DNA flexibility as a function of allomorphic conformation and of base sequence.

Systematic theoretical modeling of symmetric DNA oligomers, carried out earlier for the B conformation, is now extended to A-DNA. In contrast to the previous results, it is found that A-DNA shows no multiplicity of low-energy substate conformations. The possibilities of the Jumna algorithm are subsequently applied to studying deformations of the oligomers. Controlled winding and stretching deformations are used to study how the two allomorphs and different base sequences absorb such external stress. The results help explain the internal mechanics of the DNA double helix and the extent to which fine structure influences this behavior. The results point to some differences between the A and B double helices, but also to many similarities. Sequence effects on flexibility are relatively limited compared to their impact on optimal energy conformations. It is also shown that the conformational substates detected for B-DNA oligomers are preserved under deformation, but have little influence on its energetics.     

DNA structure and polymerase fidelity.

The accuracy of DNA replication results from both the intrinsic DNA polymerase fidelity and the DNA sequence. Although the recent structural studies on polymerases have brought new insights on polymerase fidelity, the role of DNA sequence and structure is less well understood. Here, the analysis of the crystal structures of hotspots for polymerase slippage including (CA)n and (A)n tracts in different intermolecular contexts reveals that, in the B-form, these sequences share common structural alterations which may explain the high rate of replication errors. In particular, a two-faced "Janus-like" structure with shifted base-pairs in the major groove but an apparent normal geometry in the minor groove constitutes a molecular decoy specifically suitable to mislead the polymerases. A model of the rat polymerase beta bound to this structure suggests that an altered conformation of the nascent template-primer duplex can interfere with correct nucleotide incorporation by affecting the geometry of the active site and breaking the rules of base-pairing, while at the same time escaping enzymatic mechanisms of error discrimination which scan for the correct geometry of the minor groove.In contrast, by showing that the A-form greatly attenuates the sequence-dependent structural alterations in hotspots, this study suggests that the A-conformation of the nascent template-primer duplex at the vicinity of the polymerase active site will contribute to fidelity. The A-form may play the role of a structural buffer which preserves the correct geometry of the active site for all sequences. The detailed comparison of the conformation of the nascent template-primer duplex in the available crystal structures of DNA polymerase-DNA complexes shows that polymerase beta, the least accurate enzyme, is unique in binding to a B-DNA duplex even close to its active site. This model leads to several predictions which are discussed in the light of published experimental data.     

A new D-DNA form of poly(dA-dT).poly(dA-dT): an A-DNA type structure with reversed Hoogsteen pairing

The D-DNA double helix model of poly(dA-dT).poly(dA-dT) proposed in the literature is not in accordance with some notable experimental facts and physicochemical conditions to which it is related. Thus, the fibre X-ray diffraction pattern of D-DNA obtained at a relative humidity lower than that giving the A-DNA form is singularly not taken into account when one assumes that there is only one D structure of B-DNA type. We rather suggest that there are actually two different forms of D-DNA, namely D(A) which partakes in the D-A-B transitions and D(B) associated with the D-B change of conformation. Although these two DNA structures have the same helical parameters (pitch and number of residues per turn), in agreement with X-ray data, their detailed conformations are considerably different. Whereas D(B) is indeed the structure generally defined as D-DNA, a critical analysis based on a comparison between different possible DNA double helices leads us to propose dihedral angles, a set of atomic coordinates and a stereo view of another new form of D-DNA, the D(A) structural model. It is a right-handed double helix with a dinucleotide as the repeat unit. The furanose rings are of the A-DNA type (C3' endo) and the bases are hydrogen bonded according to the reversed Hoogsteen pairing. Such a disposition renders the D(A) model unsuitable for poly(dI-dC).poly(dI-dC), the other alternating polynucleotide observed in the D(B) structure. The consistency of these two different D-DNA structures of poly(dA-dT).poly(dA-dT) with the general aspects of hydration and helix-helix transitions of DNA, as well as with the conformational variability of AT base sequences, is discussed.     

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.     

Sodium and chlorine ions as part of the DNA solvation shell.

