Crystal structure analysis of the B-DNA dodecamer CGTGAATTCACG.

The crystal structure of the DNA dodecamer C-G-T-G-A-A-T-T-C-A-C-G has been determined at a resolution of 2.5 A, with a final R factor of 15.8% for 1475 nonzero reflections measured at 0 degrees C. The structure is isomorphous with that of the Drew dodecamer, with the space group P2(1)2(1)2(1) and cell dimensions of a = 24.94 A, b = 40.78 A, and c = 66.13 A. The asymmetric unit contains all 12 base pairs of the B-DNA double helix and 36 water molecules. The structure of C-G-T-G-A-A-T-T-C-A-C-G is very similar to that of C-G-C-G-A-A-T-T-C-G-C-G, with no major alterations in helix parameters. Water peaks in the refined structure appear to represent a selection of peaks that were observed in the Drew dodecamer. The minor-groove spine of hydration at 2.5 A is fragmentary, but as Narendra et al. (1991) [Biochemistry (following paper in this issue)] have observed, lowering the temperature leads to a more complete representation of the spine.     

Crystal structure of a B-DNA dodecamer containing inosine, d(CGCIAATTCGCG), at 2.4 A resolution and its comparison with other B-DNA dodecamers.

The crystal structure of the dodecamer, d(CGCIAATTCGCG), has been determined at 2.4 A resolution by molecular replacement, and refined to an R-factor of 0.174. The structure is isomorphous with that of the B-DNA dodecamer, d(CGCGAATTCGCG), in space group P2(1)2(1)2(1) with cell dimensions of a = 24.9, b = 40.4, and c = 66.4 A. The initial difference Fourier maps clearly indicated the presence of inosine instead of guanine. The structure was refined with 44 water molecules, and compared to the parent dodecamer. Overall the two structures are very similar, and the I:C forms Watson-Crick base pairs with similar hydrogen bond geometry to the G:C base pairs. The propeller twist angle is low for I4:C21 and relatively high for the I16:C9 base pair (-3.2 degrees compared to -23.0 degrees), and the buckle angles alter, probably due to differences in the contacts with symmetry related molecules in the crystal lattice. The central base pairs of d(CGCIAATTCGCG) show the large propeller twist angles, and the narrow minor groove that characterize A-tract DNA, although I:C base pairs cannot form the major groove bifurcated hydrogen bonds that are possible for A:T base pairs.     

Crystal structure of a Z-DNA hexamer d(CGCICG) at 1.7 A resolution: inosine.cytidine base-pairing, and comparison with other Z-DNA structures.

The crystal structure of the deoxyhexamer, d(CGCICG), has been determined and refined to a resolution of 1.7A. The DNA hexamer crystallises in space group P2(1)2(1)2(1) with unit cell dimensions of a = 18.412 +/- .017 A, b = 30.485 +/- .036A, and c = 43.318 +/- .024 A. The structure has been solved by rotation and translation searches and refined to an R-factor of 0.148 using 2678 unique reflections greater than 1.0 sigma (F) between 10.0-1.7 A resolution. Although the crystal parameters are similar to several previously reported Z-DNA hexamers, this inosine containing Z-DNA differs in the relative orientation, position, and crystal packing interactions compared to d(CGCGCG) DNA. Many of these differences in the inosine form of Z-DNA can be explained by crystal packing interactions, which are responsible for distortions of the duplex at different locations. The most noteworthy features of the inosine form of Z-DNA as a result of such distortions are: (1) sugar puckers for the inosines are of C4'-exo type, (2) all phosphates have the Zl conformation, and (3) narrower minor grove and compression along the helical axis compared to d(CGCGCG) DNA. In addition, the substitution of guanosine by inosine appears to have resulted in Watson-Crick type base-pairing between inosine and cytidine with a potential bifurcated hydrogen bond between inosine N1 and cytidine N3 (2.9 A) and O2 (3.3-3.A).     

