Refined crystal structure of an octanucleotide duplex with G . T mismatched base-pairs

Single crystal X-ray diffraction techniques have been used to determine the structure of the DNA octamer d(G-G-G-G-C-T-C-C) at a resolution of 2.25 A. The asymmetric unit consists of two strands coiled about each other to produce an A-type DNA helix. The double helix contains six G . C Watson-Crick base-pairs and two G . T mismatched base-pairs. The mismatches adopt a "wobble" type structure in which both bases retain their major tautomer forms. The double helix is able to accommodate this G . T pairing with little distortion of the overall helical conformation. Crystals of this octamer melt at a substantially lower temperature than do those of a related octamer also containing two G . T base-pairs. We attribute this destabilization to disruption of the hydration network around the mismatch site combined with changes in intermolecular packing. Full details are given of conformational parameters, base stacking, intermolecular contacts and hydration involving 52 solvent molecules.     

Solution structure of a DNA double helix incorporating four consecutive non-Watson-Crick base-pairs

A series of DNA 21-mers containing a variety of the 4 x 4 internal loop sequence 5'-CAAG-3'/3'-ACGT-5' were studied using nuclear magnetic resonance (NMR) methodology and distance geometry (DG)/molecular dynamics (MD) approaches. Such oligomers exhibit excellent resolution in the NMR spectra and reveal many unusual NOEs (nuclear Overhauser effect) that allow for the detailed characterization of a DNA hairpin incorporating a track of four different non-Watson-Crick base-pairs in the stem. These include a wobble C.A base-pair, a sheared A.C base-pair, a sheared A.G base-pair, and a wobble G.T base-pair. Significantly different twisting angles were observed between the base-pairs in internal loop that results with excellent intra-strand and inter-strand base stacking within the four consecutive mismatches and the surrounding canonical base-pairs. This explains why it melts at 52 degrees C even though five out of ten base-pairs in the stem adopt non-Watson-Crick pairs. However, the 4 x 4 internal loop still fits into a B-DNA double helix very well without significant change in the backbone torsion angles; only zeta torsion angles between the tandem sheared base-pairs are changed to a great extent from the gauche(-) domain to the trans domain to accommodate the cross-strand base stacking in the internal loop. The observation that several consecutive non-canonical base-pairs can stably co-exist with Watson-Crick base-pairs greatly increases the limited repertoire of irregular DNA folds and reveals the possibility for unusual structural formation in the functionally important genomic regions that have potential to become single-stranded.     

Molecular and crystal structure of d(CGCGmo4CG): N4-methoxycytosine.guanine base-pairs in Z-DNA

The base analogue N4-methoxycytosine (mo4C) is ambivalent in its hydrogen-bonding potential, since it forms stable base-pairs with both adenine and guanine in oligomer duplexes. To investigate the base-pair geometry, the structure of d(CGCGmo4CG) has been determined by single-crystal X-ray diffraction techniques. The d(CGCGmo4CG)2 crystallized in a left-handed double helical structure (Z-type). Refinement using 2559 reflections between 10 and 1.7 A converged with a final R = 0.181 (Rw = 0.130) including 68 solvent molecules. The orthorhombic crystals are in the space group P2(1)2(1)2(1), with cell dimensions a = 18.17 A, b = 30.36 A, c = 43.93 A. The mo4C.G base-pair is of the wobble type, with mo4C in the imino form, and the methoxy group in the syn configuration.     

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.     

Structure of d(T)n.d(A)n.d(T)n: the DNA triple helix has B-form geometry with C2'-endo sugar pucker.

