A molecular simulation picture of DNA hydration around A- and B-DNA

Recent results from molecular dynamics (MD) simulations on hydration of DNA with respect to conformation are reviewed and compared with experimental data. MD simulations of explicit solvent around DNA can now give a detailed model of DNA that not only matches well with the experimental data but provides additional insight beyond current experimental limitations. Such simulation results are analyzed with a focus on differential hydration properties between A- and B-DNA and between C/G and A/T base pairs. The extent of hydration is determined from the number of waters in the primary shell and compared to experimental numbers from different measurements. High-resolution hydration patterns around the whole DNA are shown and correlated with the conformations. The role of ions associating with DNA is discussed with respect to changes in the hydration structure correlating with DNA conformation.     

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.     

RNA sequence analysis using covariance models.

We describe a general approach to several RNA sequence analysis problems using probabilistic models that flexibly describe the secondary structure and primary sequence consensus of an RNA sequence family. We call these models 'covariance models'. A covariance model of tRNA sequences is an extremely sensitive and discriminative tool for searching for additional tRNAs and tRNA-related sequences in sequence databases. A model can be built automatically from an existing sequence alignment. We also describe an algorithm for learning a model and hence a consensus secondary structure from initially unaligned example sequences and no prior structural information. Models trained on unaligned tRNA examples correctly predict tRNA secondary structure and produce high-quality multiple alignments. The approach may be applied to any family of small RNA sequences.     

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.     

A 5-nanosecond molecular dynamics trajectory for B-DNA: analysis of structure, motions, and solvation.

We report the results of four new molecular dynamics (MD) simulations on the DNA duplex of sequence d(CGCGAATTCGCG)2, including explicit consideration of solvent water, and a sufficient number of Na+ counterions to provide electroneutrality to the system. Our simulations are configured particularly to characterize the latest MD models of DNA, and to provide a basis for examining the sensitivity of MD results to the treatment of boundary conditions, electrostatics, initial placement of solvent, and run lengths. The trajectories employ the AMBER 4.1 force field. The simulations use particle mesh Ewald summation for boundary conditions, and range in length from 500 ps to 5.0 ns. Analysis of the results is carried out by means of time series for conformationalm, helicoidal parameters, newly developed indices of DNA axis bending, and groove widths. The results support a dynamically stable model of B-DNA for d(CGCGAATTCGCG)2 over the entire length of the trajectory. The MD results are compared with corresponding crystallographic and NMR studies on the d(CGCGAATTCGCG)2 duplex, and placed in the context of observed behavior of B-DNA by comparisons with the complete crystallographic data base of B-form structures. The calculated distributions of mobile solvent molecules, both water and counterions, are displayed. The calculated solvent structure of the primary solvation shell is compared with the location of ordered solvent positions in the corresponding crystal structure. The results indicate that ordered solvent positions in crystals are roughly twice as structured as bulk water. Detailed analysis of the solvent dynamics reveals evidence of the incorporation of ions in the primary solvation of the minor groove B-form DNA. The idea of localized complexation of otherwise mobile counterions in electronegative pockets in the grooves of DNA helices introduces an additional source of sequence-dependent effects on local conformational, helicoidal, and morphological structure, and may have important implications for understanding the functional energetics and specificity of the interactions of DNA and RNA with regulatory proteins, pharmaceutical agents, and other ligands.     

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.     

Origin of DNA helical structure and its sequence dependence

Conformational analysis of DNA shows that the origin of the B-form double helix can be attributed in large part to the atomic charge pattern in the base pairs. The charge patterns favor specific helical stacking of the base pairs. Base pairs alone--without backbones--have a strong tendency to form helix, indicating that the backbones play a rather passive role in determining the basic helical structure of DNA. It is mainly the electrostatic interactions determined by the charge pattern on base pairs that stabilize a particular helical conformation. The charge pattern in the base pairs appears to be responsible for much of the sequence dependence of DNA conformation, rather than steric clashes.     

Structural prediction of A- and B-DNA duplexes based on coordinates of the phosphorus atoms.

The sequence-dependent structure of DNA double helices was studied extensively during the past 10 years. How the backbone structure correlates with the base structure in a duplex conformation is still an important yet open question. Using a set of reduced coordinates and a least-squares fitting procedure, we have developed a method to predict structures for B-DNA duplexes based on coordinates of the phosphorus atoms. This method can be used to predict all-atom structures for both bent and straight molecules. We estimated the accuracies of the predictions by studying a set of 10 oligonucleotides with their structures available from the Protein Data Bank. We used this method to construct a modeled structure for the bacteriophage lambda cro operator for which the phosphorus coordinates were known from 3.5-angstrum resolution crystal data (4CRO).     

