The role of molecular structure of sugar‐phosphate backbone and nucleic acid bases in the formation of single‐stranded and double‐stranded DNA structures

Our previous DFT computations of deoxydinucleoside monophosphate complexes with Na⁺‐ions (dDMPs) have demonstrated that the main characteristics of Watson‐Crick (WC) right‐handed duplex families are predefined in the local energy minima of dDMPs. In this work, we study the mechanisms of contribution of chemically monotonous sugar‐phosphate backbone and the bases into the double helix irregularity. Geometry optimization of sugar‐phosphate backbone produces energy minima matching the WC DNA conformations. Studying the conformational variability of dDMPs in response to sequence permutation, we found that simple replacement of bases in the previously fully optimized dDMPs, e.g. by constructing Pyr‐Pur from Pur‐Pyr, and Pur‐Pyr from Pyr‐Pur sequences, while retaining the backbone geometry, automatically produces the mutual base position characteristic of the target sequence. Based on that, we infer that the directionality and the preferable regions of the sugar‐phosphate torsions, combined with the difference of purines from pyrimidines in ring shape, determines the sequence dependence of the structure of WC DNA. No such sequence dependence exists in dDMPs corresponding to other DNA conformations (e.g., Z‐family and Hoogsteen duplexes). Unlike other duplexes, WC helix is unique by its ability to match the local energy minima of the free single strand to the preferable conformations of the duplex. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 640–650, 2014.     

Sequence-specific binding of echinomycin to DNA: evidence for conformational changes affecting flanking sequences.

The technique of DNAase I footprinting has been used to investigate preferred binding sites for echinomycin on a 160-base-pair DNA fragment from E. coli containing the tyr T promoter sequence. Six binding sites have been precisely located in the sequence; a seventh has been partially identified. The minimum site-size is six base pairs. All the binding sites contain the dinucleotide sequence CpG but no other regularities can be discerned. When the protected regions on each complementary strand are compared it is evident that they are staggered by 2-3 base-pairs towards the 3' end at each site. Footprinting with DNAase II reports a similar, though less precise, pattern of protection. Cutting by both enzymes is markedly enhanced at AT-rich regions flanking the antibiotic-binding sites. This increased susceptibility to nuclease attack can be attributed to an altered helix conformation in the vicinity of the bis-intercalated echinomycin molecule. It seems that certain sequences, mainly runs of A or runs of T, switch from a nuclease-resistant to a nuclease-sensitive form when echinomycin binds nearby.     

Two dimensional NMR studies on the solution structure of d-CTCGAGCTCGAG

Two dimensional (2D) FT-NMR investigations have been carried out on the self-complementary dodecanucleotide d-CTCGAGCTCGAG, which has cleavage sites for the restriction enzyme Xho I (between C and T). The central TCG portion is also known to show a preference for DNAase activity. Complete resonance assignments have been obtained for the non-exchangeable sugar and base protons of the oligonucleotide. Information regarding sugar geometries, glycosidic torsion angles and other structural parameters has been obtained from the relative intensities of the cross peaks in the COSY and NOESY spectra. The results indicate that deoxyribose rings of C1 and C7 adopt a conformation different from the remaining sugars in the double helical oligonucleotide. The central TCG portion also exhibits variations in the backbone structure. The base stacking in the double helix shows interesting sequence dependent effects suggesting that the sequence effects are not localised to nearest neighbours but extended over longer stretches.     

The structure of the XPF-ssDNA complex underscores the distinct roles of the XPF and ERCC1 helix- hairpin-helix domains in ss/ds DNA recognition

Human XPF/ERCC1 is a structure-specific DNA endonuclease that nicks the damaged DNA strand at the 5' end during nucleotide excision repair. We determined the structure of the complex of the C-terminal domain of XPF with 10 nt ssDNA. A positively charged region within the second helix of the first HhH motif contacts the ssDNA phosphate backbone. One guanine base is flipped out of register and positioned in a pocket contacting residues from both HhH motifs of XPF. Comparison to other HhH-containing proteins indicates a one-residue deletion in the second HhH motif of XPF that has altered the hairpin conformation, thereby permitting ssDNA interactions. Previous nuclear magnetic resonance studies showed that ERCC1 in the XPF-ERCC1 heterodimer can bind dsDNA. Combining the two observations gives a model that underscores the asymmetry of the human XPF/ERCC1 heterodimer in binding at an ss/ds DNA junction.     

Sequence-specific recognition of DNA: NMR studies of the imino protons of a synthetic RNA polymerase promoter

We have synthesized both strands of a DNA duplex containing the consensus Pribnow promoter sequence TATAATG , flanked by GC base pairs to stabilize the ends of the helix. The stability of this duplex has been studied by using 1H nuclear magnetic resonance. The imino protons have been assigned by using the sequential nuclear Overhauser effect approach. Exchange rates have been monitored by using selective inversion recovery measurements. The helix is relatively unstable in the center of the AT-rich region even when surrounded by GC base pairs, and there is considerable asymmetry in the melting of the helix.     

Structural junctions in DNA: the influence of flanking sequence on nuclease digestion specificities.

