Structural studies of DNA fragments: the G.T wobble base pair in A, B and Z DNA; the G.A base pair in B-DNA

The crystal structures of five double helical DNA fragments containing non-Watson-Crick complementary base pairs are reviewed. They comprise four fragments containing G.T base pairs: two deoxyoctamers d(GGGGCTCC) and d(GGGGTCCC) which crystallise as A type helices; a deoxydodecamer d(CGCGAATTTGCG) which crystallises in the B-DNA conformation; and the deoxyhexamer d(TGCGCG), which crystallises as a Z-DNA helix. In all four duplexes the G and T bases form wobble base pairs, with bases in the major tautomer forms and hydrogen bonds linking N1 of G with O2 of T and O6 of G with N3 of T. The X-ray analyses establish that the G.T wobble base pair can be accommodated in the A, B or Z double helix with minimal distortion of the global conformation. There are, however, changes in base stacking in the neighbourhood of the mismatched bases. The fifth structure, d(CGCGAATTAGCG), contains the purine purine mismatch G.A where G is in the anti and A in the syn conformation. The results represent the first direct structure determinations of base pair mismatches in DNA fragments and are discussed in relation to the fidelity of replication and mismatch recognition.     

Solution structure of an oncogenic DNA duplex containing a G.A mismatch

The DNA duplex 5'-d(GCCACAAGCTC).d(GAGCTGGTGGC), which contains a central G.A mismatch has been studied by one and two-dimensional NMR techniques. The duplex corresponds to the sequence 29-39 of the K-ras gene. The mismatch position is that of the first base of the Gly12 codon, a hot spot for mutations. The observed NOEs of the nonexchangeable protons show that both of the bases of the mismatched pair are intrahelical over a wide range of pH. However, the structure of the G.A mispair and the conformation of the central part of the duplex change with pH. This structural change shows a pK of 6.0. At low pH, the G.A bases are base paired with hydrogen bonds between the keto group of the G residue and the amino group of the A residue and, secondly, between the N7 of the G and a proton on N1 of A. This causes the G residue to adopt a syn conformation. On raising the pH, the N1-H proton of the protonated A residue is removed, and the base pair rearranges. In the neutral G.A base pair both residues adopt an anti conformation, and the mismatch is stabilized by hydrogen bonds. Our results on the exchangeable and A(H2) protons of the mismatched pair indicate a shift from a classical face-to-face two hydrogen-bonded structure to a slipped structure stabilized by bifurcated hydrogen bonds. This may be a particular characteristics of this oncogenic sequence in which the G.A error is poorly repaired.     

A novel structure observed in d-GAATTCCCGAATTC by 2D NMR

Sequential resonance assignments of the non exchangeable base and sugar protons in d-GAATTCCCGAATTC have been obtained using two dimensional NMR experiments at 500 MHz. The chemical shifts and the NOEs have been used to determine the structure in the base-pair mismatch region which is located in the central portion of the molecule. It is observed that the molecule adopts a novel unsymmetrical loop structure in this section which is characterised by sugar geometries which are significantly different compared to the rest of the molecular. The base-paired portion of the molecule conforms to a right handed B-DNA type of structure.     

Kinetics for exchange of imino protons in the d(C-G-C-G-A-A-T-T-C-G-C-G) double helix and in two similar helices that contain a G . T base pair, d(C-G-T-G-A-A-T-T-C-G-C-G), and an extra adenine, d(C-G-C-A-G-A-A-T-T-C-G-C-G)