The distribution of sodium and chlorine ions around DNA is presented from two molecular dynamics simulations of the DNA fragment d(C(5)T(5)). (A(5)G(5)) in explicit solvent with 0.8 M additional NaCl salt. One simulation was carried out for 10 ns with the CHARMM force field that keeps the DNA structure close to A-DNA, the other for 12 ns with the AMBER force field that preferentially stabilizes B-DNA conformations (, Biophys. J. 75:134-149). From radial distributions of sodium and chlorine ions a primary ion shell is defined. The ion counts and residence times of ions within this shell are compared between conformations and with experiment. Ordered sodium ion sites were found in minor and major grooves around both A and B-DNA conformations. Changes in the surrounding hydration structure are analyzed and implications for the stabilization of A-DNA and B-DNA conformations are discussed.     

Crystal structures of A-DNA duplexes

All crystal structures of A-DNA duplexes exhibit a typical crystal packing, with the termini of one molecule abutting the shallow grooves of symmetry related neighbors, while all other forms (B, Z, and RNA) tend to form infinitely stacked helices. The A-DNA arrangement leads to the formation of shallow groove base multiples that have implications for the structure of DNA in compacted states. The characteristic packing leaves big solvent channels, which can be sometimes occupied by B-DNA duplexes. Comparisons of the structures of the same oligomer crystallizing in two different space groups and of different sequences crystallizing in the same space group show that the lattice forces dominate the A-DNA conformation in the crystals, complicating the effort to elucidate the influence of the base sequence on the structures. Nevertheless, in both alternating and nonalternating fragments some sequence effects can still be uncovered. Furthermore, several studies have started to define the minimal sequence changes or chemical modifications that can interconvert the oligomers between different double-helical conformers (A-, B-, and Z-form). Overall, it is seen that the rigid nucleotide principle applies to the oligomeric fragments. Besides the structures of the naked DNAs, their interactions with water, polyamines, and metal ions have attracted considerable attention. There are conserved patterns in the hydration, involving both the grooves and the backbone, which are different from those of B-DNA or Z-DNA. Overall, A-DNA seems to be more economically hydrated than B-DNA, particularly around the sugar-phosphate backbone. Spermine was found to be able to bind exclusively to either of the grooves or to the phosphate groups of the backbone, or exhibit a mixed binding mode. The located metal cations prefer binding to guanine bases and phosphate groups. The only mispairs investigated in A-DNA are the wobble pairs, yielding structural insight into their effects on helix stabilities and hydration. G · T wobble pairs have been determined in various sequence contexts, where they differentially affect the conformations and stableness of the duplexes. The structure of a G · ^m5C base pair, which surprisingly also adopted the wobble conformation, suggests that a similar geometry may transiently exist for G · C pairs. These results from the crystalline state will be compared to the solution state and discussed in relation to their relevance in biology. © 1997 John Wiley & Sons, Inc. Biopoly 44: 45–63, 1997     

Influence of counter-ions on the crystal structures of DNA decamers: binding of [Co(NH3)6]3+ and Ba2+ to A-DNA.

A-DNA is a stable alternative right-handed double helix that is favored by certain sequences (e.g., (dG)n.(dC)n) or under low humidity conditions. Earlier A-DNA structures of several DNA oligonucleotides and RNA.DNA chimeras have revealed some conformational variation that may be the result of sequence-dependent effects or crystal packing forces. In this study, four crystal structures of three decamer oligonucleotides, d(ACCGGCCGGT), d(ACCCGCGGGT), and r(GC)d(GTATACGC) in two crystal forms (either the P6(1)22 or the P2(1)2(1)2(1) space group) have been analyzed at high resolution to provide the molecular basis of the structural difference in an experimentally consistent manner. The study reveals that molecules crystallized in the same space group have a more similar A-DNA conformation, whereas the same molecule crystallized in different space groups has different (local) conformations. This suggests that even though the local structure is influenced by the crystal packing environments, the DNA molecule adjusts to adopt an overall conformation close to canonical A-DNA. For example, the six independent CpG steps in these four structures have different base-base stacking patterns, with their helical twist angles (omega) ranging from 28 degrees to 37 degrees. Our study further reveals the structural impact of different counter-ions on the A-DNA conformers. [Co(NH3)6]3+ has three unique A-DNA binding modes. One binds at the major groove side of a GpG step at the O6/N7 sites of guanine bases via hydrogen bonds. The other two modes involve the binding of ions to phosphates, either bridging across the narrow major groove or binding between two intra-strand adjacent phosphates. Those interactions may explain the recent spectroscopic and NMR observations that [Co(NH3)6]3+ is effective in inducing the B- to A-DNA transition for DNA with (G)n sequence. Interestingly, Ba2+ binds to the same O6/N7 sites on guanine by direct coordinations.     

