The structure of guanosine-thymidine mismatches in B-DNA at 2.5-A resolution

The structure of the deoxyoligomer d(C-G-C-G-A-A-T-T-T-G-C-G) was determined at 2.5-A resolution by single crystal x-ray diffraction techniques. The final R factor is 18% with the location of 71 water molecules. The oligomer crystallizes in a B-DNA-type conformation, with two strands interacting to form a dodecamer duplex. The double helix consists of four A X T and six G X C Watson-Crick base pairs and two G X T mismatches. The G X T pairs adopt a "wobble" structure with the thymine projecting into the major groove and the guanine into the minor groove. The mispairs are accommodated in the normal double helix by small adjustments in the conformation of the sugar phosphate backbone. A comparison with the isomorphous parent compound containing only Watson-Crick base pairs shows that any changes in the structure induced by the presence of G X T mispairs are highly localized. The global conformation of the duplex is conserved. The G X T mismatch has already been studied by x-ray techniques in A and Z helices where similar results were found. The geometry of the mispair is essentially identical in all structures so far examined, irrespective of the DNA conformation. The hydration is also similar with solvent molecules bridging the functional groups of the bases via hydrogen bonds. Hydration may be an important factor in stabilizing G X T mismatches. A characteristic of Watson-Crick paired A X T and G X C bases is the pseudo 2-fold symmetry axis in the plane of the base pairs. The G X T wobble base pair is pronouncedly asymmetric. This asymmetry, coupled with the disposition of functional groups in the major and minor grooves, provides a number of features which may contribute to the recognition of the mismatch by repair enzymes.     

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.     

Water and monovalent ions in the minor groove of B-DNA oligonucleotides as seen by NMR

During the past 8 years, two complementary nmr techniques-magnetic relaxation dispersion and nuclear Overhauser effect spectroscopy-have been applied extensively to the study of water and monovalent ions in the minor groove of B-DNA oligonucleotides in solution. In this review, the possibilities and limitations of the two methods are outlined, with emphasis on the interpretational steps whereby molecular-level information is extracted from the primary data. The results on sequence-dependent hydration and ion-DNA interactions obtained so far by these methods is summarized and critically assessed. The nmr results are also compared with structural data from x-ray crystallography.     

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 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.     

Intermolecular interactions and water structure in a condensed phase B-DNA crystal

By controlled dehydration, the unit cells of dodecamer DNA–drug crystals have been shrunk from 68 000 (normal state) to 60 000 (partially dehydrated intermediate state) to 51 000 ų (fully dehydrated state), beyond which no further solvent loss occurs. The total solvent content in the normal crystals is ~40% by volume, reducing to ~20% in the fully dehydrated phase. The 25% reduction in cell volume induced a dramatic enhancement in the resolution of the X-ray diffraction data (from 2.6 to beyond 1.5 Å). We have determined the structures of the normal, partially dehydrated and fully dehydrated crystals. Details of the ligand binding have been presented in the preceding article. The present paper describes the unique features of the structure of the fully dehydrated phase. This structure was refined with 9015 unique observed reflections to R = 14.9%, making it one of the most reliable models of B-form DNA available. The crystals exist as infinite polymeric networks, in which neighbouring dodecamer duplexes are crosslinked through phosphate oxygens via direct bonding to magnesium cations. The DNA is packed so tightly that there is essentially only a single layer of solvent between adjacent molecules. The details of the crystal packing, magnesium bridging, DNA hydration and DNA conformation are described and compared with other experimental evidence related to DNA condensation.     

Structure of a B-DNA dodecamer. II. Influence of base sequence on helix structure

