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.     

Hexagonal crystal structure of the A-DNA octamer d(GTGTACAC) and its comparison with the tetragonal structure: correlated variations in helical parameters

The alternating DNA octamer d(GTGTACAC) has been grown in a novel hexagonal crystal form. The structure has been determined and refined to a 2-A resolution, with 51 water molecules. The A-DNA conformation is a variant of that observed for the tetragonal form of the same sequence (Jain et al., 1989) containing a bound spermine. The crystals belong to the space group P6(1)22, a = b = 32.40 A and c = 79.25 A, with one strand in the asymmetric unit. The new hexagonal structure was solved by rotation and translation searches in direct space and refined to a final R value of 12.7% by using 1561 unique reflections greater than 1.5 sigma (I). The electron density clearly shows that the penultimate A7 sugar had flipped into the alternative C2'-endo pucker. This dent in the molecule can be attributed to close intermolecular contacts. In contrast, in the tetragonal structure, the DNA is distorted in the central TA step, where the A5 backbone bonds C4'-C5' and O5'-P assume trans conformations. The hexagonal double helix more closely resembles the fiber diffraction A-DNA, compared to the tetragonal form. For instance, the tilt angle is higher (16 degrees vs 10 degrees), which is correlated with a larger displacement from the helix axis (3.5 vs 3.3), a lower rise per residue (2.9 vs 3.2), and a smaller major-groove width (6.1 vs 8.7), thus indicating that the variations in these global helical parameters are correlated. The propeller twist angles in both forms are higher for the G-C base pairs (15.3 degrees, 12.14 degrees) than for the A-T base pairs (10.8 degrees, 9.1 degrees), which is the reverse of the expected order. Unlike the tetragonal structure, the hexagonal crystal structure interestingly does not contain a bound spermine molecule. Our analysis reveals that the conformational differences between the tetragonal and hexagonal forms are not entirely due to the spermine binding, and crystal packing seems to play an important role.     

Molecular structure of an A-DNA decamer d(ACCGGCCGGT)

The molecular structure of the DNA decamer d(ACCGGCCGGT) has been solved and refined by single-crystal X-ray-diffraction analysis at 0.20 nm to a final R-factor of 18.0%. The decamer crystallizes as an A-DNA double helical fragment with unit-cell dimensions of a = b = 3.923 nm and c = 7.80 nm in the space group P6(1)22. The overall conformation of this A-DNA decamer is very similar to that of the fiber model for A-DNA which has a large average base-pair tilt and hence a wide and shallow minor groove. This structure is in contrast to that of several A-DNA octamers in which the molecules all have low base-pair-tilt angles (8-12 degrees) resulting in an appearance intermediate between B-DNA and A-DNA. The average helical parameters of this decamer are typical of A-DNA with 10.9 base pairs/turn of helix, an average helical twist angle of 33.1 degrees, and a base-pair-tilt angle of 18.2 degrees. However, the CpG step in this molecule has a low local-twist angle of 24.5 degrees, similar to that seen in other A-DNA oligomers, and therefore appears to be an intrinsic stacking pattern for this step. The molecules pack in the crystal using a recurring binding motif, namely, the terminal base pair of one helix abuts the surface of the shallow minor groove of another helix. In addition, the GC base pairs have large propeller-twist angles, unlike those found most other A-DNA structures.     

Crystal structure of the self-complementary 5''-purine start decamer d(GCACGCGTGC) in the A-DNA conformation. II.

The crystal structure of the alternating 5'-purine start decamer d(GCGCGCGCGC) was found to be in the left-handed Z-DNA conformation. Inasmuch as the A.T base pair is known to resist Z-DNA formation, we substituted A.T base pairs in the dyad-related positions of the decamer duplex. The alternating self-complementary decamer d(GCACGCGTGC) crystallizes in a different hexagonal space group, P6(1)22, with very different unit cell dimensions a = b = 38.97 and c = 77.34 A compared with the all-G.C alternating decamer. The A.T-containing decamer has one strand in the asymmetric unit, and because it is isomorphous to some other A-DNA decamers it was considered also to be right-handed. The structure was refined, starting with the atomic coordinates of the A-DNA decamer d(GCGGGCCCGC), by use of 2491 unique reflections out to 1.9-A resolution. The refinement converged to an R value of 18.6% for a total of 202 nucleotide atoms and 32 water molecules. This research further demonstrates that A.T base pairs not only resist the formation of Z-DNA but can also assist the formation of A-DNA by switching the helix handedness when the oligomer starts with a 5'-purine; also, the length of the inner Z-DNA stretch (d(CG)n) is reduced from an octamer to a tetramer. It may be noted that these oligonucleotide properties are in crystals and not necessarily in solutions.     