X-ray crystallographic analysis of the hydration of A- and B-form DNA at atomic resolution

We have determined single crystal structures of an A-DNA decamer and a B-DNA dodecamer at 0.83 and 0.95 A, respectively. The resolution of the former is the highest reported thus far for any right-handed nucleic acid duplex and the quality of the diffraction data allowed determination of the structure with direct methods. The structures reveal unprecedented details of DNA fine structure and hydration; in particular, we have reexamined the overall hydration of A- and B-form DNA, the distribution of water around phosphate groups, and features of the water structure that may underlie the B to A transition.     

Comparison of the solution and crystal conformations of (G + C)-rich fragments of DNA.

DNA fragments crystallize in an unpredictable manner, and relationships between their crystal and solution conformations still are not known. We have studied, using circular dichroism spectroscopy, solution conformations of (G + C)-rich DNA fragments, the crystal structures of which were solved in the laboratory of one of the present authors. In aqueous trifluorethanol (TFE) solutions, all of the examined oligonucleotides adopted the same type of double helix as in the crystal. Specifically, the dodecamer d(CCCCCGCGGGGG) crystalized as A-DNA and isomerized into A-DNA at high TFE concentrations. On the other hand, the hexamer d(CCGCGG) crystallized in Z-form containing tilted base pairs, and high TFE concentrations cooperatively transformed it into the same Z-form as adopted by the RNA hexamer r(CGCGCG), although d(CCGCGG) could isomerize into Z-DNA in the NaCl + NiCl2) aqueous solution. The fragments crystallizing as B-DNA remained B-DNA, regardless of the solution conditions, unless they denatured or aggregated. Effects on the oligonucleotide conformation of 2-methyl-2,4-pentanediol and other crystallization agents were also studied. 2-Methyl-2,4-pentanediol induced the same conformational transitions as TFE but, in addition, caused an oligonucleotide condensation that was also promoted by the other crystallization agents. The present results indicate that the crystal double helices of DNA are stable in aqueous TFE rather than aqueous solution.     

Neomycin, spermine and hexaamminecobalt (III) share common structural motifs in converting B- to A-DNA.

The (dG)n.(dC)n-containing 34mer DNA duplex [d(A2G15C15T2)]2 can be effectively converted from the B-DNA to the A-DNA conformation by neomycin, spermine and Co(NH3)6(3+). Conversion is demonstrated by a characteristic red shift in the circular dichroism spectra and dramatic NMR spectral changes in chemical shifts. Additional support comes from the substantially stronger CH6/GH8-H3'NOE intensities of the ligand-DNA complexes than those from the native DNA duplex. Such changes are consistent with a deoxyribose pucker transition from the predominate C2'-endo (S-type) to the C3'-endo (N-type). The changes for all three ligand-DNA complexes are identical, suggesting that those three complex cations share common structural motifs for the B- to A-DNA conversion. The A-DNA structure of the 4:1 complex of Co(NH3)6(3+)/d(ACCCGCGGGT) has been analyzed by NOE-restrained refinement. The structural basis of the transition may be related to the closeness of the two negatively charged sugar-phosphate backbones along the major groove in A-DNA, which can be effectively neutralized by the multivalent positively charged amine functions of these ligands. In addition, ligands like spermine or Co(NH3)6(3+) can adhere to guanine bases in the deep major groove of the double helix, as is evident from the significant direct NOE cross-peaks from the protons of Co(NH3)6(3+) to GH8, GH1 (imino) and CH4 (amino) protons. Our results point to future directions in preparing more potent derivatives of Co(NH3)6(3+) for RNA binding or the induction of A-DNA.     

Crystallographic studies of metal ion-DNA interactions: different binding modes of cobalt(II), copper(II) and barium(II) to N7 of guanines in Z-DNA and a drug-DNA complex.

