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.     

Solvent-accessible surfaces of nucleic acids

Static solvent-accessible surface areas were calculated for DNA and RNA double helices of varied conformation, composition and sequence, for the single helix of poly(rC), and for a transfer RNA. The results show that for DNA and RNA double helices, two thirds of the water-accessible surface area become buried on double helix formation; phosphate oxygens retain near maximal exposure while the bases are 80% buried. Transfer RNA exposes slightly less surface per residue than does double-helical RNA, despite the presence of several additional “modified” groups, all of which are exposed significantly.When a probe corresponding to a single water molecule is used, both the total and atom type exposures are very similar for A-DNA and B-DNA, although marked differences appear in the major and minor groove exposures between the two conformations. For a given base-pair, the accessible surface area buried upon double-helical stacking is nearly constant (within 5%) for different sequences of neighboring base-pairs.For probes larger than single water molecules, there exist considerable differences in the total and atom type exposures of A-DNA and B-DNA. Conformational transitions between the A-DNA and B-DNA helical forms can thus be related to differences in the accessible areas for “structured” water, or a secondary hydration shell, rather than to interactions with individual water molecules of the primary hydration shell. The base-composition dependence of DNA helical conformation can be explained in terms of the opposing effects of thymine methyl groups of A · T base-pairs and the amino groups of G · C base-pairs upon the solvent within the grooves.The area calculations show that primarily the major groove of B-DNA and the minor groove of A-DNA have sufficient accessible surface area to be recognized by a probe size corresponding to the side-chains of amino acids.     

Transcription of the his3 gene region in Saccharomyces cerevisiae

The dodecamer d(CpGpCpGpApApTpTpCpGpCpG) or C-G-C-G-A-A-T-T-C-G-C-G crystallizes as slightly more than one full turn of right-handed B-DNA. It is surrounded in the crystal by one bound spermine molecule and 72 ordered water molecules, most of which associate with polar N and O atoms at the exposed edges of base-pairs. Hydration within the major groove is principally confined to a monolayer of water molecules associated with exposed N and O groups on the bases, with most association being monodentate. Waters hydrating backbone phosphate oxygens tend not to be ordered, except where they are immobilized by 5-methyl groups from nearby thymines. In contrast, the minor groove is hydrated in an extensive and regular manner, with a zigzag “spine” of first- and second-shell hydration along the floor of the groove serving as a foundation for less-regular outer shells extending beyond the radius of the phosphate backbone. This spine network bridges purine N-3 and pyrimidine O-2 atoms in adjacent base-pairs. It is particularly regular in the A-A-T-T center, and is disrupted at the C-G-C-G ends, in part by the presence of the N-2 amino groups on guanine residues. The minor groove hydration spine may be responsible for the stability of the B form of polymers containing only A · T and I · C base-pairs, and its disruption may explain the ease of transition to the A form of polymers with G · C pairs.     

Solution conformation of DNA

Optical observations on linear B-form DNA by the method of electric linear dichroism show that the value of the limiting reduced dichroism is molecular weight-dependent, increasing with molecular weight to a limit of about ?1.41 ± 0.02 in aqueous solution. These data and the rotational relaxation times obtained from the decay of the dichroism when the orienting field is instantaneously removed, imply the existence of a non-linear tertiary equilibrium structure for DNA. The data indicate that the essential B-form parameters of the double-stranded DNA are retained in this tertiary structure, and are not consistent with a DNA structure in which the base-pairs have a 34 ° propeller-like twist (Hogan et al., 1978). The interpretation of the dichroism data is supported by a clear demonstration that the magnitude of dichroism change in ethanol corresponds to that expected for the B to A-form structural transition as determined from X-ray diffraction. We propose that the tertiary structure of B-DNA is a helical coil and suggest the limits of the structural parameters of the coil consistent with the observations.     

Entamoeba histolytica: uptake of purine bases and nucleosides during axenic growth.