The relaxation lifetimes of imino protons from individual base pairs were measured in (I) a perfect helix, d(C-G-C-G-A-A-T-T-C-G-C-G), (II) this helix with a G . C base pair replaced with a G . T base pair, d(C-G-T-G-A-A-T-T-C-G-C-G), and (III) the perfect helix with an extra adenine base in a mismatch, d(C-G-C-A-G-A-A-T-T-C-G-C-G). The lifetimes were measured by saturation recovery proton nuclear magnetic resonance experiments performed on the imino protons of these duplexes. The measured lifetimes of the imino protons were shown to correspond to chemical exchange lifetimes at higher temperatures and spin-lattice relaxation times at lower temperatures. Comparison of the lifetimes in these duplexes showed that the destabilizing effect of the G . T base pair in II affected the opening rate of only the nearest-neighbor base pairs. For helix III, the extra adenine affected the opening rates of all the base pairs in the helix and thus was a larger perturbation for opening of the base pairs than the G . T base pair. The temperature dependence of the exchange rates of the imino proton in the perfect helix gives values of 14-15 kcal/mol for activation energies of A . T imino protons. These relaxation rates were shown to correspond to exchange involving individual base pair opening in this helix, which means that one base-paired imino proton can exchange independent of the others. For the other two helices that contain perturbations, much larger activation energies for exchange of the imino protons were found, indicating that a cooperative transition involving exchange of at least several base pairs was the exchange mechanism of the imino protons. The effects of a perturbation in a helix on the exchange rates and the mechanisms for exchange of imino protons from oligonucleotide helices are discussed.     

NMR studies for identification of dI:dG mismatch base-pairing structure in DNA.

One- and two-dimensional NMR experiments have been undertaken to investigate deoxyinosine:deoxyguanosine (dI:dG) base pairing in a self-complementary dodecadeoxyribonucleotide, d(C1-G2-C3-I4-A5-A6-T7-T8-G9-G10-G11-G12) (designated IG-12), duplex. The NMR data indicate formation of a dI(syn):dG(anti) base pair in a B-DNA helix. This unusual base pairing results in altered NOE patterns between the base protons (H8 and H2) of the I4 residue and the sugar protons of its own and the 5'-flanking C3 residues. The dI(syn):dG(anti) base pair is accommodated in the B-DNA duplex with only a subtle distortion of the local conformation. Identification of the dI:dG base pairing in this study confirms that a hypoxanthine base can form hydrogen-bonded base pairs with all of the four normal bases, C, A, T, and G, in DNA.     

Base pair mismatches and carcinogen-modified bases in DNA: an NMR study of G.T and G.O4meT pairing in dodecanucleotide duplexes

High-resolution two-dimensional NMR studies have been completed on the self-complementary d(C-G-C-G-A-G-C-T-T-G-C-G) duplex (designated G.T 12-mer) and the self-complementary d(C-G-C-G-A-G-C-T-O4meT-G-C-G) duplex (designated G.O4meT 12-mer) containing G.T and G.O4meT pairs at identical positions four base pairs in from either end of the duplex. The exchangeable and nonexchangeable proton resonances have been assigned from an analysis of two-dimensional nuclear Overhauser enhancement (NOESY) spectra for the G.T 12-mer and G.O4meT 12-mer duplexes in H2O and D2O solution. The guanosine and thymidine imino protons in the G.T mismatch resonate at 10.57 and 11.98 ppm, respectively, and exhibit a strong NOE between themselves and to imino protons of flanking base pairs in the G.T 12-mer duplex. These results are consistent with wobble pairing at the G.T mismatch site involving two imino proton-carbonyl hydrogen bonds as reported previously [Hare, D. R., Shapiro, L., & Patel, D. J. (1986) Biochemistry 25, 7445-7456]. In contrast, the guanosine imino proton in the G.O4meT pair resonates at 8.67 ppm. The large upfield chemical shift of this proton relative to that of the imino proton resonance of G in the G.T mismatch or in G.C base pairs indicates that hydrogen bonding to O4meT is either very weak or absent. This guanosine imino proton has an NOE to the OCH3 group of O4meT across the pair and NOEs to the imino protons of flanking base pairs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational properties of the G.G mismatch in d(CGCGAATTGGCG)2 determined by NMR.

The conformational properties of the DNA duplex d(CGCGAATTGGCG)2, which contains two noncomplementary G.G base pairs, have been examined in aqueous solution by 1H and 31P NMR as a function of temperature. The G.G mismatch is highly destabilizing, with a Tm value 35 K below that observed for the native EcoRI dodecamer. The dodecamer appears symmetric in the NMR spectra and exists largely as an average B-type DNA conformation. However, the 1H and 31P NMR spectra give evidence of considerable conformational heterogeneity at the mismatched nucleotides and their nearest neighbors, which increases with increasing temperature. There is no evidence for a significant population of the syn purine conformation. The imino protons of the mispaired bases G4 and G9 are degenerate, resonate at high field, and exchange readily with solvent. These results indicate that the mispaired bases are only weakly hydrogen-bonded and are only partially stacked into the helix. On raising the temperature, the duplex shows increasing exchange between two or more conformations originating from the mismatch sites. However, these additional conformations maintain their Watson-Crick hydrogen bonding. The increase in chemical exchange is consistent with a quasimelting process for which the G.G sites provide local nuclei. Extensive modeling studies by dynamic annealing have confirmed that the G(anti).G(anti) conformation is favored and that the mispairs are poorly stacked within the helix. The results explain both the poor thermal stability and low hypochromicity of this duplex.     