Detailed examination of the structure of the B-DNA dodecamer C-G-C-G-A-A-T-T-C-G-C-G, obtained by single-crystal X-ray analysis (Drew et al., 1981), reveals that the local helix parameters, twist, tilt and roll, are much more strongly influenced by base sequence than by crystal packing or any other external forces. The central EcoRI restriction endonuclease recognition site, G-A-A-T-T-C, is a B helix with an average of 9.8 base-pairs per turn. It is flanked on either side by single-base-pair steps having aspects of an A-like helix character. The dodecamer structure suggests several general principles, whose validity must be tested by other B-DNA analyses. (1) When an external bending moment is applied to a B-DNA double helix, it bends smoothly, without kinks or breaks, and with relatively little effect on local helix parameters. (2) Purine-3′,5′-pyrimidine steps open their base planes towards the major groove, pyrimidine-purine steps open toward the minor groove, and homopolymer (Pur-Pur, Pyr-Pyr) steps resist rolling in either direction. This behavior is related to the preference of pyrimidines for more negative glycosyl torsion angles. (3) CpG steps have smaller helical twist angles than do GpC, as though in compensation for their smaller intrinsic base overlap. Data on A-T steps are insufficient for generalization. (4) G.C base-pairs have smaller propellor twist than A · T, and this arises mainly from interstrand base overlap rather than the presence of the third hydrogen bond. (5) DNAase I cuts preferentially at positions of high helical twist, perhaps because of increased exposure of the backbone to attack. The correlation of the digestion patterns in solution and helical twist in the crystal argues for the essential identity of the helix structure in the two environments. (6) In the two places where the sequence TpCpG occurs, the C slips from under T in order to stack more efficiently over G. At the paired bases of this CpG step, the G and C are tilted so the angle between base planes is splayed out to the outside of the helix. This TpC is the most favored cutting site for DNAase I by a factor of 4.5 (Lomonossoff et al., 1981). (7) The EcoRI restriction endonuclease and methylase both appear to prefer a cutting site of the type purine-purine-A-T-T-pyrimidine, involving two adjacent homopolymer triplets, and this may be a consequence of the relative stiffness of homopolymer base-stacking observed in the dodecamer.     

The high resolution crystal structure of recombinant Crithidia fasciculata tryparedoxin-I.

Tryparedoxin-I is a recently discovered thiol-disulfide oxidoreductase involved in the regulation of oxidative stress in parasitic trypanosomatids. The crystal structure of recombinant Crithidia fasciculata tryparedoxin-I in the oxidized state has been determined using multi-wavelength anomalous dispersion methods applied to a selenomethionyl derivative. The model comprises residues 3 to 145 with 236 water molecules and has been refined using all data between a 19- and 1.4-A resolution to an R-factor and R-free of 19.1 and 22.3%, respectively. Despite sharing only about 20% sequence identity, tryparedoxin-I presents a five-stranded twisted beta-sheet and two elements of helical structure in the same type of fold as displayed by thioredoxin, the archetypal thiol-disulfide oxidoreductase. However, the relationship of secondary structure with the linear amino acid sequences is different for each protein, producing a distinctive topology. The beta-sheet core is extended in the trypanosomatid protein with an N-terminal beta-hairpin. There are also differences in the content and orientation of helical elements of secondary structure positioned at the surface of the proteins, which leads to different shapes and charge distributions between human thioredoxin and tryparedoxin-I. A right-handed redox-active disulfide is formed between Cys-40 and Cys-43 at the N-terminal region of a distorted alpha-helix (alpha1). Cys-40 is solvent-accessible, and Cys-43 is positioned in a hydrophilic cavity. Three C-H...O hydrogen bonds donated from two proline residues serve to stabilize the disulfide-carrying helix and support the correct alignment of active site residues. The accurate model for tryparedoxin-I allows for comparisons with the family of thiol-disulfide oxidoreductases and provides a template for the discovery or design of selective inhibitors of hydroperoxide metabolism in trypanosomes. Such inhibitors are sought as potential therapies against a range of human pathogens.     

Potassium channel, ions, and water: simulation studies based on the high resolution X-ray structure of KcsA

Interactions of Na(+), K(+), Rb(+), and Cs(+) ions within the selectivity filter of a potassium channel have been investigated via multiple molecular dynamics simulations (total simulation time, 48 ns) based on the high resolution structure of KcsA, embedded in a phospholipid bilayer. As in simulations based on a lower resolution structure of KcsA, concerted motions of ions and water within the filter are seen. Despite the use of a higher resolution structure and the inclusion of four buried water molecules thought to stabilize the filter, this region exhibits a significant degree of flexibility. In particular, pronounced distortion of filter occurs if no ions are present within it. The two most readily permeant ions, K(+) and Rb(+), are similar in their interactions with the selectivity filter. In contrast, Na(+) ions tend to distort the filter by binding to a ring of four carbonyl oxygens. The larger Cs(+) ions result in a small degree of expansion of the filter relative to the x-ray structure. Cs(+) ions also appear to interact differently with the gate region of the channel, showing some tendency to bind within a predominantly hydrophobic pocket. The four water molecules buried between the back of the selectivity filter and the remainder of the protein show comparable mobility to the surrounding protein and do not exchange with water molecules within the filter or the central cavity. A preliminary comparison of the use of particle mesh Ewald versus cutoff protocols for the treatment of long-range electrostatics suggests some difference in the kinetics of ion translocation within the filter.     