A comparison was made of the uptake mechanisms of selected purine bases and nucleosides by axenically grown Entamoeba histolytica. Adenine, adenosine, and guanosine were taken up, in part, by a “carrier”-mediated system. Guanine, hypoxanthine, and inosine entered amoebas via diffusion. Inhibitor studies support the presence of individual transport sites for adenine-adenosine and adenosine-guanosine. Additional sites for transport of adenine, adenosine, and guanosine are implied by “non-productive binding” involving guanine, hypoxanthine, and inosine. Uptake of adenine, adenosine, and guanosine was reduced by iodoacetate and N-ethylmaleimide. Ribose failed to inhibit uptake of purine nucleosides.     

A base-centred explanation of the B-to-A transition in DNA

In the traditional view, the bistable feature responsible for the switch between the B and A forms of DNA was the sugar-phosphate backbone. Several recent assays of the sequence-dependent structure of DNA are not compatible with that hypothesis. Here we show that certain kinds of base-pair step, mainly those of the pyrimidine-purine variety, can stack in a “bistable” fashion so as to produce one of two overall helix shapes A or B. Further, we suggest that the passive, elastic stiffness of the backbone is responsible for communicating the stacking configuration from bistable steps to their “neutral” neighbours. The role of water molecules, in stabilizing the B form of DNA over the A, may simply be to form hydrogen-bonded bridges with the minor-groove edges of neutral steps in the B configuration.     

Possible Specificity of Cellular Interactions Due to Electrostatic Forces

It is known that the cells from a mixed population in a culture medium will finally segregate. This “social behavior” of the cells is a direct consequence of both physical and chemical interactions between cells. The physical forces involved in cell-cell interactions are considered to be the electrostatic, van der Waals, and very-short-range hydration forces.

It has been believed until now that the electrostatic forces acting between identical cells were always repulsive and nonspecific. In the present work, we try to suggest that even the electrostatic forces could manifest a slightly specific character, so that a completely random Brownian motion of the cells could be influenced, corrected, and transformed into a “docking maneuver,” which could favor the specific interactions between cells suspended in a culture medium. The main point of our approach is that the peculiar patterns of electric charge distribution on the cell surfaces act like specific electrostatic fingerprints that could be responsible for the preferential interactions which precede cellular segregation (1).     

Conformation and dynamics in a Z-DNA tetramer

Two kinds of conformational variability have been reported for left-handed Z-DNA: the Z to Z′ transition, which involves a change in guanine sugar pucker from C-3′-endo to C-1′-exo, and the Z_Ito Z_II transition, which corresponds to a simple three-atom phosphate-group rotation. Neither of these motions substantially affects base stacking or helical twist, and this is because the degree of independent motion of phosphate groups, sugar molecules and base-pairs is greater in the left-handed Z helix than in right-handed B-DNA. Detailed considerations of Z helix geometry suggest that Z_I, Z_IIand Z′ are not separate species, but only samplings of the full range of conformation open to Z-DNA.     

Sequence-dependent variation in the conformation of DNA

The specificity of action of the enzyme DNAase I on double-stranded DNA polymers of defined sequence has been investigated. The results obtained with the alternating copolymers poly[d(A-T)] · poly[d(A-T)] and poly[d(G-C)] · poly[d(G-C)] support the suggestion of Klug et al. (1979) that regions of double-stranded DNA containing alternating purine-pyrimidine sequences may exist as structural variants of the classical B-form under physiological salt conditions. Digestion of defined oligomers containing alternating dG-dC sequences indicate that these too exist in some “alternating-B” structure in solution under similar conditions. The results obtained with the oligomers also provide a number of insights into the mode of action of DNAase I.In the case of the B-DNA dodecamer d(C-G-C-G-A-A-T-T-C-G-C-G), for which the crystal structure has been solved (Dickerson &; Drew, 1981), there is a very good correlation between the sites of rapid DNAase I cutting and positions of high local helical twist.     