Metal ion coordination to nucleic acids is not only required for charge neutralization, it is also essential for the biological function of nucleic acids. The structural impact of different metal ion coordinations of DNA helices is an open question. We carried out X-ray diffraction analyses of the interactions of the two transition metal ions Co(II) and Cu(II) and an alkaline earth metal ion Ba(II), with DNA of different conformations. In crystals, Co(II) ion binds exclusively at the N7 position of guanine bases by direct coordination. The coordination geometry around Co(II) is octahedral, although some sites have an incomplete hydration shell. The averaged Co-N7 bond distance is 2.3 A. The averaged Co-N7-C8 angle is 121 degrees, significantly smaller than the value of 128 degrees if the Co-N7 vector were to bisect the C5-N7-C8 bond angle. Model building of Co(II) binding to guanine N7 in B-DNA indicates that the coordinated waters in the axial positions would have a van der Waals clash with the neighboring base on the 5' side. In contrast, the major groove of A-DNA does not have enough room to accommodate the entire hydration shell. This suggests that Co(II) binding to either B-DNA or A-DNA may induce significant conformational changes. The Z-DNA structure of Cu(II)-soaked CGCGTG crystal revealed that the Cu(II) ion is bis-coordinated to N7 position of G10 and #G12 (# denotes a symmetry-related position) bases with a trigonal bipyramid geometry, suggesting a possible N7-Cu-N7 crosslinking mechanism. A similar bis-coordination to two guanines has also been seen in the interaction of Cu(II) in m5CGUAm5CG Z-DNA crystal and of Ba(II) with two other Z-DNA crystals.     

Parallel nucleic acid helices with hoogsteen base pairing: Symmetry and structure

Molecular structures for parallel DNA and RNA double helices with Hoogsteen pairing are proposed for the first time. The DNA helices have sugars in the C2′-endo region and the phosphodiester conformations are (trans, gauche^?), and the RNA helices have sugars in the C3′-endo region and the phosphodiester conformations are (gauche^?, gauche^?). A pseudorotational symmetry relates the two parallel strands of DNA helices and a screw symmetry relates the two strands of RNA helices, which have an associated tilt of the The conformational space of parallel helices with Hoogsteen base pairing, unlike the Watson-Crick duplex, is highly restricted due to the unique positioning of the symmetry axis in the former case. The features of the parallel double helix with Hoogsteen pairing are compared with the Watson-Crick duplex and the corresponding triple helix. © 1994 John Wiley & Sons, Inc.     

Triple helical DNA in a duplex context and base pair opening

It is fundamental to explore in atomic detail the behavior of DNA triple helices as a means to understand the role they might play in vivo and to better engineer their use in genetic technologies, such as antigene therapy. To this aim we have performed atomistic simulations of a purine-rich antiparallel triple helix stretch of 10 base triplets flanked by canonical Watson–Crick double helices. At the same time we have explored the thermodynamic behavior of a flipping Watson–Crick base pair in the context of the triple and double helix. The third strand can be accommodated in a B-like duplex conformation. Upon binding, the double helix changes shape, and becomes more rigid. The triple-helical region increases its major groove width mainly by oversliding in the negative direction. The resulting conformations are somewhere between the A and B conformations with base pairs remaining almost perpendicular to the helical axis. The neighboring duplex regions maintain a B DNA conformation. Base pair opening in the duplex regions is more probable than in the triplex and binding of the Hoogsteen strand does not influence base pair breathing in the neighboring duplex region.     

The structure and hydration of the A-DNA fragment d(GGGTACCC) at room temperature and low temperature.

The DNA fragment d(GGGTACCC) was crystallized as an A-DNA duplex in the hexagonal space group P6(1). The structure was analyzed at room temperature and low temperature (100K) at a resolution of 2.5 A. The helical conformations at the two temperatures are similar but the low-temperature structure is more economically hydrated than the room-temperature one. The structure of d(GGGTACCC) is compared to those of d(GGGTGCCC) and d(GGGCGCCC). This series of molecules, which consists of a mismatched duplex and its two Watson-Crick analogues, exhibits three conformational variants of the A-form of DNA, which are correlated with the specific intermolecular interactions observed in the various crystals. The largest differences in local conformation are displayed by the stacking geometries of the central pyrimidine-purine and the flanking purine-pyrimidine sites in each of the three duplexes. Stacking energy calculations performed on the crystal structures show that the mismatched duplex is destabilized with respect to each of the error-free duplexes, in accordance with helix-coil transition measurements.     

