Influence of sequence on the conformation of the B-DNA helix.

We have tried to ascertain whether the variability found in the conformational features of the 10 base steps in B-DNA is mainly due to the flanking sequences or to interactions with the environment. From an analysis of the twist parameter of the base-pair steps available from crystals of oligonucleotides and protein/oligonucleotide complexes, we conclude that in most cases the flanking sequences show little influence: the conformation of a DNA region results from the combination of the independent intrinsic features of each base step (average conformation and intrinsic variability), modulated by their interactions with the environment. Only in some cases (YR steps, in particular CG and CA/TG) does it appear that flanking sequences have an influence on the conformation of the central base step. The values obtained allow an approximation to the parameters expected for repetitive DNA sequences. In particular, it is found that poly[d(AG/CT)] should have a strongly alternating conformation, in agreement with recently reported oligonucleotide structures.     

Crystal structure analysis of the B-DNA dodecamer CGTGAATTCACG.

The crystal structure of the DNA dodecamer C-G-T-G-A-A-T-T-C-A-C-G has been determined at a resolution of 2.5 A, with a final R factor of 15.8% for 1475 nonzero reflections measured at 0 degrees C. The structure is isomorphous with that of the Drew dodecamer, with the space group P2(1)2(1)2(1) and cell dimensions of a = 24.94 A, b = 40.78 A, and c = 66.13 A. The asymmetric unit contains all 12 base pairs of the B-DNA double helix and 36 water molecules. The structure of C-G-T-G-A-A-T-T-C-A-C-G is very similar to that of C-G-C-G-A-A-T-T-C-G-C-G, with no major alterations in helix parameters. Water peaks in the refined structure appear to represent a selection of peaks that were observed in the Drew dodecamer. The minor-groove spine of hydration at 2.5 A is fragmentary, but as Narendra et al. (1991) [Biochemistry (following paper in this issue)] have observed, lowering the temperature leads to a more complete representation of the spine.     

Structural features of B-DNA dodecamer crystal structures: influence of crystal packing versus base sequence

We have analyzed the set of inter and intra base pair parameters for each dinucleotide step in single crystal structures of dodecamers, solved at high and medium resolution and all crystallized in P2(1)2(1)2(1) space group. The objective was to identify whether all the structures which have either the Drew-Dickerson (DD) sequence d[CGCGAATTCGCG] with some base modification or related sequence (non-DD), would display the same sequence dependent structural variability about its palindromic sequence, despite the molecule being bent at one end because of similar crystal lattice packing effect. Most of the local doublet parameters for base pairs steps G2-C3 and G10-C11 positions, symmetrically situated about the lateral two-fold, were significantly correlated between themselves. In non-DD sequences, significant correlations between these positional parameters were absent. The different range of local step parameter values at each sequence position contributed to the gross feature of smooth helix axis bending in all structures. The base pair parameters in some of the positions, for medium resolution DD sequence, were quite unlike the high-resolution set and encompassed a higher range of values. Twist and slide are the two main parameters that show wider conformational range for the middle region of non-DD sequence structures in comparison to DD sequence structures. On the contrary, the minor and major groove features bear good resemblance between DD and non-DD sequence crystal structure datasets. The sugar-phosphate backbone torsion angles are similar in all structures, in sharp contrast to base pair parameter variation for high and low resolution DD and non-DD sequence structures, consisting of unusual (epsilon = g-, xi = t) BII conformation at the 10th position of the dodecamer sequence. Thus examining DD and non-DD sequence structures packed in the same crystal lattice arrangement, we infer that inter and intra base pair parameters are as symmetrically equivalent in its value as the symmetry related step for the palindromic DD sequence about lateral two-fold axis. This feature would lead us to agree with the conclusion that DNA conformation is not substantially affected by end-to-end or lateral inter-molecular interaction due to crystal lattice packing effect. Non-DD sequence structures acquire step parameter values which reflect the altered sequence at each of the dodecamer sequence position in the orthorhombic lattice while showing similar gross features of DD sequence structures.     