Refinement of the solution structure of the DNA dodecamer 5'd(CGCGPATTCGCG)2 containing a stable purine-thymine base pair: combined use of nuclear magnetic resonance and restrained molecular dynamics

The solution structure of the self-complementary dodecamer 5'd(CGCGPATTCGCG)2, containing a purine-thymine base pair within the hexameric canonical recognition site GAATTC for the restriction endonuclease EcoRI, is investigated by nuclear magnetic resonance spectroscopy and restrained molecular dynamics. Nonexchangeable and exchangeable protons are assigned in a sequential manner. A set of 228 approximate interproton distance restraints are derived from two-dimensional nuclear Overhauser enhancement spectra recorded at short mixing times. These distances are used as the basis for refinement using restrained molecular dynamics in which the interproton distance restraints are incorporated into the total energy function of the system in the form of effective potentials. Eight calculations are carried out, four starting from classical A-DNA and four from classical B-DNA. In all cases convergence to very similar B-type structures is achieved with an average atomic root mean square (rms) difference between the eight converged structures of 0.7 +/- 0.2 A, compared to a value of 6.5 A for that between the two starting structures. It is shown that the introduction of the purine-thymine mismatch does not result in any significant distortion of the structure. The variations in the helical parameters display a clear sequence dependence. The variation in helix twist and propeller twist follows Calladine's rules and can be attributed to the relief of interstrand purine-purine clash at adjacent base pairs. Overall the structure is straight. Closer examination, however, reveals that the central 5 base pair steps describe a smooth bend directed toward the major groove with a radius of curvature of approximately 38 A, which is compensated by two smaller kinks in the direction of the minor groove at base pair steps 3 and 9. These features can be explained in terms of the observed variation in roll and slide.     

Conformation, hydrogen bonding and aggregate formation of guanosine 5'-monophosphate and guanosine in dimethylsulfoxide.

The tetrabutylammonium salt of guanosine 5'-monophosphate (5'-GMP) dissolves in DMSO-d6 forming aggregated species which exhibit some properties of reverse micelles. 1H NOESY experiments show that the 5'-GMP adopts the syn conformation about the glycosidic bond. Molecular mechanics calculations reveal a stable structure with this conformation in which the phosphate group and the amino group of the base are in close enough proximity to hydrogen bond. In contrast inosine 5'-monophosphate in DMSO-d6, which has no NH2 group for hydrogen bond stabilization of the syn conformation, is shown by NMR to have the anti structure. Guanosine in DMSO-d6 behaves differently from 5'-GMP. Guanosine adopts the anti conformation and forms a symmetric dimer via hydrogen bonding between the N3 and NH2 of the bases.     

2D NMR studies and 3D structure of the parallel-stranded duplex oligonucleotide Acrm5-alpha-d(TCTAAACTC)-beta-d(AGATTTGAG) via complete relaxation matrix analysis of the NOE effects and molecular mechanics calculations

The three-dimensional structure of the duplex formed by the association of the unnatural oligonucleotide alpha-d(TCTAAACTC) covalently linked to an acridine derivative (m5Acr) with its natural and parallel complementary sequence beta-d(AGATTTGAG) was investigated by nuclear magnetic resonance spectroscopy and constrained molecular mechanics calculations. All the nonexchangeable and exchangeable resonances were assigned in this duplex. The structure was refined by using interproton distances determined by NOE measurements. The NOE values were converted into distances by using the complete 190 x 190 relaxation matrix. The unnatural duplex Acrm5-alpha-d(TCTAAACTC)-beta-d(AGATTTGAG) forms a parallel right-handed helix with Watson-Crick base pairing; the alpha and beta deoxyriboses adopt a 3'-exo conformation. The acridine moiety was found stacked up the C9-G9 base pair. The structure of the first seven base pairs of this duplex was found similar to that of the duplex alpha-d(TCTAAAC)-beta-d(AGATTTG), which we had already investigated [Lancelot, G., et al. (1989) Biochemistry 28, 7871-7878]. Since these structures were generated by using experimental NOE values obtained independently on macromolecules whose global correlation time was different (3.8 and 2.2 ns), we conclude that this comparison is a good test of the viability of our method to generate three-dimensional structures of oligonucleotides in solution. Starting from different initial conformations, we show that the NOE constraints allow one to reach the same final restrained conformation, taking into account implicitly the solvent effect.     