Water structure in a protein crystal: rubredoxin at 1.2 A resolution

The model for rubredoxin based on X-ray diffraction data has been extensively refined with a 1.2 Å resolution data set. Water oxygen atoms were deleted from the model if B exceeded 50 Ų and occupancy was less than 0.3 eÅ^?3. The final water model consists of 127 sites with B values ranging from 15 to $\text{6}\text{?}$0 Ų and occupancies from unity down to 0.3, the most tightly bound water oxygen atoms being hydrogen bonded to two or more main-chain nitrogen or oxygen atoms. The water forms extensive hydrogen bond networks bridging the crevices on the molecular surfaces and between adjacent molecules. The minimum distances of the water sites from the protein surface are distributed about two distinct maxima, the major one at 2.5 to 3 Å and a minor one at 4 to 4.5 Å. Beyond $\text{5}\text{?}$ to 6 Å from the protein surface, the discrete water merges into the aqueous continuum.     

Structure of hydrated Na+ ions around a region of A- or B-DNA helix

A molecular-dynamics simulation was used to carry out an introductory study of the hydration of a section of a rigid single A- or B-DNA helix with one Na⁺ counterion per nucleotide. Four Na⁺ ions and four nucleotides and periodic boundary conditions were used to mimic an infinite helix. The atoms of the helix and the Na⁺ ions were assumed to be Lennard-Jones spheres that also carried charges. Stillinger four-point charge model water molecules were used. We carried out five calculations, for 26 and 46 water molecules in B-DNA and 20, 32, and 46 in A-DNA fragments. The arrangements of the Na⁺ ions are found to have some similarities to those obtained by Clementi and Corongiu. In the calculations with 46 water molecules, we found that two Na⁺ ions can be bridged by about two water molecules and form a hydrated bound pair, which in turn forms a bridge between the guanine N7 and a near phosphate group. These bound pairs may be important in stabilizing the helix structure of DNA molecules.     

Conformational sub-states in B-DNA.

Theoretical studies of the sequence-dependent conformation of B-DNA have been carried out using Jumna, a helicoidal co-ordinate minimization algorithm. The results obtained for a series of six oligomers with repetitive sequences show that, with the exception of the homopolymers (dA)n.(dT)n and (dG)n.(dC)n, all sequences can adopt a variety of conformations characterized by considerable changes in helicoidal parameters and also in sugar puckers which adopt C(2')-endo (falling into 2 classes) or, in the case of pyrimidine nucleotides, O(1')-endo forms. These studies lead to an improved understanding of the role of base sequence on DNA conformation and point to a number of interesting correlations between the various structural parameters describing the double helix.     

BI-BII transitions in B-DNA.

Molecular modelling is used to study the conformational and energetic aspects of BI-BII transitions within the backbone of a B-DNA dodecamer d(CATGACGTCATG) whose fine structure has previously been determined by molecular modelling combined with NMR spectroscopy. It is shown that while the dodecamer under investigation does not contain any BII junctions, the central CpG step can most easily undergo the transition. More generally, it is also found that the base sequence and hence the backbone geometry of a DNA segment, strongly influences both the conformational impact of the transition, the associated energy barrier and the stability of the resulting BII state.     

Inosine.adenine base pairs in a B-DNA duplex.

