Sequence-dependent anisotropic flexibility of B-DNA. A conformational study

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 degree, 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 degree 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 degree. the backbone dihedral angles vary by no more than 15 degree. 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-dimensional 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.     

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.     

Sequence-dependent bending of DNA and phasing of nucleosomes

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 oligopyrimidine 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.     

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.     

Selective DNA bending by a variety of bZIP proteins.

We have investigated DNA bending by bZIP family proteins that can bind to the AP-1 site. DNA bending is widespread, although not universal, among members of this family. Different bZIP protein dimers induced distinct DNA bends. The DNA bend angles ranged from virtually 0 to greater than 40 degrees as measured by phasing analysis and were oriented toward both the major and the minor grooves at the center of the AP-1 site. The DNA bends induced by the various heterodimeric complexes suggested that each component of the complex induced an independent DNA bend as previously shown for Fos and Jun. The Fos-related proteins Fra1 and Fra2 bent DNA in the same orientation as Fos but induced smaller DNA bend angles. ATF2 also bent DNA toward the minor groove in heterodimers formed with Fos, Fra2, and Jun. CREB and ATF1, which favor binding to the CRE site, did not induce significant DNA bending. Zta, which is a divergent member of the bZIP family, bent DNA toward the major groove. A variety of DNA structures can therefore be induced at the AP-1 site through combinatorial interactions between different bZIP family proteins. This diversity of DNA structures may contribute to regulatory specificity among the plethora of proteins that can bind to the AP-1 site.     

DNA minor groove electrostatic potential: influence of sequence-specific transitions of the torsion angle gamma and deoxyribose conformations

The structural adjustments of the sugar-phosphate DNA backbone (switching of the γ angle (O5′–C5′–C4′–C3′) from canonical to alternative conformations and/or C2′-endo → C3′-endo transition of deoxyribose) lead to the sequence-specific changes in accessible surface area of both polar and non-polar atoms of the grooves and the polar/hydrophobic profile of the latter ones. The distribution of the minor groove electrostatic potential is likely to be changing as a result of such conformational rearrangements in sugar-phosphate DNA backbone. Our analysis of the crystal structures of the short free DNA fragments and calculation of their electrostatic potentials allowed us to determine: (1) the number of classical and alternative γ angle conformations in the free B-DNA; (2) changes in the minor groove electrostatic potential, depending on the conformation of the sugar-phosphate DNA backbone; (3) the effect of the DNA sequence on the minor groove electrostatic potential. We have demonstrated that the structural adjustments of the DNA double helix (the conformations of the sugar-phosphate backbone and the minor groove dimensions) induce changes in the distribution of the minor groove electrostatic potential and are sequence-specific. Therefore, these features of the minor groove sizes and distribution of minor groove electrostatic potential can be used as a signal for recognition of the target DNA sequence by protein in the implementation of the indirect readout mechanism.     

Mutual backbone phosphate group interactions promote DNA double helix bending at high salt concentrations in solution

Results of free energy calculations connected with the backbone phosphate group interactions upon local bending and helical twist modifications of A-, B- or Z-DNA at high salt concentrations have been reported recently (Jursa and Kypr 1990). Here we calculate energies necessary for DNA bending, using three models based on experimentally determined persistence length values. A comparison of energies following from the two quite different approaches suggests that high salt concentrations induce A- and mainly B-DNA bending into the double helix minor groove at least up to 10 degrees.     

Tet repressor binding induced curvature of tet operator DNA.

Tet repressor dimer binds to two tet operator sites spaced by 30 bp in the Tn10 encoded tet regulatory DNA. The effect of repressor binding on the gel mobility of circular permutated DNA fragments containing either one or both operator sequences is reported. The EcoRI induced bending of DNA is used to compare the results with other protein binding induced structural perturbations of DNA. Tet repressor bends a DNA fragment with a single tet operator to an angle of 42 degrees +/- 7 degrees. The apparent bend angle of DNA fragments containing the tandem tet operator arrangement occupied by two Tet repressor dimers turns out to be 52 degrees +/- 9 degrees. These results are interpreted with respect to the end to end distances of the bent DNA fragments. They indicate that either the intervening tet regulatory DNA between the operators or the bound operator sequences themselves contain additional perturbations from the canonical B-DNA structure. This finding is discussed in the light of previously obtained results from CD, neutron scattering, and electrooptical studies.     