When a protein binds to DNA, the affinity of this protein for its primary site of interaction may be influenced by the nature of flanking sequences. This is thought to be a consequence of local cooperativity in the DNA molecule, where the conformation at one point along the helix can influence the conformation at another, and thereby modulate the free energy of protein-DNA recognition. In order to learn more about this process, we have carried out experiments of two sorts. First, we have constructed sequences of the type (dA)11 (dG)8, where the conformational preferences of the DNA molecule switch from one extreme to another over just a single base pair, and subjected them to digestion by DNAase I and DNAase II. This is to learn whether the structure changes abruptly at the junction point, or more gradually with an influence extending into residues on either side. Secondly, we have subjected long plasmid DNA to digestion by restriction enzymes Fnu DII, Hae III, Hha I and Msp I, to look for correlations between cutting rate and the identity of nucleotides on either side of the restriction site. The influence of flanking sequence on nuclease digestion specificities is clearly evident in both kinds of experiment, but the rules governing this seem complex and not easily formulated. The best that can be done at present is to divide the problem into two parts, "analogue" and "digital", representing sugar-phosphate and base components of recognition.     

DNA bending and its relation to nucleosome positioning

X-ray and solution studies have shown that the conformation of a DNA double helix depends strongly on its base sequence. Here we show that certain sequence-dependent modulations in structure appear to determine the rotational positioning of DNA about the nucleosome. Three different experiments are described. First, a piece of DNA of defined sequence (169 base-pairs long) is closed into a circle, and its structure examined by digestion with DNAase I: the helix adopts a highly preferred configuration, with short runs of (A, T) facing in and runs of (G, C) facing out. Secondly, the same sequence is reconstituted with a histone octamer: the angular orientation around the histone core remains conserved, apart from a small uniform increase in helix twist. Finally, it is shown that the average sequence content of DNA molecules isolated from chicken nucleosome cores is non-random, as in a reconstituted nucleosome: short runs of (A, T) are preferentially positioned with minor grooves facing in, while runs of (G, C) tend to have their minor grooves facing out. The periodicity of this modulation in sequence content (10.17 base-pairs) corresponds to the helix twist in a local frame of reference (a result that bears on the change in linking number upon nucleosome formation). The determinants of translational positioning have not been identified, but one possibility is that long runs of homopolymer (dA) X (dT) or (dG) X (dC) will be excluded from the central region of the supercoil on account of their resistance to curvature.     

Oligonucleotide labeling methods. 3. Direct labeling of oligonucleotides employing a novel, non-nucleosidic, 2-aminobutyl-1,3-propanediol backbone.

Novel CE-phosphoramidite (7a-e) and CPG (8a, c, d, e) reagents have been prepared from a unique 2-aminobutyl-1,3-propanediol backbone. The reagents have been used to directly label oligonucleotides with fluorescein, acridine, and biotin via automated DNA synthesis. The versatile 2-aminobutyl-1,3-propanediol backbone allows for labeling at any position (5', internal, and 3') during solid phase oligonucleotide synthesis. Multiple labels can be achieved by repetitive coupling cycles. Furthermore, the 3-carbon atom internucleotide phosphate distance is retained when inserted internally. Using this method, individual oligonucleotides possessing two and three different reporter molecules have been prepared.     

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.     

O6-methylguanine and A.C and G.T mismatches cause asymmetric structural defects in DNA that are affected by DNA sequence

Mismatched and modified base pairs are central to questions of DNA mutation and repair. NMR and X-ray crystallography of mispairs indicate little to no local helical distortion, but these techniques are not sensitive to more global distortions of the DNA molecule. We used polyacrylamide gel electrophoresis and thermal denaturation to examine A.C, G.T, and O6-methylG.T and O6-methylG.C mismatches synthesized in place of either of two adjacent G.C base pairs in synthetic DNA duplexes. Substitution for G.C at either position decreased the stability of the duplex; O6-methylguanine was more destabilizing in place of the 5'G than in place of the 3'G. Comparisons between polymers synthesized so that lesions occurred regularly spaced on the same side of the helix and polymers synthesized so that the lesions alternated from side to side on the helix showed that these lesions introduced helical distortion composed of (i) a symmetric frictional component, probably caused by localized bubble formation, and (ii) an asymmetric component indicative of a more global effect on the DNA molecule. Comparisons between these effects at the two adjacent positions show that the extent of structural perturbation depends on sequence context.     