The structure of the synthetic deoxydodecamer d(C-G-C-I-A-A-T-T-A-G-C-G) has been determined by single crystal X-ray diffraction techniques at 2.5A resolution. The refinement converged with a crystallographic residual, R = 0.19 and the location of 64 solvent molecules. The sequence crystallises as a B-DNA helix with 10 Watson-Crick base-pairs (4 A.T. and 6 G.C) and 2 inosine.adenine (I.A) pairs. The present work shows that in the purine.purine base-pairs the adenine adopts syn orientation with respect to the furanose moiety while the inosine is in the trans (anti) orientation. Two hydrogen bonds link the I.A. base-pair, one between N-1(I) and N-7(A), the other between O-6(I) and N-6(A). This bulky purine.purine base-pair is incorporated in the double helix at two positions with little distortion of either local or global conformation. The pairing observed in this study is presented as a model for I.A base-pairs in RNA codon-anticodon interactions and may help explain the thermodynamic stability of inosine containing base-pairs. Conformational parameters and base stacking interactions are presented and where appropriate compared with those of the native compound, d(C-G-C-G-A-A-T-T-C-G-C-G) and with other studies of oligonucleotides containing purine.purine base-pairs.     

Ordered water structure around a B-DNA dodecamer: A quantitative study

The crystal structure of the double-helical B-DNA dodecamer of sequence C-G-C-G-A-A-T-T-C-G-C-G has been solved and refined independently in three forms: (1) the parent sequence at room temperature; (2) the same sequence at 16 K; and (3) the 9-bromo variant C-G-C-G-A-A-T-T^BrC-G-C-G at 7 °C in 60% (v/v) 2-methyl-2.4-pentanediol. The latter two structures show extensive hydration along the phosphate backbone, a feature that was invisible in the native structure because of high temperature factors (indicating thermal or static disorder) of the backbone atoms. Sixty-five solvent peaks are associated with the phosphate backbone, or an average of three per phosphate group. Nineteen other molecules form a first shell of hydration to base edge N and O atoms within the major groove, and 36 more are found in upper hydration layers. The latter tend to occur in strings or clusters spanning the major groove from one phosphate group to another. A single spermine molecule also spans the major groove. In the minor groove, the zig-zag spine of hydration that we believe to be principally responsible for stabilizing the B form of DNA is found in all three structures. Upper level hydration in the minor groove is relatively sparse, and consists mainly of strings of water molecules extending across the groove, with few contacts to the spine below. Sugar O-1′ atoms are closely associated with water molecules, but these are chiefly molecules in the spine, so the association may reflect the geometry of the minor groove rather than any intrinsic attraction of O-1′ atoms for hydration. The phosphate O-3′ and O-5′ atoms within the backbone chain are least hydrated of all, although no physical or steric impediment seems to exist that would deny access to these oxygen atoms by water molecules.     

Bending and curvature calculations in B-DNA.

A simple program, BEND, has been written to calculate the magnitude of local bending and macroscopic curvature at each point along an arbitrary B-DNA sequence, using any desired bending model that specifies values of twist, roll and tilt as a function of sequence. The program has been used to evaluate six different DNA bending models in three categories. Two are bent non-A-tract models: (a) A new model based on the nucleosome positioning data of Satchwell et al 1986 (J. Mol. Biol. 191, 659-675), (b) The model of Calladine et al 1988 (J. Mol. Biol. 201, 127-137). Three are bent A-tract models: (c) The wedge model of Bolshoy et al 1991 (Proc. Natl. Acad. Sci. USA 88, 2312-2316), (d) The model of Cacchione et al 1989 (Biochem. 28, 8706-8713), (e) A reversed version of model (b). The last is a junction model: (f) The model of Koo & Crothers 1988 (Proc. Natl. Acad. Sci. USA 85, 1763-1767). Although they have widely different assumptions and values for twist, roll and tilt, all six models correctly predict experimental A-tract curvature as measured by gel retardation and cyclization kinetics, but only the new nucleosome positioning model is successful in predicting curvature in regions containing phased GGGCCC sequences. This model--showing local bending at mixed sequence DNA, strong bends at the sequence GGC, and straight, rigid A-tracts--is the only model consistent with both solution data from gel retardation and cyclization kinetics and structural data from x-ray crystallography.     

Atomic-resolution crystal structures of B-DNA reveal specific influences of divalent metal ions on conformation and packing.