Ordered water structure around a B-DNA dodecamer: A quantitative study

The crystal structure of the double-helical B-DNA dodecamer of sequence C-G-C-G-A-A-T-T-C-G-C-G has been solved and refined independently in three forms: (1) the parent sequence at room temperature; (2) the same sequence at 16 K; and (3) the 9-bromo variant C-G-C-G-A-A-T-T^BrC-G-C-G at 7 °C in 60% (v/v) 2-methyl-2.4-pentanediol. The latter two structures show extensive hydration along the phosphate backbone, a feature that was invisible in the native structure because of high temperature factors (indicating thermal or static disorder) of the backbone atoms. Sixty-five solvent peaks are associated with the phosphate backbone, or an average of three per phosphate group. Nineteen other molecules form a first shell of hydration to base edge N and O atoms within the major groove, and 36 more are found in upper hydration layers. The latter tend to occur in strings or clusters spanning the major groove from one phosphate group to another. A single spermine molecule also spans the major groove. In the minor groove, the zig-zag spine of hydration that we believe to be principally responsible for stabilizing the B form of DNA is found in all three structures. Upper level hydration in the minor groove is relatively sparse, and consists mainly of strings of water molecules extending across the groove, with few contacts to the spine below. Sugar O-1′ atoms are closely associated with water molecules, but these are chiefly molecules in the spine, so the association may reflect the geometry of the minor groove rather than any intrinsic attraction of O-1′ atoms for hydration. The phosphate O-3′ and O-5′ atoms within the backbone chain are least hydrated of all, although no physical or steric impediment seems to exist that would deny access to these oxygen atoms by water molecules.     

Base sequence, local helix structure, and macroscopic curvature of A-DNA and B-DNA

Given a specified DNA sequence and starting with an idealized conformation for the double helix (A-DNA or B-DNA), the dependence of conformational energy on variations in the local geometry of the double helix can be examined by computer modeling. By averaging over all thermally accessible states, it is possible to determine 1) how the optimum local structure differs from the initial idealized conformation and 2) the energetic costs of small structural deformations. This paper describes such a study. Tables are presented for the prediction of helix twist angles and base pair roll angles for both A-DNA and B-DNA when the sequence has been specified. Local deviations of helix parameters from their average values can accumulate to produce a net curvature of the molecule, a curvature that can be sharp enough to be experimentally detectable. As an independent check on the method, the calculations provide predictions for the longitudinal compressibility (Young's modulus) and the average torsional stiffness, both of which are in good agreement with experimental values. In examining the role of sequence-dependent variations in helix structure for the recognition of specific sequences by proteins, we have calculated the energy needed to deform the self-complementary hexanucleotide d(CAATTG) to match the local geometry of d(GAATTC), which is the sequence recognized by the EcoRI restriction endonuclease. That energy would be sufficient to reduce the binding of the incorrect sequence to the protein by over 2 orders of magnitude relative to the correct sequence.     

Influence of base sequence on the stability of the double helix of DNA.

On the basis of published measurements of the melting transitions of synthetic polydeoxyribonucleotides with known sequences we have determined the parameters of the interplane (stacking) interactions of base pairs in DNA over the range of ionic strengths from 0.01 to 0.1 M Na+. We found that deviations of the stacking-interaction energy from the mean value of 7-8 kcal/mole were extremely small and did not exceed 0.2 kcal/mole. We report an analysis of the influence of the heterogeneity of the stacking interactions on the melting parameters of polynucleotides with random sequences (models of natural DNA's). Inclusion of this effect does not significantly distort the linear dependence of the melting temperature on the relative content of G-C pairs and insignificantly affects the width of the helix-coil transition in DNA under normal conditions. However it is the heterogeneity of the stacking interactions that plays the crucial role in the melting of DNA under conditions where the difference between the relative stabilities of the A-T and G-C pairs tends to zero, as in concentrated solutions of tetraethylammonium and tetramethylammonium salts.     

Dependence of the Raman signature of genomic B-DNA on nucleotide base sequence.