The polynucleotide helix d(T)n.d(A)n.d(T)n is the only deoxypolynucleotide triple helix for which a structure has been published, and it is generally assumed as the structural basis for studies of DNA triplexes. The helix has been assigned to an A-form conformation with C3'-endo sugar pucker by Arnott and Selsing [1974; cf. Arnott et al. (1976)]. We show here by infrared spectroscopy in D2O solution that the helix is instead B-form and that the sugar pucker is in the C2'-endo region. Distamycin A, which binds only to B-form and not to A-form helices, binds to the triple helix without displacement of the third strand, as demonstrated by CD spectroscopy and gel electrophoresis. Molecular modeling shows that a stereochemically satisfactory structure can be build using C2'-endo sugars and a displacement of the Watson-Crick base-pair center from the helix axis of 2.5 A. Helical constraints of rise per residue (h = 3.26 A) and residues per turn (n = 12) were taken from fiber diffraction experiments of Arnott and Selsing (1974). The conformational torsion angles are in the standard B-form range, and there are no short contacts. In contrast, we were unable to construct a stereochemically allowed model with A-form geometry and C3'-endo sugars. Arnott et al. (1976) observed that their model had short contacts (e.g., 2.3 A between the phosphate-dependent oxygen on the A strand and O2 in the Hoogsteen-paired thymine strand) which are generally known to be outside the allowed range.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence-dependent conformation of an A-DNA double helix. The crystal structure of the octamer d(G-G-T-A-T-A-C-C)

The crystal structures of the synthetic self-complementary octamer d(G-G-T-A-T-A-C-C) and its 5-bromouracil-containing analogue have been refined to R values of 20% and 14% at resolutions of 1.8 and 2.25 A, respectively. The molecules adopt and A-DNA type double-helical conformation, which is minimally affected by crystal forces. A detailed analysis of the structure shows a considerable influence of the nucleotide sequence on the base-pair stacking patterns. In particular, the electrostatic stacking interactions between adjacent guanine and thymine bases produce symmetric bending of the double helix and a major-groove widening. The sugar-phosphate backbone appears to be only slightly affected by the base sequence. The local variations in the base-pair orientation are brought about by correlated adjustments in the backbone torsion angles and the glycosidic orientation. Sequence-dependent conformational variations of the type observed here may contribute to the specificity of certain protein-DNA interactions.     

The crystal structure of N4-methylcytosine.guanosine base-pairs in the synthetic hexanucleotide d(CGCGm4CG).

The structure of d(CGCGm4CG) were m4C = N4-methylcytosine has been determined by crystallographic methods. The crystals are multifaced prisms, with orthorhombic space group P2(1)2(1)2(1) and unit cell dimensions of a = 17.98, b = 30.77 and c = 44.75A. The asymmetric unit consists of one duplex of hexanucleotide and 49 waters. The R-factor is 0.189 for 1495 reflections with F > or = sigma(F) to a resolution limit of 1.8A. The double helix has a Z-DNA type structure which appears to be intermediate in structure to the two previously characterised structure types for Z-DNA hexamers. The two m4C.G base-pairs adopt structures that are very similar to those of the equivalent base-pairs in the structure of the native sequence d(CGCGCG) except for the presence of the methyl groups which are trans to the N3 atoms of their parent nucleotides and protrude into the solvent region. The introduction of the modified base-pairs into the d(CGCGCG) duplex appears to have a minimal effect on the overall base-pair morphology of the Z-DNA duplex.     

G.T wobble base-pairing in Z-DNA at 1.0 A atomic resolution: the crystal structure of d(CGCGTG).

The DNA oligomer d(CGCGTG) crystallizes as a Z-DNA double helix containing two guanine-thymine base pair mismatches of the wobble type. The crystal diffracts to 1 A resolution and the structure has been solved and refined. At this resolution, a large amount of information is revealed about the organization of the water molecules in the lattice generally and more specifically around the wobble base pairs. By comparing this structure with the analogous high resolution structure of d(CGCGCG) we can visualize the structural changes as well as the reorganization of the solvent molecules associated with wobble base pairing. There is only a small distortion of the Z-DNA backbone resulting from introduction of the GT mismatched base pairs. The water molecules cluster around the wobble base pair taking up all of the hydrogen bonding capabilities of the bases due to wobble pairing. These bridging water molecules serve to stabilize the base-base interaction and, thus, may be generally important for base mispairing either in DNA or in RNA molecules.     