Non-cognate enzyme-DNA complex: structural and kinetic analysis of EcoRV endonuclease bound to the EcoRI recognition site GAATTC

The crystal structure of EcoRV endonuclease bound to non-cognate DNA at 2.0 angstroms resolution shows that very small structural adaptations are sufficient to ensure the extreme sequence specificity characteristic of restriction enzymes. EcoRV bends its specific GATATC site sharply by 50 degrees into the major groove at the center TA step, generating unusual base-base interactions along each individual DNA strand. In the symmetric non-cognate complex bound to GAATTC, the center step bend is relaxed to avoid steric hindrance caused by the different placement of the exocyclic thymine methyl groups. The decreased base-pair unstacking in turn leads to small conformational rearrangements in the sugar-phosphate backbone, sufficient to destabilize binding of crucial divalent metal ions in the active site. A second crystal structure of EcoRV bound to the base-analog GAAUTC site shows that the 50 degrees center-step bend of the DNA is restored. However, while divalent metals bind at high occupancy in this structure, one metal ion shifts away from binding at the scissile DNA phosphate to a position near the 3'-adjacent phosphate group. This may explain why the 10(4)-fold attenuated cleavage efficiency toward GAATTC is reconstituted by less than tenfold toward GAAUTC. Examination of DNA binding and bending by equilibrium and stopped-flow florescence quenching and fluorescence resonance energy transfer (FRET) methods demonstrates that the capacity of EcoRV to bend the GAATTC non-cognate site is severely limited, but that full bending of GAAUTC is achieved at only a threefold reduced rate compared with the cognate complex. Together, the structural and biochemical data demonstrate the existence of distinct mechanisms for ensuring specificity at the bending and catalytic steps, respectively. The limited conformational rearrangements observed in the EcoRV non-cognate complex provide a sharp contrast to the extensive structural changes found in a non-cognate BamHI-DNA crystal structure, thus demonstrating a diversity of mechanisms by which restriction enzymes are able to achieve specificity.     

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.     

Structural features and hydration of a dodecamer duplex containing two C.A mispairs.

X-ray diffraction techniques have been used to characterise the crystal and molecular structure of the deoxyoligomer d(C-G-C-A-A-A-T-T-C-G-C-G) at 2.5A resolution. The final R factor is 0.19 with the location of 78 solvent molecules. The oligomer crystallises in a B-DNA type conformation with two strands coiled about each other to produce a duplex. This double helix consists of four A.T and six G.C Watson-Crick base pairs and two C.A mispairs. The mismatched base pairs adopt a "wobble" type structure with the cytosine displaced laterally into the major groove, the adenine into the minor groove. We have proposed that the two close contacts observed in the C.A pairing represent two hydrogen bonds one of which results from protonation of adenine. The mispairs are accommodated in the double helix with small adjustments in the conformation of the sugar-phosphate backbone. Details of the backbone conformation, base stacking interactions, thermal parameters and the hydration are now presented and compared with those of the native oligomer d(C-G-C-G-A-A-T-T-C-G-C-G) and with variations of this sequence containing G.T and G.A mispairs.     

Left-handed DNA crossovers. Implications for DNA-DNA recognition and structural alterations

The close approach of DNA segments participates in many biological functions including DNA condensation and DNA processing. Previous crystallographic studies have shown that B-DNA self-fitting by mutual groove-backbone interaction produces right-handed DNA crossovers. These structures have opened new perspectives on the role of close DNA-DNA interactions in the architecture and activity the DNA molecule. In the present study, the analysis of the crystal packing of two B-DNA decamer duplexes d(CCIIICCCGG) and d(CCGCCGGCGG) reveals the existence of new modes of DNA crossing. Symmetric left-handed crossovers are produced by mutual fitting of DNA grooves at the crossing point. New sequence patterns contribute to stabilize longitudinal fitting of the sugar-phosphate backbone into the major groove. In addition, the close approach of DNA segments greatly influences the DNA conformation in a sequence dependent manner. This study provides new insights into the role of DNA sequence and structure in DNA-DNA recognition. In providing detailed molecular views of DNA crossovers of opposite chirality, this study can also help to elucidate the role of symmetry and chirality in the recognition of complex DNA structures by protein dimers or tetramers, such as topoisomerase II and recombinase enzymes. These results are discussed in the context of the possible relationships between DNA condensation and DNA processing.     

DNA bending by small, mobile multivalent cations.