The vibrational spectra of four genomic and two synthetic DNAs, encompassing a wide range in base composition [poly(dA-dT). poly(dA-dT), 0% G + C; Clostridium perfringens DNA, 27% G + C; calf thymus DNA, 42% G + C; Escherichia coli DNA, 50% G + C; Micrococcus luteus DNA, 72% G + C; poly(dG-dC).poly(dG-dC), 100% G + C] (dA: deoxyadenosine; dG: deoxyguanosine; dC: deoxycytidine; dT: thymidine), have been analyzed using Raman difference methods of high sensitivity. The results show that the Raman signature of B DNA depends in detail upon both genomic base composition and sequence. Raman bands assigned to vibrational modes of the deoxyribose-phosphate backbone are among the most sensitive to base sequence, indicating that within the B family of conformations major differences occur in the backbone geometry of AT- and GC-rich domains. Raman bands assigned to in-plane vibrations of the purine and pyrimidine bases-particularly of A and T-exhibit large deviations from the patterns expected for random base distributions, establishing that Raman hypochromic effects in genomic DNA are also highly sequence dependent. The present study provides a basis for future use of Raman spectroscopy to analyze sequence-specific DNA-ligand interactions. The demonstration of sequence dependency in the Raman spectrum of genomic B DNA also implies the capability to distinguish genomic DNAs by means of their characteristic Raman signatures.     

Crystal structure of a B-DNA dodecamer containing inosine, d(CGCIAATTCGCG), at 2.4 A resolution and its comparison with other B-DNA dodecamers.

The crystal structure of the dodecamer, d(CGCIAATTCGCG), has been determined at 2.4 A resolution by molecular replacement, and refined to an R-factor of 0.174. The structure is isomorphous with that of the B-DNA dodecamer, d(CGCGAATTCGCG), in space group P2(1)2(1)2(1) with cell dimensions of a = 24.9, b = 40.4, and c = 66.4 A. The initial difference Fourier maps clearly indicated the presence of inosine instead of guanine. The structure was refined with 44 water molecules, and compared to the parent dodecamer. Overall the two structures are very similar, and the I:C forms Watson-Crick base pairs with similar hydrogen bond geometry to the G:C base pairs. The propeller twist angle is low for I4:C21 and relatively high for the I16:C9 base pair (-3.2 degrees compared to -23.0 degrees), and the buckle angles alter, probably due to differences in the contacts with symmetry related molecules in the crystal lattice. The central base pairs of d(CGCIAATTCGCG) show the large propeller twist angles, and the narrow minor groove that characterize A-tract DNA, although I:C base pairs cannot form the major groove bifurcated hydrogen bonds that are possible for A:T base pairs.     

Structure of hydrated Na+ ions around a region of A- or B-DNA helix

A molecular-dynamics simulation was used to carry out an introductory study of the hydration of a section of a rigid single A- or B-DNA helix with one Na⁺ counterion per nucleotide. Four Na⁺ ions and four nucleotides and periodic boundary conditions were used to mimic an infinite helix. The atoms of the helix and the Na⁺ ions were assumed to be Lennard-Jones spheres that also carried charges. Stillinger four-point charge model water molecules were used. We carried out five calculations, for 26 and 46 water molecules in B-DNA and 20, 32, and 46 in A-DNA fragments. The arrangements of the Na⁺ ions are found to have some similarities to those obtained by Clementi and Corongiu. In the calculations with 46 water molecules, we found that two Na⁺ ions can be bridged by about two water molecules and form a hydrated bound pair, which in turn forms a bridge between the guanine N7 and a near phosphate group. These bound pairs may be important in stabilizing the helix structure of DNA molecules.     