Crystal and solution structures of the B-DNA dodecamer d(CGCAAATTTGCG) probed by Raman spectroscopy: heterogeneity in the crystal structure does not persist in the solution structure

The self-complementary dodecamer d(CGCAAATTTGCG) crystallizes as a double helix of the B form and manifests a Raman spectrum with features not observed in Raman spectra of either DNA solutions or wet DNA fibers. A number of Raman bands are assigned to specific nucleoside sugar and phosphodiester conformations associated with this model B-DNA crystal structure. The Raman bands proposed as markers of the crystalline B-DNA structure are compared and contrasted with previously proposed markers of Z-DNA and A-DNA crystals. The results indicate that the three canonical forms of DNA can be readily distinguished by Raman spectroscopy. However, unlike Z-DNA and A-DNA, which retain their characteristic Raman fingerprints in aqueous solution, the B-DNA Raman spectrum is not completely conserved between crystal and solution states. The Raman spectra reveal greater heterogeneity of nucleoside conformations (sugar puckers) in the DNA molecules of the crystal structure than in those of the solution structure. The results are consistent with conversion of one-third of the dG residues from the C2'-endo/anti conformation in the solution structure to another conformation, deduced to be C1'-exo/anti, in the crystal. The dodecamer crystal also exhibits unusually broad Raman bands at 790 and 820 cm-1, associated with the geometry of the phosphodiester backbone and indicating a wider range of (alpha, zeta) backbone torsion angles in the crystal than in the solution structure. The results suggest that backbone torsion angles in the CGC and GCG sequences, which flank the central AAATTT sequence, are significantly different for crystal and solution structures, the former containing the greater diversity.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Crystal structure of d(GCGCGCG) with 5''-overhang G residues.

The crystal structure of the DNA heptamer d(GCGCGCG) has been solved at 1.65 A resolution by the molecular replacement method and refined to an R-value of 0.184 for 3598 reflections. The heptamer forms a Z-DNA d(CGCGCG)2 with 5'-overhang G residues instead of an A-DNA d(GCGCGC)2 with 3'-overhang G residues. The overhang G residues from parallel strands of two adjacent duplexes form a trans reverse Hoogsteen G x G basepair that stacks on the six Z-DNA basepairs to produce a pseudocontinuous helix. The reverse Hoogsteen G x G basepair is unusual in that the displacement of one G base relative to the other allows them to participate in a bifurcated (G1)N2 . . . N7(G8) and an enhanced (G8)C8-H . . . O6(G1) hydrogen bond, in addition to the two usual hydrogen bonds. The 5'-overhang G residues are anti and C2'-endo while the 3'-terminal G residues are syn and C2'-endo. The conformations of both G residues are different from the syn/C3'-endo for the guanosine in a standard Z-DNA. The two cobalt hexammine ions bind to the phosphate groups in both GpC and CpG steps in Z(I) and Z(II) conformations. The water structure motif is similar to the other Z-DNA structures.     

Alternative structures for alternating poly(dA-dT) tracts: the structure of the B-DNA decamer C-G-A-T-A-T-A-T-C-G.

The X-ray crystal structure of the decamer C-G-A-T-A-T-A-T-C-G has been solved with two contrasting cations, Ca2+ and Mg2+. Crystals with calcium are space group P2(1)2(1)2(1), cell dimensions a = 38.76 A, b = 40.06 A, and c = 33.73 A, and diffract to 1.7-A resolution. Crystals with magnesium have the same space group, cell dimensions a = 38.69 A, b = 39.56 A, and c = 33.64 A, and diffract to 2.0 A. Their structures were solved independently by molecular replacement, beginning with idealized Arnott B-DNA geometry. The calcium structure refined to R = 17.8% for the 3683 reflections greater than 2 sigma, with 404 DNA atoms, 95 solvent peaks, and 1 Ca(H2O)7(2+) ion. The magnesium structure refined to R = 16.5% for the 1852 reflections greater than 2 sigma, with 404 DNA atoms, 62 solvent peaks, and 1 Mg(H2O)6(2+) ion. The two structures are virtually identical and are isostructural with C-G-A-T-C-G-A-T-C-G [Grzeskowiak et al. (1991) J. Biol. Chem. 266, 8861-8883] and C-G-A-T-T-A-A-T-C-G [Quintana et al. (1992) J. Mol. Biol. 225, 375-395]. Comparison of C-G-A-T-A-T-A-T-C-G with C-G-C-A-T-A-T-A-T-G-C-G [Yoon et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 6332-6336] shows that the expected alternation of twist angles is found in the central A-T-A-T-A-T region of the decamer (A-T small, T-A large), but the minor groove remains wide at the center, rather than narrow. Minor groove narrowing is produced, in these two structures, not by overwinding of the helix, but by an increase in base pair propeller. This analysis confirms the concept that poly(dA-dT).poly(dA-dT) is polymorphous, with different local conformations possible in different local environments.     