Solution structure of the parallel-stranded duplex oligonucleotide alpha-d(TCTAAAC)-beta-d(AGATTTG) via complete relaxation matrix analysis of the NOE effects and molecular mechanics calculations

The solution structure of the duplex formed by the association of the unnatural oligonucleotide alpha-d(TCTAAAC) with its natural and parallel complementary sequence beta-d(AGATTTG) was investigated by nuclear magnetic resonance spectroscopy and constrained molecular mechanics calculations. The structure was refined on the basis of interproton distances determined by NOE measurements for a series of mixing times. The NOE values were converted to distances by using the complete 134 x 134 relaxation matrix including all proton dipole-dipole interactions and spin diffusion. The computation of the relaxation matrix requires the Cartesian coordinates of the oligonucleotide, which are not known, a priori. To avoid this ambiguity, we used an iterative procedure in which the new distance constraints are obtained by using the complete relaxation matrix calculated from the previous structure. After three iterations, the process converged. The unnatural duplex alpha-d(TCTAAAC)-beta-d(AGATTTG) adopts in solution a right-helical structure with Watson-Crick base pairing, an anti conformation on the glycosyl linkage on the beta-strand, a syn conformation on the alpha-strand, and a 3'-exo conformation of the deoxyriboses for both sugar anomers. The three-dimensional structure obtained allowed us to describe the local heterogeneity of the duplex.     

Structural Studies of DNA Fragments: The G·T Wobble Base Pair in A,B and Z DNA; The G·A Base Pair in B-DNA

Abstract

The crystal structures of five double helical DNA fragments containing non-Watson-Crick complementary base pairs are reviewed. They comprise four fragments containing G·T base pairs: two deoxyoctamers d(GGGGCTCC) and d(GGGGTCCC) which crystallise as A type helices; a deoxydodecamer d(CGCGAATTTGCG) which crystallises in the B-DNA conformation; and the deoxyhexamer d(TGCGCG), which crystallises as a Z-DNA helix. In all four duplexes the G and T bases form wobble base pairs, with bases in the major tautomer forms and hydrogen bonds linking N1 of G with 02 of T and 06 of G with N3 of T. The X-ray analyses establish that the G·T wobble base pair can be accommodated in the A, B or Z double helix with minimal distortion of the global conformation. There are, however, changes in base stacking in the neighbourhood of the mismatched bases. The fifth structure, d(CGCGAATTAGCG), contains the purine purine mismatch G·A where G is in the anti and A in the syn conformation. The results represent the first direct structure determinations of base pair mismatches in DNA fragments and are discussed in relation to the fidelity of replication and mismatch recognition.     

NMR structures of r(GCAGGCGUGC)2 and determinants of stability for single guanosine-guanosine base pairs

Nucleotides in RNA that are not Watson-Crick-paired form unique structures for recognition or catalysis, but determinants of these structures and their stabilities are poorly understood. A single noncanonical pair of two guanosines (G) is more stable than other noncanonical pairs and can potentially form pairing structures with two hydrogen bonds in four different ways. Here, the energetics and structure of single GG pairs are investigated in several sequence contexts by optical melting and NMR. The data for r(5'GCAGGCGUGC3')(2), in which G4 and G7 are paired, are consistent with a model in which G4 and G7 alternate syn glycosidic conformations in a two-hydrogen-bond pair. The two distinct structures are derived from nuclear Overhauser effect spectroscopic distance restraints coupled with simulated annealing using the AMBER 95 force field. In each structure, the imino and amino protons of the anti G are hydrogen bonded to the O6 and N7 acceptors of the syn G, respectively. An additional hydrogen-bond connects the syn G amino group to the 5' nonbridging pro-R(p) phosphate oxygen. The GG pair fits well into a Watson-Crick helix. In r(5'GCAGGCGUGC3')(2), the G4(anti), G7(syn) structure is preferred over G4(syn), G7(anti). For single GG pairs in other contexts, exchange processes make interpretation of spectra more difficult but the pairs are also G(syn), G(anti). Thermodynamic data for a variety of duplexes containing pairs of G, inosine, and 7-deazaguanosine flanked by GC pairs are consistent with the structural and energetic interpretations for r(5'GCAGGCGUGC3')(2), suggesting similar GG conformations.     