Solution structure of the dodecamer d-(CATGGGCC-CATG)2 is B-DNA. Experimental and molecular dynamics study

The DNA duplex d-(CATGGGCCCATG)2 has been studied in solution by FTIR, NMR and CD. The experimental approaches have been complemented by series of large-scale unrestrained molecular dynamics simulation with explicit inclusion of solvent and counterions. Typical proton-proton distances extracted from the NMR spectra and the CD spectra are completely in agreement with slightly modified B-DNA. By molecular dynamics simulation, starting from A-type sugar pucker, a spontaneous repuckering to B-type sugar pucker was observed. Both experimental and theoretical approaches suggest for the dodecamer d-(CATGGGCCCATG)2 under solution conditions puckering of all 2'-deoxyribose residues in the south conformation (mostly C2'-endo) and can exclude significant population of sugars in the north conformation (C3'-endo). NMR, FTIR and CD data are in agreement with a B-form of the dodecamer in solution. Furthermore, the duplex shows a cooperative B-A transition in solution induced by addition of trifluorethanol. This contrasts a recently published crystal structure of the same oligonucleotide found as an intermediate between B- and A-DNA where 23 out of 24 sugar residues were reported to adopt the north (N-type) conformation (C3'-endo) like in A-DNA (Ng, H. L., Kopka, M. L. and Dickerson, R. E., Proc. Natl. Acad. Sci. U S A 97, 2035-2039 (2000)). The simulated structures resemble standard B-DNA. They nevertheless show a moderate shift towards A-type stacking similar to that seen in the crystal, despite the striking difference in sugar puckers between the MD and X-ray structures. This is in agreement with preceding MD reports noticing special stacking features of G-tracts exhibiting a tendency towards the A-type stacking supported by the CD spectra also reflecting the G-tract stacking. MD simulations reveal several noticeable local conformational variations, such as redistribution of helical twist and base pair roll between the central GpC steps and the adjacent G-tract segments, as well as a substantial helical twist variability in the CpA(TpG) steps combined with a large positive base pair roll. These local variations are rather different from those seen in the crystal.     

The primary mode of binding of cisplatin to a B-DNA dodecamer: C-G-C-G-A-A-T-T-C-G-C-G

When cisplatin [cis- diamminodichloroplatinum (II)] is diffused into pre-grown crystals of the B-DNA double-helical dodecamer C-G-C-G-A-A-T-T-C-G-C-G, it binds preferentially to the N7 positions of guanines, with what probably is an aquo bridge between Pt and the adjacent O6 atom of the same guanine. The entire guanine ring moves slightly toward the platinum site, into the major groove. Only three of the eight potential cisplatin binding sites on guanines actually are occupied, and this differential reactivity can be explained in terms of the relative freedom of motion of guanines toward the major groove. This shift of guanines upon ligation may weaken the glycosyl bond and assist in the depurination that leads to mismatch SOS repair and G.C. to T.A. transversion.     

Inosine.adenine base pairs in a B-DNA duplex.

The structure of the synthetic deoxydodecamer d(C-G-C-I-A-A-T-T-A-G-C-G) has been determined by single crystal X-ray diffraction techniques at 2.5A resolution. The refinement converged with a crystallographic residual, R = 0.19 and the location of 64 solvent molecules. The sequence crystallises as a B-DNA helix with 10 Watson-Crick base-pairs (4 A.T. and 6 G.C) and 2 inosine.adenine (I.A) pairs. The present work shows that in the purine.purine base-pairs the adenine adopts syn orientation with respect to the furanose moiety while the inosine is in the trans (anti) orientation. Two hydrogen bonds link the I.A. base-pair, one between N-1(I) and N-7(A), the other between O-6(I) and N-6(A). This bulky purine.purine base-pair is incorporated in the double helix at two positions with little distortion of either local or global conformation. The pairing observed in this study is presented as a model for I.A base-pairs in RNA codon-anticodon interactions and may help explain the thermodynamic stability of inosine containing base-pairs. Conformational parameters and base stacking interactions are presented and where appropriate compared with those of the native compound, d(C-G-C-G-A-A-T-T-C-G-C-G) and with other studies of oligonucleotides containing purine.purine base-pairs.     