Crystal and molecular structure of a DNA fragment: d(CGTGAATTCACG)

The crystal structure of the dodecanucleotide d(CGTGAATTCACG) has been determined to a resolution of 2.7 A and refined to an R factor of 17.0% for 1532 reflections. The sequence crystallizes as a B-form double helix, with Watson-Crick base pairing. This sequence contains the EcoRI restriction endonuclease recognition site, GAATTC, and is flanked by CGT on the 5'-end and ACG on the 3'-end, in contrast to the CGC on the 5'-end and GCG on the 3'-end in the parent dodecamer d(CGCGAATTCGCG). A comparison with the isomorphous parent compound shows that any changes in the structure induced by the change in the sequence in the flanking region are highly localized. The global conformation of the duplex is conserved. The overall bend in the helix is 10 degrees. The average helical twist values for the present and the parent structures are 36.5 degrees and 36.4 degrees, respectively, corresponding to 10 base pairs per turn. The buckle at the substituted sites are significantly different from those seen at the corresponding positions in the parent dodecamer. Step 2 (GpT) is underwound with respect to the parent structure (27 degrees vs 36 degrees) and step 3 (TpG) is overwound (34 degrees vs 27 degrees). There is a spine of hydration in the narrow minor groove. The N3 atom of adenine on the substituted A10 and A22 bases are involved in the formation of hydrogen bonds with other duplexes or with water; the N3 atom of guanine on G10 and G22 bases in the parent structure does not form hydrogen bonds.     

Molecular structure of the B-DNA dodecamer d(CGCAAATTTGCG)2. An examination of propeller twist and minor-groove water structure at 2.2 A resolution.

The crystal structure of the dodecanucleotide duplex d(CGCAAATTTGCG)2 has been solved to 2.2 A resolution and refined to an R-factor of 18.1% with the inclusion of 71 water molecules. The structure shows propeller twists of up to -20 degrees for the A.T base-pairs, although there is probably only one (weak) three-centre hydrogen bond in the six base-pair AT narrow minor-groove region. An extensive ribbon of hydration has been located in this groove that has features distinctive from the classic "spine of hydration". Solvation around phosphate groups is described, with several instances of water molecules bridging between phosphates.     

Crystal structure of (Gly-Pro-Hyp)(9) : implications for the collagen molecular model

Collagens have long been believed to adopt a triple‐stranded molecular structure with a 10/3 symmetry (ten triplet units in three turns) and an axial repeat of 29 Å. This belief even persisted after an alternative structure with a 7/2 symmetry (seven triplet units in two turns) with an axial repeat of 20 Å had been proposed. The uncertainty regarding the helical symmetry of collagens is attributed to inadequate X‐ray fiber diffraction data. Therefore, for better understanding of the collagen helix, single‐crystal analyses of peptides with simplified characteristic amino acid sequences and similar compositions to collagens have long been awaited. Here we report the crystal structure of (Gly‐Pro‐Hyp)₉ peptide at a resolution of 1.45 Å. The repeating unit of this peptide, Gly‐Pro‐Hyp, is the most typical sequence present in collagens, and it has been used as a basic repeating unit in fiber diffraction analyses of collagen. The (Gly‐Pro‐Hyp)₉ peptide adopts a triple‐stranded structure with an average helical symmetry close to the ideal 7/2 helical model for collagen. This observation strongly suggests that the average molecular structure of collagen is not the accepted Rich and Crick 10/3 helical model but is a 7/2 helical conformation. © 2012 Wiley Periodicals, Inc. Biopolymers 97: 607–616, 2012.     

Crystal structure at 1.5-A resolution of d(CGCICICG), an octanucleotide containing inosine, and its comparison with d(CGCG) and d(CGCGCG) structures.