Characterization of anti-Z-DNA antibody binding sites on Z-DNA by nuclear magnetic resonance spectroscopy

Two monoclonal anti-Z-DNA antibodies, Z22 and Z44, were shown to bind to the oligonucleotides, d(CG)2 and d(CG)3, and to interact with different parts of the helix. 1H nuclear magnetic resonance spectroscopy showed that Fab fragments stabilize an ordered structure in the tetranucleotide d(CG)2. Nuclear Overhauser effects measured in the presence of Z22 Fab indicate a syn conformation of guanine residues of d(CG)2. Intermolecular transfer of saturation between the Fabs and bound d(CG)3 was detected by a saturation of the protein spectrum and observation of changes in the DNA spectrum. Antibodies with deuterated aromatic amino acids were prepared to eliminate the protein aromatic resonances and thereby allow a more detailed analysis of the transfer to the DNA base protons. The greatest transfer with Z44 was to the dC-5 protons although all of the base protons interact with this antibody. Little, if any, transfer to the DNA base protons was observed with Z22. These results are consistent with a Z44 binding site on the convex surface of the Z-helix (analogous to the major groove of B-DNA) and a Z22 binding site on the sugar-phosphate backbone.     

Structural and thermodynamic studies on the adenine.guanine mismatch in B-DNA.

The structure of the synthetic dodecamer d(CGCAAATTGGCG) has been shown by single crystal X-ray diffraction methods to be that of a B-DNA helix containing two A(anti).G(syn) base pairs. The refinement, based on data to a resolution of 2.25 A shows that the mismatch base pairs are held together by two hydrogen bonds. The syn-conformation of the guanine base of the mismatch is stabilised by hydrogen bonding to a network of solvent molecules in both the major and minor grooves. A pH-dependent ultraviolet melting study indicates that the duplex is stabilised by protonation, suggesting that the bases of the A.G mispair are present in their most common tautomeric forms and that the N(1)-atom of adenine is protonated. The structure refinement shows that there is some disorder in the sugar-phosphate backbone.     

Crystal structure and stability of a DNA duplex containing A(anti).G(syn) base-pairs

The synthetic dodecanucleotide d(CGCAAATTGGCG) has been analysed by single-crystal X-ray diffraction techniques and the structure refined to R = 0.16 and 2.25 A resolution, with the location of 94 solvent molecules. The sequence crystallizes as a full turn of a B-DNA helix with ten Watson-Crick base-pairs and two adenine-guanine mispairs. The analysis clearly shows that the mismatches are of the form A(anti).G(syn). Thermal denaturation studies indicate that the stability of the duplex is strongly pH dependent, with a maximum at pH 5.0, suggesting that the base-pair is stabilized by protonation. Three different arrangements have been observed for base-pairs between guanine and adenine and it is likely that A.G mismatch conformation is strongly influenced by dipole-dipole interactions with adjacent base-pairs.     

NMR studies of exocyclic 1,N2-propanodeoxyguanosine adducts (X) opposite purines in DNA duplexes: protonated X(syn).A(anti) pairing (acidic pH) and X(syn).G(anti) pairing (neutral pH) at the lesion site