Spatial translational motions of base pairs in DNA molecules: Application of the extended matrix generator method

We have used the elementary generator matrices outlined in the preceding paper to examine the conformational plasticity of the nucleic acid double helix. Here we investigate kinked DNA structures made up of alternating B- and A-type helices and intrinsically curved duplexes perturbed by the intercalation of ligands. We model the B-to-A transition by the lateral translation of adjacent base pairs, and the intercalation of ligands by the vertical displacement of neighboring residues. We report a complete set of average configuration-dependent parameters, ranging from scalars (i.e., persistence lengths) to first- and second-order tensor parameters (i.e., average second moments of inertia), as well as approximations of the associated spatial distributions of the DNA and their angular correlations. The average structures of short chains (of lengths less than 100 base pairs) with local kinks or intrinsically curved sequences are essentially rigid rods. At the smallest chain lengths (10 base pairs), the kinked and curved chains exhibit similar average properties, although they are structurally perturbed compared to the standard B-DNA duplex. In contrast, at lengths of 200 base pairs, the curved and kinked chains are more compact on average and are located in a different space from the standard B- or A-DNA helix. While A-DNA is shorter and thicker than B-DNA in x-ray models, the long flexible A-DNA helix is thinner and more extended on average than its B-DNA counterpart because of more limited fluctuations in local structure. Curved polymers of 50 base pairs or longer also show significantly greater asymmetry than other DNAs (in terms of the distribution of base pairs with respect to the center of gravity of the chain). The intercalation of drugs in the curved DNA straightens and extends the smoothly deformed template. The dimensions of the average ellipsoidal boundaries defining the configurations of the intercalated polymers are roughly double those of the intrinsically curved chain. The altered proportions and orientations of these density functions reflect the changing shape and flexibility of the double helix. The calculations shed new light on the possible structural role of short A-DNA fragments in long B-type duplexes and also offer a model for understanding how GC-specific intercalative ligands can straighten naturally curved DNA. The mechanism is not immediately obvious from current models of DNA curvature, which attribute the bending of the chain to a perturbed structure in repeating tracts of A · T base pairs. © 1994 John Wiley & Sons, Inc.     

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.     

Hydration and recognition of methylated CpG steps in DNA.

The analysis of the hydration pattern around methylated CpG steps in three high resolution (1.7, 2.15 and 2.2 A) crystal structures of A-DNA decamers reveals that the methyl groups of cytosine residues are well hydrated. In comparing the native structure with two structurally distinct forms of the decamer d(CCGCCGGCGG) fully methylated at its CpG steps, this study shows also that in certain structural and sequence contexts, the methylated cytosine base can be more hydrated that the unmodified one. These water molecules seem to be stabilized in front of the methyl group through the formation C-H...O interactions. In addition, these structures provide the first observation of magnesium cations bound to the major groove of A-DNA and reveal two distinct modes of metal binding in methylated and native duplexes. These findings suggest that methylated cytosine bases could be recognized by protein or DNA polar residues through their tightly bound water molecules.     

Molecular dynamics of a DNA Holliday junction: the inverted repeat sequence d(CCGGTACCGG)₄

All-atom molecular dynamics (MD) computer simulations have been applied successfully to duplex DNA structures in solution for some years and found to give close accord with observed results. However, the MD force fields have generally not been parameterized against unusual DNA structures, and their use to obtain dynamical models for this class of systems needs to be independently validated. The four-way junction (4WJ), or Holliday junction, is a dynamic DNA structure involved in central cellular processes of homologous replication and double strand break repair. Two conformations are observed in solution: a planar open-X form (OPN) with a mobile center and four duplex arms, and an immobile stacked-X (STX) form with two continuous strands and two crossover strands, stabilized by high salt conditions. To characterize the accuracy of MD modeling on 4WJ, we report a set of explicit solvent MD simulations of ～100 ns on the repeat sequence d(CCGGTACCGG)₄ starting from the STX structure (PDB code 1dcw), and an OPN structure built for the same sequence. All 4WJ MD simulations converged to a stable STX structure in close accord with the crystal structure. Our MD beginning in the OPN form converts to the STX form spontaneously at both high and low salt conditions, providing a model for the conformational transition. Thus, these simulations provide a successful account of the dynamical structure of the STX form of d(CCGGTACCGG)₄ in solution, and provide new, to our knowledge, information on the conformational stability of the junction and distribution of counterions in the junction interior.