Thermodynamics of triple helix formation: spectrophotometric studies on the d(A)10.2d(T)10 and d(C+3T4C+3).d(G3A4G3).d(C3T4C3) triple helices.

We have stabilized the d(A)10.2d(T)10 and d(C+LT4C+3).d(G3A4G3).d(C3T4C3) triple helices with either NaCl or MgCl2 at pH 5.5. UV mixing curves demonstrate a 1:2 stoichiometry of purine to pyrimidine strands under the appropriate conditions of pH and ionic strength. Circular dichroic titrations suggest a possible sequence-independent spectral signature for triplex formation. Thermal denaturation profiles indicate the initial loss of the third strand followed by dissociation of the underlying duplex with increasing temperature. Depending on the base sequence and ionic conditions, the binding affinity of the third strand for the duplex at 25 degrees C is two to five orders of magnitude lower than that of the two strands forming the duplex. Thermodynamic parameters for triplex formation were determined for both sequences in the presence of 50 mM MgCl2 and/or 2.0 M NaCl. Hoogsteen base pairs are 0.22-0.64 kcal/mole less stable than Watson-Crick base pairs, depending on ionic conditions and base composition. C+.G and T.A Hoogsteen base pairs appear to have similar stability in the presence of Mg2+ ions at low pH.     

Association of DNA quadruplexes through G:C:G:C tetrads. Solution structure of d(GCGGTGGAT)

The structure formed by the DNA sequence d(GCGGTGGAT) in a 100 mM Na(+) solution has been determined using molecular dynamics calculations constrained by distance and dihedral restraints derived from NMR experiments performed at isotopic natural abundance. The sequence folds into a dimer of dimers. Each symmetry-related half contains two parallel stranded G:G:G:G tetrads flanked by an A:A mismatch and by four-stranded G:C:G:C tetrads. Each of the two juxtaposed G:C:G:C tetrads is composed of alternating antiparallel strands from the two halves of the dimer. For each single strand, a thymine intersperses a double chain reversal connecting the juxtaposed G:G:G:G tetrads. This architecture has potential implications in genetic recombination. It suggests a pathway for oligomerization involving association of quadruplex entities through GpC steps.     

Amine free crystal structure: the crystal structure of d(CGCGCG)2 and methylamine complex crystal

We succeeded in the crystallization of d(CGCGCG)2 and methylamine Complex. The crystal was clear and of sufficient size to collect the X-ray crystallographic data up to 1.0 A resolution using synchrotron radiation. As a result of X-ray crystallographic analysis of 2Fo-Fc map was much clear and easily traced. It is the first time monoamine co-crystallizes with d(CGCGCG)2. However, methylamine was not found from the complex crystal of d(CGCGCG)2 and methylamine. Five Mg ions were found around d(CGCGCG)2 molecules. These Mg ions neutralized the anion of 10 values of the phosphate group of DNA with five Mg2+. DNA stabilized only by a metallic ion and there is no example of analyzing the X-ray crystal structure like this. Mg ion stabilizes the conformation of Z-DNA. To use monoamine for crystallization of DNA, we found that we can get only d(CGCGCG)2 and Mg cation crystal. Only Mg cation can stabilize the conformation of Z-DNA. The method of using the monoamine for the crystallization of DNA can be applied to the crystallization of DNA of long chain of length in the future like this.     

Genetic conversion of G.C base-pairs to A.U base-pairs in a transfer RNA

The cloverleaf stem segments of the suppressor gene of bacteriophage T4 tRNA(Gln) contain ten G.C and ten A.U base-pairs. To gain a better appreciation of the G.C base-pair requirement, we isolated multiple mutants of this suppressor gene in which base-pairs of G.C were replaced by A.U. One active suppressor gene contained only A.U base-pairs on the anticodon stem, indicating that G.C base-pairs in this region of tRNA(Gln) are not essential for function. In contrast, replacement was not possible at two base-pairs on the D stem and at one base-pair on the T stem.     