Effect of a single 3''-methylene phosphonate linkage on the conformation of an A-DNA octamer double helix.

The three-dimensional structure of the self-complementary DNA octamer d(GCCCGpGGC) has been determined in the crystalline state using X-ray diffraction data to a nominal resolutoin of 2.12 measured from a very small crystal at DESY, Hamburg. The structure was refined with stereochemical restraints to an R value of 17.1%. d(GCCCGpGGC), containing one single 3'-methylene phosphonate linkage (denoted p), forms an A-DNA double helix with strict dyad symmetry, that is distinct from canonical A-DNA by a wide open major groove and a small average base-pair inclination against the helix axis. The conformation of the unmodified control d(GCCCGGGC) is known from an X-ray analysis of isomorphous crystals (Heinemann et al. (1987) Nucleic Acids Res. 15, 9531-9550). Comparison of the two structures reveals only minor conformational differences, most notably in the pucker of the reduced deoxyribose. It is suggested that oligonucleotides with charged 3'-methylene phosphonate groups may form stable duplexes with complementary DNA or RNA strands rendering them candidates for use as gene-regulatory antisense probes.     

A note on crystal packing and global helix structure in short A-DNA duplexes

A simple relation exists between the packing density in crystals of short A-DNA duplexes and their global double-helical structure. The volume per nucleotide pair shows a linear inverse correlation with the mean displacement of base pairs from the best straight helix axis. The mean displacement is a measure of major groove depth and varies between -3.3 A and -4.9 A in A-form oligonucleotides analysed in the crystalline state. Since the mean displacement of base pairs from the helix axis determines other helical parameters such as base-pair longitudinal slide, its correlation with crystal packing is of considerable interest. The displacement-packing correlation is very clear for octamer duplexes which crystallize in three different lattices. Longer A-helical fragments sometimes deviate from the rule. It may be speculated whether A-form duplexes not completing a full helical turn are especially prone to distortions due to packing in crystals or arising from intermolecular contacts in solution.     

Base only binding of spermine in the deep groove of the A-DNA octamer d(GTGTACAC)

The crystal structure of a complex of spermine with the DNA octamer d(GTGTACAC) has been determined at 2.0-A resolution. The alternating sequence adopts an A-DNA conformation with a novel purine-purine extra-Watson-Crick hydrogen bond involving the central guanine G3 (G11) and adenine A13 (A5) in the deep groove. The oligocation spermine binds in the floor of the deep groove by interacting with the bases and assumes an S-shape. Its dyad is coincident with that of the DNA, reminiscent of repressor binding to B-DNA. The terminal and central ammonium groups of the top half of spermine form hydrogen-bonding interactions to the 5'-bases, GTG, of one strand; then the spermine winds across the groove to interact with the corresponding set of bases on the other strand. The methylene groups of spermine form a hydrophobic cluster with the methyl groups of the thymines and the O6 atoms of the guanines of the TGT sequences on either side of the dyad. The observed mode of binding of spermine to A-DNA can serve as a model for deep groove binding in RNA and DNA-RNA hybrids that show a propensity also for the A-conformation. It will be of interest to see if base binding of spermine to DNA is involved in the regulation of gene expression, since spermine and other oligocations are ubiquitous in cells and their concentration is coupled to stages in cell cycle.     

Sequence-dependent conformation of DNA duplexes. The AATT segment of the d(G-G-A-A-T-T-C-C) duplex in aqueous solution

The nonexchangeable base and sugar protons of the octanucleotide d(G-G-A-A-T-T-C-C) have been assigned by two-dimensional correlated (COSY) and nuclear Overhauser effect (NOESY) methods in aqueous solution. The assignments are based on distance connectivities of less than 4.5 A established from NOE effects between base and sugar protons on the same strand and occasionally between strands, as well as, coupling connectivities within the protons on each sugar ring. We observe the NOEs to exhibit directionality and are consistent with the d(G-G-A-A-T-T-C-C) duplex adopting a right-handed helix in solution. The relative magnitude of the NOEs between base and sugar H2' protons of the same and 5'-adjacent sugars characterizes the AATT segment to the B-helix type in solution.     