The octadeoxyribonucleotide d(CGCICICG) has been crystallized in space group P(6)5(22) with unit cell dimensions of a = b = 31.0 A and c = 43.7 A, and X-ray diffraction data have been collected to 1.5-A resolution. Precession photographs and the self-Patterson function indicate that 12 base pairs of Z-conformation DNA stack along the c-axis, and the double helices pack in a hexagonal array similar to that seen in other crystals of Z-DNA. The structure has been solved by both Patterson deconvolution and molecular replacement methods and refined in space group P(6)5 to an R factor of 0.225 using 2503 unique reflections greater than 3.0 sigma (F). Comparison of the molecules within the hexagonal lattice with highly refined crystal structures of other Z-DNA reveals only minor conformational differences, most notably in the pucker of the deoxyribose of the purine residues. The DNA has multiple occupancy of C:I and C:G base pairs, and C:I base pairs adopt a conformation similar to that of C:G base pairs.     

Spectroscopic evidence for an intermediate in the T6 to R6 allosteric transition of the Co(II)-substituted insulin hexamer.

The phenol-induced conformational transition in the insulin hexamer is known to involve a large change in structure wherein residues 1-8 of the insulin B-chain are transformed from an extended coil (T-state) to a helix (R-state). This change in protein conformation both exposes a cryptic protein pocket on each subunit to which phenol binds and forces the HisB10 zinc sites to undergo a change in coordination geometry from octahedral to tetrahedral [Derewenda, U., Derewenda, Z., Dodson, E. J., Dodson, G. G., Reynolds, C. D., Smith, G. D., Sparks, C., & Swensen, D. (1989) Nature 338, 593-596]. Substitution of Co(II) for Zn(II) at the HisB10 sites introduces a sensitive chromophoric probe of the structural and chemical events that occur during this allosteric transition [Roy, M., Brader, M. L., Lee, R. W.-K., Kaarsholm, N. C., Hansen, J. F., & Dunn, M. F. (1989) J. Biol. Chem. 264, 19081-19085]. In this study, using rapid-scannig stopped-flow (RSSF) UV-visible spectroscopic studies, we demonstrate that a transient chemical intermediate is formed during the phenol-induced conversion of Co(II)-substituted hexamer from the T-state to the R-state. Decomposition of the RSSF spectra gave a spectrum for the intermediate with d-d transitions consistent with the assignment of the intermediate as either a distorted tetrahedral or a 5-coordinate Co(II) species. Possible structures for the intermediate and the implications of these findings to the allosteric mechanism are considered.     

Vibrational circular dichroism studies of the A-to-B conformational transition in DNA.

The vibrational circular dichroism (VCD) spectra of several natural DNAs as well as tRNA, poly(dG-dC).poly(dG-dC), and poly(dA-dT).poly(dA-dT) are reported for the base deformation modes in the IR region from 1700 to 1550 cm-1 for the polymers in D2O as well as in high alcohol dehydrating conditions. Spectra of both the B- and A-forms were identified. The A-form DNA VCD, not previously reported, has characteristics that can be found in the VCD spectra of RNAs as would be expected from the similarity of their structures. The VCD is sequence-dependent. Under the dehydrating conditions studied, poly(dA-dT)poly(dA-dT),poly(dA).poly(dT), and a high-A-T fraction natural DNA had a different bandshape from the other DNAs, which was similar to that of poly(rA).poly(rU). Poly(dG-dC).poly-(dG-dC) did not form an A-form in high-alcohol conditions but instead had a VCD spectrum much like that of its high-salt-induced Z-form. Qualitative differences seen experimentally between A- and B-form DNA VCD were suggested by the differences in the coupled oscillator VCD calculated for the two forms.     

Elasticity theory of the B-DNA to S-DNA transition.

We propose in this note a simple model--the two-state Worm Like Chain--to describe the elasticity of the recently discovered stress-induced transformation from B-DNA to S-DNA. The model reduces for low tractions to the well-known Worm Like chain theory, which is used to describe the elastic properties of B-DNA, while in the limit of high chain-bending moduli it reduces to the two-state Ising model proposed by Cluzel et al. for the B-S transition [Cluzel, P., A. Lebrun, C. Heller, R. Lavery, J-L. Viovy, D. Chatenay, and F. Caron. 1996. DNA: an extensible molecule. Science. 271:792-794]. Our model can be treated analytically to produce an explicit form of the force-extension relationship which agrees reasonably with the observations. We use the model to show that conformational fluctuations of the chain play a role also for the B to S transformation.     

Crystal structure of the 2:1 complex between d(GAAGCTTC) and the anticancer drug actinomycin D.