Computer analysis of conformational and physicochemical percularities of sequences cleaved by DNA topoisomerase I

After complexation of DNA with enzymes a specific adaptation of DNA structure including its partial or nearly complet melting, change of sugar-phosphate backbone structure, stretching, compression, bending or kinking, flipping out of nucleotides from the DNA helix, etc. take place. The full set of such changes is specific for each individual enzyme and is a very important for effective adjustment of reacting orbitals of enzyme and specific DNA atoms with accuracy up to 10-15 degrees. Efficiency of DNA sequence adaptation in the direction providing by enzyme depends on many specific structural characteristics of DNA. Maximal adjustment of DNA structure can be achieved only for specific sequences, therefore on going from nonspecific to specific DNAs the increase of the catalytic rate by 4-8 orders of magnitude takes place. DNA topoisomerase I is a sequence-dependent enzyme, but it can cleave with lower efficiency DNA sequences, which are significantly different from an optimal one. We have carried out the computer analysis of structural characteristics of many DNA sequences utilizing by topoisomerase using the method which is based on the analysis of conformational and physico-chemical characteristics of DNA helix and gives a detailed information about similarities or differences of DNA structural units. In addition to such characteristics as base tilt angle, shift of base pair, helix steering angle, and helix step for all cleaved sequences the presence of sterically disadvantageous contacts in small grove between N3 and NH2 of guanines and N3 of adenines were detected which corresponds to the presence Py-Pu dinucleotides in the cleavaged site. In addition, for optimal sequences bending of DNA helix toward major groove is characterized. The proposed method seems to be a very perspective for the analysis of an efficiency of nucleic acids cleavage by different DNA- and RNA-dependent enzymes.     

Molecular Dynamics Simulation in Solvent of the Estrogen Receptor Protein DNA Binding Domain in Complex with a Non-Consensus Estrogen Response Element DNA Sequence

Abstract

We investigated protein/DNA interactions, using molecular dynamics simulations computed between a 10 Angstom water layer model of the estrogen receptor (ER) protein DNA binding domain (DBD) amino acids and DNA of a non-consensus estrogen response element (ERE) consisting of 29 nucleotide base pairs. This ERE nucleotide sequence occurs naturally upstream of the Xenopus laevis Vitelligenin AI gene. The ER DBD is encoded by three exons. Namely, exons 2 and 3 which encode the two zinc binding motifs and a sequence of exon 4 which encodes a predicted alpha helix. We generated a computer model of the ER DBD using atomic coordinates derived from the average of 30 nuclear magnetic resonance (NMR) spectroscopy coordinate sets. Amino acids on the carboxyl end of the ER DBD were disordered in both X-ray crystallography and NMR determinations and no coordinates were reported. This disordered region includes 10 amino acids of a predicted alpha helix encoded in exon 4 at the exon 3/4 splice junction. These amino acids are known to be important in DNA binding and are also believed to function as a nuclear translocation signal sequence for the ER protein. We generated a computer model of the predicted alpha helix consisting of the 10 amino acids encoded in exon 4 and attached this helix to the carboxyl end of the ER DBD at the exon 3/4 splice junction site. We docked the ER DBD model within the DNA major groove halfsites of the 29 base pair non-consensus ERE and flanking nucleotides. We constructed a solvated model with the ER DBD/ERE complex surrounded by a ten Angstrom water layer and conducted molecular dynamics simulations. Hydrogen bonding interactions were monitored. In addition, van der Waals and electrostatic interaction energies were calculated. Amino acids of the ER DBD DNA recognition helix formed both direct and water mediated hydrogen bonds at cognate codon-anticodon nucleotide base and backbone sites within the ERE DNA right major groove halfsite. Amino acids of the ER DBD exon 4 encoded predicted alpha helix formed direct and water mediated H-bonds with base and backbone sites of their cognate codon-anticodon nucleotides within the minor grooves flanking the ERE DNA major groove halfsites. These interactions together induced bending of the DNA into the protein.     

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.     

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

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

DNA-induced alpha-helix capping in conserved linker sequences is a determinant of binding affinity in Cys(2)-His(2) zinc fingers

High-affinity, sequence-specific DNA binding by Cys(2)-His(2) zinc finger proteins is mediated by both specific protein-base interactions and non-specific contacts between charged side-chains and the phosphate backbone. In addition, in DNA complexes of multiple zinc fingers, protein-protein interactions between the finger units contribute to the binding affinity. We present NMR evidence for another contribution to high- affinity binding, a highly specific DNA-induced helix capping involving residues in the linker sequence between fingers. Capping at the C terminus of the alpha-helix in each zinc finger, incorporating a consensus TGEKP linker sequence that follows each finger, provides substantial binding energy to the DNA complexes of zinc fingers 1-3 of TFIIIA (zf1-3) and the four zinc fingers of the Wilms' tumor suppressor protein (wt1-4). The same alpha-helix C-capping motif is observed in the X-ray structures of four other protein-DNA complexes. The structures of each of the TGEKP linkers in these complexes can be superimposed on the linker sequences in the zf1-3 complex, revealing a remarkable similarity in both backbone and side-chain conformations. The canonical linker structures from the zinc-finger-DNA complexes have been compared to the NMR structure of the TGEKP linker connecting fingers 1 and 2 in zf1-3 in the absence of DNA. This comparison reveals that additional stabilization likely arises in the DNA complexes from hydrogen bonding between the backbone amide of E3 and the side-chain O(gamma) of T1 in the linker. We suggest that these DNA-induced C-capping interactions provide a means whereby the multiple-finger complex, which must necessarily be domain-flexible in the unbound state as it searches for the correct DNA sequence, can be "snap-locked" in place once the correct DNA sequence is encountered. These observations provide a rationale for the high conservation of the TGEKP linker sequences in Cys(2)-His(2) zinc finger proteins.     

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

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