The solution structure of d(G(4)T(4)G(3))(2): a bimolecular G-quadruplex with a novel fold

The G-rich 11-mer oligonucleotide d(G(4)T(4)G(3)) forms a bimolecular G-quadruplex in the presence of sodium ions with a topology that is distinct from the folds of the closely related and well-characterized sequences d(G(4)T(4)G(4)) and d(G(3)T(4)G(3)). The solution structure of d(G(4)T(4)G(3))(2) has been determined using a combination of NMR spectroscopy and restrained molecular dynamics calculations. d(G(4)T(4)G(3))(2) forms an asymmetric dimeric fold-back structure consisting of three stacked G-quartets. The two T(4) loops that span diagonally across the outer faces of the G-quartets assume different conformations. The glycosidic torsion angle conformations of the guanine bases are 5'-syn-anti-syn-anti-(T(4) loop)-anti-syn-anti in one strand and 5'-syn-anti-syn-anti-(T(4) loop)-syn-anti-syn in the other strand. The guanine bases of the two outer G-quartets exhibit a clockwise donor-acceptor hydrogen-bonding directionality, while those of the middle G-quartet exhibit the anti-clockwise directionality. The topology of this G-quadruplex, like other bimolecular fold-back structures with diagonal loops, places each strand of the G-quartet region next to a neighboring parallel and an anti-parallel strand. The two guanine residues not involved in G-quartet formation, G4 and G12 (i.e. the fourth guanine base of one strand and the first guanine base of the other strand), adopt distinct conformations. G4 is stacked on top of an adjacent G-quartet, and this base-stacking continues along with the bases of the loop residues T5 and T6. G12 is orientated away from the core of G-quartets; stacked on the T7 base and apparently involved in hydrogen-bonding interactions with the phosphodiester group of this same residue. The cation-dependent folding of the d(G(4)T(4)G(3))(2) quadruplex structure is distinct from that observed for similar sequences. While both d(G(4)T(4)G(4)) and d(G(3)T(4)G(3)) form bimolecular, diagonally looped G-quadruplex structures in the presence of Na(+), K(+) and NH(4)(+), we have observed this folding to be favored for d(G(4)T(4)G(3)) in the presence of Na(+), but not in the presence of K(+) or NH(4)(+). The structure of d(G(4)T(4)G(3))(2) exhibits a "slipped-loop" element that is similar to what has been proposed for structural intermediates in the folding pathway of some G-quadruplexes, and therefore provides support for the feasibility of these proposed transient structures in G-quadruplex formation.     

Base pairing structure in the poly d(G-T) double helix: wobble base pairs.

High resolution nuclear magnetic resonance (NMR) and ethidium bromide binding studies are used to demonstrate that poly d(G-T) forms an ordered double helical structure at low temperatures (below 24 degrees C in 0.3 M NaCl) in which G and T are hydrogen bonded together in a wobble base pair hydrogen bonding scheme as proposed earlier by Lezius and Domin. Alternative hydrogen bonding schemes involving the tautomeric form of either T or G, such as have been proposed to account for mutation rates in DNA synthesis, are eliminated.     

B-form to A-form conversion by a 3'-terminal ribose: crystal structure of the chimera d(CCACTAGTG)r(G)

The crystal structure of the chimerical decamer d(CCACTAGTG)r(G), bearing a 3′-terminal ribo-guanidine, has been solved and refined at 1.8 Å resolution (R-factor 16.6%; free R-factor 22.8%). The decamer crystallizes in the orthorhombic space group P2₁2₁2₁ with unit cell constants a = 23.90 Å, b = 45.76 Å and c = 49.27 Å. The structure was solved by molecular replacement using the coordinates of the isomorphous chimera r(GCG)d(TATACGC). The final model contains one duplex and 77 water molecules per asymmetric unit. Surprisingly, all residues adopt a conformation typical for A-form nucleic acids (C3′-endo type sugar pucker) although the all-DNA analog, d(CCACTAGTGG), has been crystallized in the B-form. Comparing circular dichroism spectra of the chimera and the corresponding all-DNA sequence reveals a similar trend of the former molecule to adopt an A-like conformation in solution. The results suggest that the preference of ribonucleotides for the A-form is communicated into the 5′-direction of an oligonucleotide strand, although direct interactions of the 2′-hydroxyl group can only be discerned with nucleotides in the 3′-direction of a C3′-endo puckered ribose. These observations imply that forces like water-mediated contacts, the concerted motions of backbone torsion angles, and stacking preferences, are responsible for such long-range influences. This bi-directional structural communication originating from a ribonucleotide can be expected to contribute to the stability of the A-form within all-RNA duplexes.     