The crystal structures of the 2:1 complex of the self-complementary DNA octamer d(GAAGCTTC) with actinomycin D has been determined at 3.0 A resolution. This is the first example of a crystal structure of a DNA-drug complex in which the drug intercalates into the middle of a relatively long DNA segment. The results finally confirmed the DNA-actinomycin intercalation model proposed by Sobell & co-workers in 1971. The DNA molecule adopts a severely distorted and slightly kinked B-DNA-like structure with an actinomycin D molecule intercalated in the middle sequence, GC. The two cyclic depsipeptides, which differ from each other in overall conformation, lie in the minor groove. The complex is further stabilized by forming base-peptide and chromophore-backbone hydrogen bonds. The DNA helix appears to be unwound by rotating one of the base-pairs at the intercalation site. This single base-pair unwinding motion generates a unique asymmetrically wound helix at the binding site of the drug, i.e. the helix is loosened at one end of the intercalation site and tightened at the other end. The large unwinding of the DNA by the drug intercalation is absorbed mostly in a few residues adjacent to the intercalation site. The asymmetrical twist of the DNA helix, the overall conformation of the two cyclic depsipeptides and their interaction mode with DNA are correlated to each other and rationally explained.     

Crystal structure of RNase A complexed with d(pA)4

Co-crystals of pancreatic RNase A complexed with oligomers of d(pA)4 were grown from polyethylene glycol 4000 at low ionic strength and the X-ray diffraction data were collected to 2.5 A resolution. From a series of heavy-atom derivatives a multiple isomorphous replacement-phased electron density map of the RNase-d(pA)4 complex was calculated to 3.5 A. By inspection, the disposition of the known structure of RNase in the unit cell was determined and this was confirmed by calculation of a standard crystallographic residual, R. Refinement of the protein alone in the unit cell as a strictly rigid body yielded an R factor of 0.32 at 2.8 A resolution. From difference Fourier syntheses DNA fragments were elucidated and incorporated into a model of the complex. The entire asymmetric unit was refined using a restrained-constrained least-squares procedure (CORELS) interspersed with difference Fourier syntheses. At the present time the crystal structure has been refined to an overall R value of 0.215 at 2.5 A resolution. The asymmetric unit of the complex crystals contains four oligomers of d(pA)4 associated with each molecule of RNase. In addition, there may also be partially ordered fragments of DNA at low occupancy present in the unit cell, but these have not, at this time, been incorporated into the model. One tetramer of d(pA)4 is entirely bound by a single protein molecule and occupies a portion of the active site cleft, filling the purine binding site and the phosphate site at the catalytic center with its 5' nucleotide. Two other tetramers are partly intermolecular. One passes from near the pyrimidine binding site over the surface of the protein toward arginine 39 and into a solvent region. A third tetramer is anchored at its 5' terminus by a salt link to lysine 98, passes near arginine and then through a solvent region to terminate with its 3' end near the surface of another protein molecule in the lattice. The fourth tetramer of d(pA)4 is bound at its 5' end on the opposite side of the protein from the active site in an electropositive anion trap that includes lysines 31 and 91 as well as arginine 33. There may be a DNA-DNA interaction involving the 5' phosphate of one tetramer and the 3' bases of two other tetramers and this may help to stabilize the crystalline complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Opening of a Single Base Without Perturbations of Neighboring Nucleotides: A Study on Crystal B-DNA duplex d(CGCGAATTCGCG)2

Abstract

In this work we explore the possibility of the opening of a single base without perturbation of its neighboring nucleotides. Low energy base opening into the grooves can be accomplished by rotation of the relevant backbone and glycosidic bond torsion angles. The pathway has been determined by identifying ζ torsion angle as the reaction coordinate together with the accompanying geometric requirement that guides the displacement of other torsion angles. Our study on Dickerson dodecamer duplex d(CGCGAATTCGCG)₂ showed that all bases with normal equilibrium ζ can be rotated by ~ 30°, corresponding to ~ 3.5Å base displacement, towards the major groove. Such an opening extent is comparable with estimated amplitudes of local angular motions in DNA bases from NMR experiments, which might facilitate proton exchange. The computed base opening energy barrier is also comparable with measured base pair opening enthalpy. These results indicate possible relevance of the pathway studied in this work with experimentally observed base pair opening process. Our analysis also showed a preference for base opening along the major groove and an abnormal behavior for bases with unusual equilibrium ζ torsion angle.