Cobalt hexammine induced tautomeric shift in Z-DNA: the structure of d(CGCGCA)*d(TGCGCG) in two crystal forms

We report here the crystal structure of the DNA hexamer duplex d(CGCGCA).d(TGCGCG) at 1.71 Å resolution. The crystals, in orthorhombic space group, were grown in the presence of cobalt hexammine, a known inducer of the left-handed Z form of DNA. The interaction of this ion with the DNA helix results in a change of the adenine base from the common amino tautomeric form to the imino tautomer. Consequently the A:T base pair is disrupted from the normal Watson–Crick base pairing to a ‘wobble’ like base pairing. This change is accommodated easily within the helix, and the helical parameters are those expected for Z-DNA. When the cobalt hexammine concentration is decreased slightly in the crystallization conditions, the duplex crystallizes in a different, hexagonal space group, with two hexamer duplexes in the asymmetric unit. One of these is situated on a crystallographic 6-fold screw axis, leading to disorder. The tautomeric shift is not observed in this space group. We show that the change in inter-helix interactions that lead to the two different space groups probably arise from the small decrease in ion concentration, and consequently disordered positions for the ion.     

The structure of a full turn of an A-DNA duplex d(CGCGGGTACCCGCG)2

We report the 2.6 Å resolution crystal structure of the tetra-decamer d(CGCGGGTACCCGCG) in the tetragonal space group P4₃. This sequence contains the KpnI restriction site GGTACC in the centre which is flanked by alternating ‘CG’ sequences, and has a ‘TA’ step at the centre. These are features could favour the left-handed Z type helix. Despite this, overall the molecule has the A form. This is the first tetra-decamer crystallized in the A-DNA conformation, i.e. more than one full turn of the A helix. The crystallographic asymmetric unit consists of one tetra-decamer duplex. The helical twist and slide, as well as the base pair–base pair stacking interactions show alternations at the alternating pyrimidine–purine and purine–pyrimidine base steps. This variation is reminiscent of the dinucleotide repeat in left-handed Z-DNA helices. The crystal packing is unlike other A-DNA crystal structures, with each helix having a large number of contacts of many different types with symmetry-related neighbours.     

The conformation of the d(ACCCGGGT) duplex in aqueous solution

The nonexchangeable base and sugar protons of the octanucleotide d(ACCCGGGT)2 have been assigned using two dimensional homonuclear Hartmann-Hahn relayed spectroscopy (HOHAHA), double quantum filtered homonuclear correlation spectroscopy (DQFCOSY) and nuclear Overhauser spectroscopy (NOESY) in D2O at 12 degrees C. The observed NOE's between the base protons and their own H2' protons and between the base protons and the H2' protons of the 5' adjacent nucleotide and the observed coupling constants between the deoxyribose 1' and 2',2' protons indicate that this duplex assumes a right-handed B-type helix conformation in solution.     

Crystal and molecular structure of d(GTGCGCAC): investigation of the effects of base sequence on the conformation of octamer duplexes.

The structure of the self-complementary deoxyoctanucleotide d(GTGCGCAC), which crystallized as an A-type helix in the space group P4(3)2(1)2, with one strand in the crystallographic asymmetric unit has been determined and refined to a final R-value of 0.154 using 1.64-A diffraction data collected on an area detector. In contrast to the closely related sequence d(GTGTACAC)tet, there was no evidence for an ordered spermine molecule in the major groove of this octamer. Ordered water is found associated with almost all the exposed hydrogen bonding groups of the octamer. A pentagonal ring of water molecules is hydrogen bonded to O6 and N7 of G3 and the N4 and O6 of the C4.G13 base pair. A detailed comparison of the local helical parameters of d(GTGCGCAC) and d(GTGTACAC)tet is presented. The base sequence change at the center of the octamers affects several of the local helical parameters, via both intra- and interduplex interactions within the crystal.     

Model of the packing of DNA and histone octamer in the structure of the nucleosome

A model is proposed which describes the packing of polypeptide chains of histone molecules in the octamer (H3--H4--H2A--H2B)2, and interlocation of DNA and octamer in the nucleosome. DNA packing in the nucleosome is provided for by electrostatic interactions between DNA phosphates and cationic groups located on the globular part surface of histones octamer. The cationic groups of N- and C-end regions of the histone molecules (histones H3 and H4 in particular) additionally stabilize the nucleosome structure.     