Proton and phosphorus two-dimensional NMR studies are reported for the complementary d(C1-A2-T3-G4-X5-G6-T7-A8-C9).d(G10-T11-A12-C13-A14-C15-A 16-T17-G18) nonanucleotide duplex (designated X.A 9-mer) that contains a 1,N2-propanodeoxyguanosine exocyclic adduct, X5, opposite deoxyadenosine A14 in the center of the helix. The NMR studies detect a pH-dependent conformational transition; this paper focuses on the structure present at pH 5.8. The two-dimensional NOESY studies of the X.A 9-mer duplex in H2O and D2O solution establish that X5 adopts a syn orientation while A14 adopts an anti orientation about the glycosidic bond at the lesion site. The large downfield shift of the amino protons of A14 demonstrates protonation of the deoxyadenosine base at pH 5.8 such that the protonated X5(syn).A14(anti) pair is stabilized by two hydrogen bonds at low pH. At pH 5.8, the observed NOE between the H8 proton of X5 and the H2 proton of A14 in the X.A 9-mer duplex demonstrates unequivocally the formation of the protonated X5(syn).A14(anti) pair. The 1,N2-propano bridge of X5(syn) is located in the major groove. Selective NOEs from the exocyclic methylene protons of X5 to the major groove H8 proton of flanking G4 but not G6 of the G4-X5-G6 segment provide additional structural constraints on the local conformation at the lesion site. A perturbation in the phosphodiester backbone is detected at the C13-A14 phosphorus located at the lesion site by 31P NMR spectroscopy. The two-dimensional NMR studies have been extended to the related complementary X.G 9-mer duplex that contains a central X5.G14 lesion in a sequence that is otherwise identical with the X.A 9-mer duplex. The NMR experimental parameters are consistent with formation of a pH-independent X5(syn).G14(anti) pair stabilized by two hydrogen bonds with the 1,N2-propano exocyclic adduct of X5(syn) located in the major groove.     

Helix opening in deoxyribonucleic acid from a proton nuclear magnetic resonance study of imino and amino protons in d(CG)3.

All exchangeable protons in a short DNA helix, d(CG)3 sodium salt, have been studied by proton nuclear magnetic resonance. The cytidine and guanosine amino protons have been assigned for the first time. As a function of temperature the cytidine amino protons and the imino protons behave very similarly, their relaxation is dominated by exchange with solvent above 30 degrees C. The guanosine amino protons, however, show that helix opening can only be described by a multistate model. The most rapid process observed is probably a twist about the helix axis which lengthens or breaks the guanosine amino hydrogen bond and allows rotation of the amino group. The second fastest process is a scissor opening into the major groove which gives rise to solvent exchange with the imino and cytidine amino protons. The slowest process observed is the complete base pair opening in which the guanosine amino protons also exchange with solvent. For the ammonium salt of the oligonucleotide, a specific ammonium ion complex is observed which at low temperature may catalyze exchange of the guanosine amino protons with the protons of the ammonium ion, but retards exchange with solvent. The complex appears to be specific for the sequence d(CpG).     

Immunochemical detection of multiple conformations within a 36 base pair oligonucleotide.

A 36 base pair chimeric oligonucleotide containing a central core of DNA duplex flanked by RNA/DNA hybrid at each end was synthesized. These distinct regions of the oligonucleotide adopt different conformations which were detected with antibody probes. Enzyme linked immunosorbent assays (ELISA) and a gel electrophoresis retardation assay were used to demonstrate the binding of antibodies which recognize B-DNA, Z-DNA and RNA/DNA hybrid. The DNA duplex core of this oligonucleotide adopts the B-conformation in 0.14 M NaCl. In high salt solution (4 M NaCl) the DNA core adopts the Z-conformation. The RNA/DNA hybrid at the ends of the oligomer adopt a conformation which is distinct from both B-DNA and A-RNA.     

Structural features and hydration of d(C-G-C-G-A-A-T-T-A-G-C-G); a double helix containing two G.A mispairs

Single crystal X-ray diffraction techniques have been used to characterise the molecular structure of the title compound to 2.5A resolution. The structure consists of ten standard Watson-Crick base pairs and two G.A mismatched base pairs. The purine-purine mismatches have guanine in the usual anti orientation with respect to the sugar and adenine in syn orientation. There are two hydrogen bonds formed between the mismatch bases, N-1 and O-6 of guanine with N-7 and N-6 of adenine respectively. The bulky purine-purine mismatches are accommodated with minor perturbation of the sugar-phosphate backbone. There is a slight improvement in base pair overlap at the mismatch sites. Details of the backbone conformation, base stacking interactions and hydration are presented and compared with those of the parent compound d(C-G-C-G-A-A-T-T-C-G-C-G).