The structure of d(CGCGAAT[]TCGCG) . d(CGCGAATTCGCG); the incorporation of a thymine photodimer into a B-DNA helix

In the light of the biological significance of thymine photodimers , studies of the energetics of the dodecanucleotide fragment d( CGCGAATTCGCG )2 have been carried out using the methods of molecular mechanics, with and without incorporation of a thymine dimer in the cis-syn configuration. The results of the calculations suggest that the thymine dimerized structures show no gross distortion in the double helix with the conformational changes relative to the normal B-DNA double helix restricted largely to the dimer region. The energetics of dTp[]dT reveal a number of conformers which are energetically almost equally favorable and are, as a group, qualitatively consistent with NMR studies on this molecule. The biological implications of the results of the conformational studies, reported here, have been examined vis-a-vis the currently available models for the recognition of DNA "damage" by repair enzymes.     

Persistence analysis of the static and dynamical helix deformations of DNA oligonucleotides: Application to the crystal structure and molecular dynamics simulation of d(CGCGAATTCGCG)2

A theory and graphical presentation for the analysis of helix structure and deformations in oligonucleotides is presented. The parameters “persistence” and “flexibility” as defined in the configurational statistics of polymers of infinite length are reformulated at the oligonucleotide level in an extension of J. A. Schellman's method [(1974) Biopolymers, Vol. 17, pp. 217–226], and used as a basis for a systematic “Persistence Analysis” of the helix deformation properties for all possible subsequences in the structure. The basis for the analysis is a set of link vectors referenced to individual base pairs, and is limited to sequences exhibiting only perturbed rod-like behavior, i.e., below the threshold for supercoiling. The present application of the method is concerned with a physical model for the angular component of bending, so the link vectors are defined as the unit components of a global helix axis obtained by the procedure “Curves” of R. Lavery and H. Sklenar [(1988) J. Biomol. Struct. Dynam., Vol. 6, pp. 63–91; (1989) J. Biomol. Struct. Dynam., Vol. 6, pp. 655–667]. A discussion, of the relationship between global bending and relative orientation of base pairs is provided. Our approach is illustrated by analysis of some model oligonucleotide structures with intrinsic kinks, the crystal structure of the dodecamer d (CGCGAATTCGCG)₂, and the results of two molecular dynamics simulations on this dodecamer using two variations of the GROMOS force field. The results indicate that essentially all aspects of curvature in short oligonucleotides can be determined, such as the position and orientation of each bend, the sharpness or smoothness, and the location and linearity of subsequences. In the case of molecular dynamics simulations, where a Boltzmann ensemble of structures is analyzed, the spatial extent of the deformations (flexibility) is also considered. © 1993 John Wiley & Sons, Inc.     

Further studies on telestability in DNA. The synthesis and characterization of the duplex block polymers d(C20A10) - d(T10G20) and d(C20A15) - d(T15G20).

The synthesis and characterization of the duplex block polymers d(C20A10) - d(T10G20) and d(C20A15) - d(T15G20) are described. Thermal denaturation studies on these DNAs in the absence and presence of actinomycin, which binds only to the GC portions of these molecules, have confirmed and extended our previous observation that the properties of one region of a DNA can be influenced (telestabilized) by a remote region. In addition, the large scale synthesis of d(C15A15) - d(T15G15) is described.     

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.