exchange at a faster rate than the Watson–Crick side-chain amino protons, with exchange occurring by transient opening of the double helix. The structure of the d-CpG(pCpG)_n double helices has been probed by proton relaxation time measurements, sugar proton coupling constants, and the proton chemical shift changes associated with the helix–coil transition. The experimental data support a structural model in solution, which incorporates an anti conformation about the glycosyl bonds, C(3) exo sugar ring pucker, and base overlap geometries similar to the B-DNA helix. Rotational correlation times of 1.7 and 0.9 × 10^?9 sec have been computed for the hexanucleotide and tetranucleotide duplexes in 0.1 M salt, D₂O, pH 6.25 at 27°C. The well-resolved ³¹P resonances for the internucleotide phosphates of the tetramer and hexamer sequences at superconducting fields shift upfield by 0.2–0.5 ppm on helix formation. These shifts reflect a conformational change about the ω,ω′ phosphodiester bonds from gauche-gauche in the duplex structure to a distribution of gauche-trans states in the coil structure. Significant differences are observed in the transition width and midpoint of the chemical shift versus temperature profiles plotted in differentiated form for the various base and sugar proton and internucleotide phosphorous resonances monitoring the d-CpG(pCpG)_n helix–coil transition. The twofold symmetry of the d-CpGpCpG duplex is removed on complex formation with the antibiotic actinomycin-D. Two phosphorous resonances are shifted downfield by ～2.6 ppm and ～1.6 ppm on formation of the 1:2 Act-D:d-CpGpCpG complex in solution. Model studies on binding of the antibiotic to dinucleotides of varying sequence indicate that intercalation of the actinomycin-D occurs at the GpC site in the d-CpGpCpG duplex and that the magnitude of the downfield shifts reflects strain at the O-P-O backbone angles and hydrogen bonding between the phenoxazone and the phosphate oxygens. Actinomycin-D is known to bind to nucleic acids that exhibit a B-DNA conformation; this suggests that the d-CpG(pCpG)_n duplexes exhibit a B-DNA conformation in solution.     

Interactions of molecules with nucleic acids. I. An algorithm to generate nucleic acid structures with an application to the B-DNA structure and a counterclockwise helix.

An algorithm is developed that enables the routine determination of backbone conformations of nucleic acids. All atomic positions including hydrogen are specified in accord with experimental bond lengths and angles but with theoretically determined conformational angles. For two Watson-Crick base pairs at a separation of 3.38 Å, and perpendicular to a common helical axis, minimum energy configurations are found for all 10 combinations at helical angles of α ～ 36°–38°, corresponding to the B-DNA structure with C(2′)-endo sugar puckers. Backbone configurations exist only within the range 35.5° ? α ? 42°, which suggests the origin of the 10-fold helix. Calculated stacking energies for the B-DNA structure increases for each of the clustered groups of base pairs: G·C with G·C, G·C with A·T, and A·T with A·T, and they are in approximate agreement with experimental observations. The counter-clockwise helix is examined, and physically meaningful structures are found only when the helical axes of successive base pairs are disjointed.