We propose a purely electrostatic mechanism by which small, mobile, multivalent cations can induce DNA bending. A multivalent cation binds at the entrance to the B-DNA major groove, between the two phosphate strands, electrostatically repelling sodium counterions from the neighboring phosphates. The unscreened phosphates on both strands are strongly attracted to the groove-bound cation. This leads to groove closure, accompanied by DNA bending toward the cationic ligand. We explicitly treat the dynamic character of the cation-DNA interaction using an adiabatic approximation, noting that DNA bending is much slower than the diffusion of nonspecifically bound, mobile cations. We make semiquantitative estimates of the free energy components of bending-electrostatic (with a sigmoidal distance-dependent dielectric function), elastic, and entropic cation localization-and find that the equilibrium state is bent B-DNA stabilized with a self-localized cation. This is a bending polaron, formation of which should be critically dependent on the strength of electrostatic interaction and the concentration of highly mobile cations available for self-localization. We predict that the resultant bend will be large (approximately 20-40 degrees), smooth (because it is spread over 6 bp), and infrequent. The stability of such a bend can be variable, from transient to highly stable (static) bending, observable with standard curvature-measuring techniques. We further predict that this bending mechanism will have an unusual sequence dependence: sequences with less binding specificity will be more bent, unless the specific binding site is in the major groove.     

Left-handed DNA Crossovers. Implications for DNA-DNA Recognition and Structural Alterations

Abstract

The close approach of DNA segments participates in many biological functions including DNA condensation and DNA processing. Previous crystallographic studies have shown that B-DNA self-fitting by mutual groove-backbone interaction produces right-handed DNA crossovers. These structures have opened new perspectives on the role of close DNA-DNA interactions in the architecture and activity the DNA molecule. In the present study, the analysis of the crystal packing of two B-DNA decamer duplexes d(CCIIICCCGG) and d(CCGCCGGCGG) reveals the existence of new modes of DNA crossing. Symmetric left- handed crossovers are produced by mutual fitting of DNA grooves at the crossing point. New sequence patterns contribute to stabilize longitudinal fitting of the sugar-phosphate backbone into the major groove. In addition, the close approach of DNA segments greatly influences the DNA conformation in a sequence dependent manner. This study provides new insights into the role of DNA sequence and structure in DNA-DNA recognition. In providing detailed molecular views of DNA crossovers of opposite chirality, this study can also help to elucidate the role of symmetry and chirality in the recognition of complex DNA structures by protein dimers or tetramers, such as topoisomerase II and recombinase enzymes. These results are discussed in the context of the possible relationships between DNA condensation and DNA processing.     

1 A crystal structures of B-DNA reveal sequence-specific binding and groove-specific bending of DNA by magnesium and calcium

The 1 A resolution X-ray crystal structures of Mg(2+) and Ca(2+) salts of the B-DNA decamers CCAACGTTGG and CCAGCGCTGG reveal sequence-specific binding of Mg(2+) and Ca(2+) to the major and minor grooves of DNA, as well as non-specific binding to backbone phosphate oxygen atoms. Minor groove binding involves H-bond interactions between cross-strand DNA base atoms of adjacent base-pairs and the cations' water ligands. In the major groove the cations' water ligands can interact through H-bonds with O and N atoms from either one base or adjacent bases, and in addition the softer Ca(2+) can form polar covalent bonds bridging adjacent N7 and O6 atoms at GG bases. For reasons outlined earlier, localized monovalent cations are neither expected nor found.Ultra-high atomic resolution gives an unprecedented view of hydration in both grooves of DNA, permits an analysis of individual anisotropic displacement parameters, and reveals up to 22 divalent cations per DNA duplex. Each DNA helix is quite anisotropic, and alternate conformations, with motion in the direction of opening and closing the minor groove, are observed for the sugar-phosphate backbone. Taking into consideration the variability of experimental parameters and crystal packing environments among these four helices, and 24 other Mg(2+) and Ca(2+) bound B-DNA structures, we conclude that sequence-specific and strand-specific binding of Mg(2+) and Ca(2+) to the major groove causes DNA bending by base-roll compression towards the major groove, while sequence-specific binding of Mg(2+) and Ca(2+) in the minor groove has a negligible effect on helix curvature. The minor groove opens and closes to accommodate Mg(2+) and Ca(2+) without the necessity for significant bending of the overall helix.The program Shelxdna was written to facilitate refinement and analysis of X-ray crystal structures by Shelxl-97 and to plot and analyze one or more Curves and Freehelix output files.     