Bayesian basecalling for DNA sequence analysis using hidden Markov models

It has been shown that electropherograms of DNA sequences can be modeled with hidden Markov models. Basecalling, the procedure that determines the sequence of bases from the given eletropherogram, can then be performed using the Viterbi algorithm. A training step is required prior to basecalling in order to estimate the HMM parameters. In this paper, we propose a Bayesian approach which employs the Markov chain Monte Carlo (MCMC) method to perform basecalling. Such an approach not only allows one to naturally encode the prior biological knowledge into the basecalling algorithm, it also exploits both the training data and the basecalling data in estimating the HMM parameters, leading to more accurate estimates. Using the recently sequenced genome of the organism Legionella pneumophila we show that the MCMC basecaller outperforms the state-of-the-art basecalling algorithm in terms of total errors while requiring much less training than other proposed statistical basecallers.     

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.     

Peptide sequence analysis using exopeptidases with molecular analysis of the truncated polypeptides by mass spectrometry

Fast atom bombardment mass spectrometry is used for the analysis of the series of molecular products formed by the cleavage of polypeptide substrates with the exopeptidases carboxypeptidase Y and leucine aminopeptidase. By following the polypeptide molecular species rather than the released residues, sequence information is obtained regardless of the relative rates of cleavage of peptide bonds. In addition, unambiguous assignments of sequence can be made in the presence of multiple identical residues. The lower level of sensitivity for the analysis is in the picomole range. When carboxypeptidase Y is used, the method provides a specific and sensitive method for the sequencing of polypeptides from the C-terminus.     

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.     

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.     

Refinement of comparative models of protein structure by using multicanonical molecular dynamics simulations

Comparative modelling is a powerful method that easily predicts a considerably accurate structure of a protein by using a template structure having a similar amino-acid sequence to the target protein. However, in the region where the amino-acid sequence is different between the target and the template, the predicted structure remains unreliable. In such a case, the model has to be refined. In the present study, we explored the possibility of a molecular dynamics-based method, using the human SAP Src Homology 2 (SH2) domain as the modelling target. The multicanonical method was used to alleviate the multiple-minima problem and the generalised Born/surface area model was used to reduce the computational cost. In addition, position restraints were imposed on the atoms in the reliable regions to avoid unnecessary conformational sampling. We analyzed the conformational distribution of the ligand-recognition loop of the domain and found that the most populated conformational clusters in the ensemble of the model agreed well with one of the two major clusters in the ensemble of the reference simulation starting from the crystal structure. This demonstrates that the current refinement method can significantly improve the accuracy of an unreliable region in a comparative model.     

The dependence of the surface electrostatic potential of B-DNA on environmental factors

The electrostatic potential of B-DNA is calculated on its surface envelope for two homopolymeric base pair sequences using models representing the effects of both counterion binding and of aqueous solution. The influence of these two factors on the resulting potentials is established and the significance of calculations which omit such effects is discussed.     

Iron affects the structure of cell membrane molecular models

The effects of Fe(3+) and Fe(2+) on molecular models of biomembranes were investigated. These consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and of dimyristoylphosphatidylethanolamine (DMPE), classes of phospholipids located in the outer and inner moieties of cell membranes, respectively. X-ray studies showed that very low concentrations of Fe(3+) affected DMPC organization and 10(-3)M induced a total loss of its multilamellar periodic stacking. Experiments carried out with Fe(2+) on DMPC showed weaker effects than those induced by Fe(3+) ions. Similar experiments were performed on DMPE bilayers. Fe(3+) from 10(-7)M up to 10(-4)M had practically no effect on DMPE structure. However, 10(-3)M Fe(3+) induced a deep perturbation of the multilamellar structure of DMPE. However, 10(-3)M Fe(2+) had no effect on DMPE organization practically. Differential scanning calorimetry measurements also revealed different effects of Fe(3+) and Fe(2+) on the phase transition and other thermal properties of the examined lipids. In conclusion, the results obtained indicate that iron ions interact with phospholipid bilayers perturbing their structures. These findings are consistent with the observation that iron ions change cell membrane fluidity and, therefore, affect its functions.