Hairpin and duplex formation of the DNA octamer d(m5C-G-m5C-G-T-G-m5C-G) in solution. An NMR study.

The partly self-complementary DNA octamer d(m5C-G-m5C-G-T-G-m5C-G) was investigated by NMR spectroscopy in solution. It is demonstrated that this peculiar DNA fragment, under suitable conditions of concentration, salt and temperature, exclusively prefers to adopt a monomeric hairpin form with a stem of three Watson-Crick type base pairs and a loop of two residues. At high single strand concentration (8 mM DNA) and low temperature (i.e. below 295 K) the hairpin occurs in slow equilibrium with a B-dimer structure. At high ionic strength (greater than or equal to 100 mM Na+) and/or in the presence of methanol a third species appears, which is assigned to a Z-like dimer. In the B form, as well as in the Z dimer, the two central base pairs form G.T wobble pairs with the bases as major tautomers.     

High-resolution A-DNA crystal structures of d(AGGGGCCCCT). An A-DNA model of poly(dG) x poly(dC).

A-DNA conformation is favored by guanine-rich sequences, such as (dG)n x (dC)n, or under low-humidity conditions. Earlier A-DNA crystal structures revealed some conformational variations which may be the result of sequence-dependent effects and/or crystal packing forces. Here we report the high-resolution crystal structure of d(AGGGGCCCCT) in two crystal forms (either in the P212121 or the P6122 space group) to gain insights into the conformation and dynamics of the (dG)n x (dC)n sequence. The P212121 form has been analyzed using data to 1.1 A resolution by the anisotropic temperature factor refinement procedure of the SHELX97 program. Such analysis affords us with the detailed geometric, conformational and motional property of an A-DNA structure. The backbone torsional angles fall in a narrow range, except for the alpha/gamma angles which have two distinct combinations (gauche-/gauche+ or trans/trans). An A-DNA model of poly(dG) x poly(dC) has been constructed using the conformational parameters derived from the crystal structure of the P212121 form. In the crystal structure of the P6122 space group, the central eight base pairs of the decamer adopt A-DNA conformation with the two terminal nucleotides flipped out to form base pairs with the neighboring nucleotides. Comparison of the A-DNA structure of the same sequence from two different crystal forms, reinforced the conclusion that molecules crystallized in the same space group have a more similar conformation, whereas the same molecule crystallized in different space groups has different (local) conformations.     

Conformations of two duplex forms of d(TCGA) in slow-exchange equilibrium characterized by NMR

Two conformations adopted by the tetranucleoside triphosphate d(TCGA) in aqueous solution are in slow-exchange equilibrium on the NMR time scale. 1H and 31P NMR spectra obtained at temperatures below 25 degrees C contain two sets of signals that vary in relative proportions with changing temperature. High-field NMR techniques allow the conformations of these species to be examined. Both forms are right-handed double-helical structures, and their interconversion does not involve a single-stranded species since transfer of saturation is observed between corresponding imino protons held in the base pairs of each duplex. The form that predominates at higher temperatures resembles B-DNA, but the other, while of similar conformation at the ends of the molecule, is distorted at the C-G step. Shearing at the center of the duplex results in interstrand stacking of the two cytosines in a way that is reminiscent of Z-DNA. Distances between nonexchangeable protons in this model are consistent with nuclear Overhauser effects observed for resonances of the low-temperature form, while the 1H NMR spectrum shows cytidine H-2' resonances at unusually high field. The relative stabilities of the two forms are discussed in terms of base stacking and hydration, but the origin of the high activation energy for interconversion implicit in the slow-exchange rate is unclear. The conformation of the low-temperature form may represent a sequence-dependent structural feature important in natural DNA, although somewhat fortuitously exemplified by this tetramer. The suggested involvement in correct nucleosome phasing of the pentamer d(TTCGA), present in some eukaryotic genes, is noted.     

Variability in DNA minor groove width recognised by ligand binding: the crystal structure of a bis-benzimidazole compound bound to the DNA duplex d(CGCGAATTCGCG)2.