Crystal structures of B-form DNA have provided insights into the global and local conformational properties of the double helix, the solvent environment, drug binding and DNA packing. For example, structures of the duplex with sequence CGCGAATTCGCG, the Dickerson-Drew dodecamer (DDD), established a unique geometry of the central A-tract and a hydration spine in the minor groove. However, our knowledge of the various interaction modes between metal ions and DNA is very limited and almost no information exists concerning the origins of the different effects on DNA conformation and packing exerted by individual metal ions.Crystallization of the DDD duplex in the presence of Mg(2+)and Ca(2+)yields different crystal forms. The structures of the new Ca(2+)-form and isomorphous structures of oligonucleotides with sequences GGCGAATTCGCG and GCGAATTCGCG were determined at a maximum resolution of 1.3 A. These and the 1.1 A structure of the DDD Mg(2+)-form have revealed the most detailed picture yet of the ionic environment of B-DNA. In the Mg(2+)and Ca(2+)-forms, duplexes in the crystal lattice are surrounded by 13 magnesium and 11 calcium ions, respectively.Mg(2+)and Ca(2+)generate different DNA crystal lattices and stabilize different end-to-end overlaps and lateral contacts between duplexes, thus using different strategies for reducing the effective repeat length of the helix to ten base-pairs. Mg(2+)crystals allow the two outermost base-pairs at either end to interact laterally via minor groove H-bonds, turning the 12-mer into an effective 10-mer. Ca(2+)crystals, in contrast, unpair the outermost base-pair at each end, converting the helix into a 10-mer that can stack along its axis. This reduction of a 12-mer into a functional 10-mer is followed no matter what the detailed nature of the 5'-end of the chain: C-G-C-G-A-ellipsis, G-G-C-G-A-ellipsis, or a truncated G-C-G-A-ellipsis Rather than merely mediating close contacts between phosphate groups, ions are at the origin of many well-known features of the DDD duplex structure. A Mg(2+)coordinates in the major groove, contributing to kinking of the duplex at one end. While Ca(2+)resides in the minor groove, coordinating to bases via its hydration shell, two magnesium ions are located at the periphery of the minor groove, bridging phosphate groups from opposite strands and contracting the groove at one border of the A-tract.     

Phytoplankton communities and antecedent conditions: high resolution sampling in Esthwaite Water

1. The succession of a phytoplankton community was investigated through an intensive period of sampling and related to physical, chemical and biological conditions sampled at an equal, or higher, temporal resolution. 2. Phytoplankton samples were taken on a weekly basis from June to September 2004 and analysed for diversity, species composition, and contribution of different functional groups to total biomass. Physical and chemical data were collected on the sampling days, and physical environmental factors were also logged continuously throughout the period by automatic measuring stations. This continuous logging allowed community structure to be compared with physical data averaged over periods from a day to a week before each sampling date. 3. The Schmidt stability of the lake, a measure of the strength of stratification calculated from thermal data, showed a negative correlation with phytoplankton species diversity. This is consistent with the hypothesis that mixing was preventing exclusion by species that would otherwise dominate in stratified conditions. 4. At a functional level, stress tolerant (S‐type) species dominated during the stratified summer conditions, with small, colonising species (C types) and ruderal, disturbance tolerant species (R types) contributing little to the overall biomass. Of the stress tolerant species, the faster growing (S_C) phytoplankters were significantly favoured by more stable, stratified conditions and higher solar radiation. Increased abundance of this group resulted in decreased species diversity. Correlations were generally strongest when using the 6‐ to 7‐day averaged physical data, stressing the importance of continuous measurements of these drivers in phytoplankton studies.     

Cyanidioschyzon merolae ferredoxin: a high resolution crystal structure analysis and its thermal stability

Cyanidioschyzon merolae (Cm) is a single-cell red alga that grows under moderately thermophilic (40-50°C), acidic (pH 1-3) conditions. We purified a Cm ferredoxin (Fd) that was characterized as a plant-type [2Fe-2S] Fd by physicochemical techniques. X-ray crystallography revealed that the overall three-dimensional structure of CmFd was highly similar to, but slightly different from, the [2Fe-2S] Fd from Spinacia oleracea, whose growth temperature is 15-20°C. Therefore, slight structural differences, including non-covalent-bond number and amino acid sequence, may underlie the differential thermostabilities of the plant-type Fds.