CAP binding sites reveal pyrimidine-purine pattern characteristic of DNA bending

To investigate the intrinsic bending of DNA at sites where proteins bind, we analyzed catabolite gene activator protein (CAP) binding sites and various operators from the viewpoint of DNA bending flexibility. Theoretical conformational analysis. DNase I digestion and x-ray crystallography data indicate that bending of B-DNA is highly anisotropic and sequence-dependent. Certain dimers prefer to bend into the major groove ("major-philic") and others prefer to bend into the minor groove ("minor-philic" dimers). From these data we considered TA, CG, CA:TG and GG:CC as major-philic dimers and AT,AA:TT and GT:AC as minor-philic ones. Analysis of 31 CAP binding sites has identified strong major-philic tendencies 5-7 base pairs (bp) away from the center. In addition, we found minor-philic poly-A tracts extending 4-5 bp away from the proposed major-philic bends. Finally, to analyze the central regions we followed the lead of Shumilov and classified the DNA sites by their spacer lengths [V.Y. Shumilov, Mol. Biol. (Mosk) 21, 168-187 (1987)]. In this way, we identified two subsets of CAP binding sites: one with 6 bp between the TGTGA:TCACA consensus boxes (N6-set) and one with 8 central bp (N8-set). We discovered that the dimer at the center of an N6-set site was usually major-philic, whereas at the center of an N8-set site more often minor-philic. Analysis of phages 434, P22 lambda and trp operators revealed similar results. In conclusion, our data show that CAP binding sites have major-philic and minor-philic dimers at specific positions; the location of these dimers may facilitate wrapping of DNA around CAP. A similar pattern is seen in nucleosomes.     

Parallel DNA double helices. II. Conformational analysis of regular helices having a second order axis of symmetry

Conformational analysis of double helices of DNA with parallel arranged sugar-phosphate chains connected by twofold symmetry has been performed. Homopolymers poly(dA).poly(dA), poly(dC).poly(dC), poly(dG).poly(dG) and poly(dT).poly(dT) were studied. For each of the homopolymers all variants of H-bond pairing were checked. The maps of closing of sugar-phosphate backbone were previously computed. By the optimization of potential energy the dihedral angles and helix parameters of relatively stable conformations of parallel stranded polynucleotides were calculated. The dependence of conformational energy on the nucleic base character and the base pair type were studied. Two main conformational regions for favourable "parallel" helix of polynucleotides were found. The former of these two regions coincide with the region of typical conformational parameters of B-DNA. On an average the conformational energy of "parallel" DNA is close to the energy of canonic "antiparallel" B-DNA.     

Bent dsDNA with defined geometric characteristics in terms of complexes of bridged oligonucleotides

An opportunity of designing nontypical double-stranded DNA structures containing nonnatural inserts in a regular nucleotide DNA sequence has been investigated. The looped nucleotide inserts on the basis of adenylates and thymidilates, and nonnucleotide inserts on the basis of phosphodiesters of diethyleneglycol, 1,10-decanediol, and 3-hydroxy-2(hydroxymethyl)tetrahydrofuran were introduced into the backbone of a 32-mer native DNA duplex. These inserts formed the internal loops in the modified double-stranded DNA fragments which were shown to lead to bending of the linear duplex structure by 16 to 83 degrees. The dependencies of the bend angle of dsDNA on the composition and the length of the looped regions were determined. It was established that the bend of the irregular region of dsDNA depended on the electrostatic interaction of the phosphate residues. The tension in the complex structure could be reduced by the introduction of additional nucleotide units opposite the loop, which led to some relaxation of the bent helix. The resulting parameters of the bend values were shown to be in a good agreement with the published data obtained by NMR spectroscopy. It was demonstrated that the variation of the nature or the length of the insert allowed one to regulate the level of the local perturbation of the duplex structure and, thereby, influence both the bend level of the double helix and the destabilization of the modified complex.     

Flexibility of DNA in 2:1 drug-DNA complexes--simultaneous binding of two DAPI molecules to DNA.

Simultaneous binding of two DAPI molecules in the minor groove of (dA)15.(dT)15 B-DNA helix has been simulated by molecular mechanics calculations. The energy minimised structure shows some novel features in relation to binding of DAPI molecules as well as the flexibility of the grooves of DNA helices. The minor groove of the helix expands locally considerably (to 15 angstroms) to accommodate the two DAPI molecules and is achieved by positive propeller twisting of base pairs at the binding site concomitant with small variations in the local nucleotide stereochemistry. The expansion also brings forth simultaneously a contraction in the width of the major groove spread over to a few phosphates. These findings demonstrate another facet of the flexible stereochemistry of DNA helices in which the local features are significantly altered without being propagated beyond a few base pairs, and with the rest of the regions retaining the normal structure. Both the DAPI molecules are engaged in specific hydrogen bonds with the bases and non specific interactions with phosphates. Stacking interactions of DAPI molecules between themselves as well as with sugar-phosphate backbone contribute to the stability of the complex. The studies provide a stereochemical support to the experimental findings that under high drug-DNA ratio DAPI could bind in the 2:1 ratio.