An analogue of the DNA-binding compound Hoechst 33258, in which the piperazine ring has been replaced by an imidazoline group, has been cocrystallized with the dodecanucleotide sequence d(CGCGAATTCGCG)2. The structure has been solved by X-ray diffraction analysis and has been refined to an R-factor of 19.7% at a resolution of 2.0 A. The ligand is found to bind in the minor groove, at the central four AATT base pairs of the B-DNA double helix, with the involvement of a number of van der Waals contacts and hydrogen bonds. There are significant differences in minor groove width for the two compounds, along much of the AATT region. In particular this structure shows a narrower groove at the 3' end of the binding site consistent with the narrower cross-section of the imidazole group compared with the piperazine ring of Hoechst 33258 and therefore a smaller perturbation in groove width. The higher binding affinity to DNA shown by this analogue compared with Hoechst 33258 itself, has been rationalised in terms of these differences.     

Interaction between the Z-type DNA duplex and 1,3-propanediamine: crystal structure of d(CACGTG)2 at 1.2 A resolution

The crystal structure of a hexamer duplex d(CACGTG)(2) has been determined and refined to an R-factor of 18.3% using X-ray data up to 1.2 A resolution. The sequence crystallizes as a left-handed Z-form double helix with Watson-Crick base pairing. There is one hexamer duplex, a spermine molecule, 71 water molecules, and an unexpected diamine (Z-5, 1,3-propanediamine, C(3)H(10)N(2)) in the asymmetric unit. This is the high-resolution non-disordered structure of a Z-DNA hexamer containing two AT base pairs in the interior of a duplex with no modifications such as bromination or methylation on cytosine bases. This structure does not possess multivalent cations such as cobalt hexaammine that are known to stabilize Z-DNA. The overall duplex structure and its crystal interactions are similar to those of the pure-spermine form of the d(CGCGCG)(2) structure. The spine of hydration in the minor groove is intact except in the vicinity of the T5A8 base pair. The binding of the Z-5 molecule in the minor grove of the d(CACGTG)(2) duplex appears to have a profound effect in conferring stability to a Z-DNA conformation via electrostatic complementarity and hydrogen bonding interactions. The successive base stacking geometry in d(CACGTG)(2) is similar to the corresponding steps in d(CG)(3). These results suggest that specific polyamines such as Z-5 could serve as powerful inducers of Z-type conformation in unmodified DNA sequences with AT base pairs. This structure provides a molecular basis for stabilizing AT base pairs incorporated into an alternating d(CG) sequence.     

G . T base-pairs in a DNA helix: the crystal structure of d(G-G-G-G-T-C-C-C)

The synthetic deoxyoctanucleotide d(G-G-G-G-T-C-C-C) crystallizes as an A-type DNA double helix containing two adjacent G . T base-pair mismatches. The structure has been refined to an R-factor of 14% at 2.1 A resolution with 104 solvent molecules located. The two G . T mismatches adopt the "wobble" form of base-pairing. The mismatched bases are linked by a network of water molecules interacting with the exposed functional groups in both the major and minor grooves. The presence of two mispaired bases in the octamer has surprisingly little effect on the global structure of the helix or the backbone and glycosidic torsional angles. Base stacking around the mismatch is perturbed, but the central G-T step shows particularly good base overlap, which may contribute to the relatively high stability of this oligomer.     

The crystal structure analysis of d(CGCGAASSCGCG)2, a synthetic DNA dodecamer duplex containing four 4'-thio-2'-deoxythymidine nucleotides.

The crystal structure refinement of the synthetic dodecamer d(CGCGAASSCGCG), where S = 4'-thio-2'-deoxythymidine, has converged at R=0.201 for 2605 reflections with F > 2sigma(F) in the resolution range 8.0-2.4 A for a model consisting of the dodecamer duplex and 66 water molecules. A comparison of its structure with that of the native dodecamer d(CGCGAATTCGCG) has revealed that the major differences between the two structures is a change in the conformation of the sugar-phosphate backbone in the regions at and adjacent to the positions of the modified nucleosides. Examination of the fine structural parameters for each of the structures reveals that the thiosugars adopt a C3'-exo conformation in d(CGCGAASSCGCG), rather than the approximate C1'-exo conformation found for the analogous sugars in the structure of d(CGCGAATTCGCG). The observed differences in structure between the two duplexes may help to explain the enhanced resistance to nuclease digestion of synthetic oligonucleotides containing 4'-thio-2'-deoxynucleotides.