Structural change in a B-DNA helix with hydrostatic pressure

Study of the effects of pressure on macromolecular structure improves our understanding of the forces governing structure, provides details on the relevance of cavities and packing in structure, increases our understanding of hydration and provides a basis to understand the biology of high-pressure organisms. A study of DNA, in particular, helps us to understand how pressure can affect gene activity. Here we present the first high-resolution experimental study of B-DNA structure at high pressure, using NMR data acquired at pressures up to 200 MPa (2 kbar). The structure of DNA compresses very little, but is distorted so as to widen the minor groove, and to compress hydrogen bonds, with AT pairs compressing more than GC pairs. The minor groove changes are suggested to lead to a compression of the hydration water in the minor groove.     

A critical analysis of a left-handed double helix model for B-DNA fibers.

A search for a left-handed double helix model for B-DNA fibers has been undertaken. The model has to present good stereochemistry and also to be in agreement with X-ray and infrared data. Dihedral angles as well as atomic coordinates and calculated intensities curves are given for the best model obtained. Comparison with experimental results shows that this model must be rejected as a candidate for the representation of B-DNA fibers.     

Computer graphics program to reveal the dependence of the gross three-dimensional structure of the B-DNA double helix on primary structure.

Programs are presented to plot the gross three-dimensional structure of the DNA double helix with the base sequence as input information. The rules that determine the overall structure of the double helix are those that predict the dependence of local helix parameters (specifically, helix twist angle and relative basepair roll angle) on sequence. For this purpose, the user can select either the Calladine-Dickerson parameters or the Tung-Harvey parameters. These programs can be used as tools to investigate the variation of DNA tertiary structure with sequence, which may play an important role in the sequence-specific recognition of DNA by proteins.     

Molecular structure of the B-DNA dodecamer d(CGCAAATTTGCG)2. An examination of propeller twist and minor-groove water structure at 2.2 A resolution.

The crystal structure of the dodecanucleotide duplex d(CGCAAATTTGCG)2 has been solved to 2.2 A resolution and refined to an R-factor of 18.1% with the inclusion of 71 water molecules. The structure shows propeller twists of up to -20 degrees for the A.T base-pairs, although there is probably only one (weak) three-centre hydrogen bond in the six base-pair AT narrow minor-groove region. An extensive ribbon of hydration has been located in this groove that has features distinctive from the classic "spine of hydration". Solvation around phosphate groups is described, with several instances of water molecules bridging between phosphates.     

Simulations of the solvent structure for macromolecules. I. Solvation of B-DNA double helix at T = 300 K

Monte Carlo simulations are reported for a system of 447 water molecules enclosing a B-DNA double-helix fragment with 12 base pairs and the corresponding sugar and phosphate units. From a detailed analysis on the interaction energies and probability distributions (at a simulated temperature of 300 K), the water molecules can be partitioned into clusters strongly interacting with (1) the phosphates, (2) the sugars, (3) the sugars and the bases, and (4) the base pairs. In addition, transgroove and interphosphate filament of hydrogen-bonded water molecules have been detected. From simulations performed with variable numbers of water molecules, a theoretical isotherm has been obtained, with the characteristic sigmoidal shape, known from absorption–desorption experiments on related systems. The expected main features for the structure of water molecules solvating B-DNA with Na⁺ counterions are briefly discussed at the end of the paper.     

The structure of d(CGCGAAT[]TCGCG) . d(CGCGAATTCGCG); the incorporation of a thymine photodimer into a B-DNA helix

In the light of the biological significance of thymine photodimers , studies of the energetics of the dodecanucleotide fragment d( CGCGAATTCGCG )2 have been carried out using the methods of molecular mechanics, with and without incorporation of a thymine dimer in the cis-syn configuration. The results of the calculations suggest that the thymine dimerized structures show no gross distortion in the double helix with the conformational changes relative to the normal B-DNA double helix restricted largely to the dimer region. The energetics of dTp[]dT reveal a number of conformers which are energetically almost equally favorable and are, as a group, qualitatively consistent with NMR studies on this molecule. The biological implications of the results of the conformational studies, reported here, have been examined vis-a-vis the currently available models for the recognition of DNA "damage" by repair enzymes.