A conformational rationale for the replication of nucleic acids under prebiotic conditions

The Watson-Crick model at once gave an explanation for the mechanism of replication of DNA. But the hydrogen-bonding forces between the bases alone are not enough for the specificity of base-pairing mechanisms, since any pair of bases can be positioned to have at least two hydrogen bonds. In the present-day biological organisms, sophisticated enzymatic machinery is supposed to constrain the ribose-phosphate backbone to have regular structure, aiding the self-templating duplication. For the prebiotic stage, whence sophisticated enzymes would not have been evolved, we propose a novel double helical conformation of DNA wherein the two sugar-phosphate backbones are pulled towards each other by (CH  O) hydrogen bonds conferring stereospecificity for the formation of (A : T)- and (G : C)-pairs, in the self-templating chains of DNA. Our model-building efforts and computer calculations endorse the stereochemical feasibility of the conformation. The pairing of homologous sequences of two double helices of DNA is explained by direct hydrogen-bonding interactions in our model and it is thus relevant to the present-day biological functions also, at least in some stages of the cell-cycle.     

Analysis of conformational parameters in nucleic acid fragments. I. Single crystals of nucleosides and nucleotides

A study has been undertaken of conformational parameters in single crystal structures of nucleosides and nucleotides using the techniques of helical conformational analysis. A "quasi-helix" was generated from the geometry of base-paired structures, using published data extracted from the Cambridge structural database. A total of 54 base-pairs were found in these structures, for each of which were calculated hydrogen bond parameters, propeller twist, buckle and C1'-C1' separations. These were analysed according to various classifications. Propeller twists are found to show a wide range of values and the magnitude of twist appears to be unrelated to hydrogen bond parameters or C1'-C1' separation. The values of the buckle parameter vary over a smaller range of values and are unrelated to propeller twist magnitude. There is found to be a greater tendency to form homo-base-pairs among compounds containing adenine bases.     

Monte Carlo Simulation of Hydration of the Nucleic Acid Fragments

Abstract

Systems containing a base or a base pair and 25 water molecules, as well as a helical stack and 30 water molecules per base pair, have been simulated. Changes in the base hydration shell structure, after the bases have been included into the pair and then into the base pair stack are discussed. Hydration shells of several configurations of the base pair stacks are discussed. Probabilities of formation of the hydrogen-bonded bridges of 1, 2 and 3 water molecules between hydrophilic centres have been estimated. The hydration shell structure was shown to depend on the nature of the base pair and on the stack configuration, while dependence of the global hydration shell characteristics on the stack configuration has been proved to be rather slight. The most typical structural elements of hydration shells, in the glycosidic (minor in B-like conformation) and non-glycosidic (major) grooves, for different configurations of AU and GC stacks, have been found and discussed. The number of hydrogen bonds between water molecules and bases per water molecule was shown to change upon transformation of the stack from A to B configuration. This result is discussed in connection with the reasons for B to A conformational transition and the concept of “water economy”. Hydration shell patterns of NH₂-groups of AU and GC helical stacks differ significantly.     

Base sequence and helix structure variation in B and A DNA

The observed propeller twist in base-pairs of crystalline double-helical DNA oligomers improves the stacking overlap along each individual helix strand. But, as proposed by Calladine, it also leads to clash or steric hindrance between purines at adjacent base-pairs on opposite strands of the helix. This clash can be relieved by: (1) decreasing the local helix twist angle between base-pairs; (2) opening up the roll angle between base-pairs on the side on which the clash occurs; (3) separating purines by sliding base-pairs along their long axes so that the purines are partially pulled out of the stack (leading to equal but opposite alterations in main-chain torsion angle delta at the two ends of the base-pair); and (4) flattening the propeller twist of the offending base-pairs. Simple sum functions, sigma 1 through sigma 4, are defined, by which the expected local variation in helix twist, base roll angle, torsion angle delta and propeller twist may be calculated from base sequence. All four functions are quite successful in predicting the behavior of B DNA. Only the helix twist and base roll functions are applicable to A DNA, and the helix twist function begins to fail for an A helical RNA/DNA hybrid. Within these limits, the sequence-derived sum functions match the observed helix parameter variation quite closely, with correlation coefficients greater than 0.900 in nearly all cases. Implications of this sequence-derived helix parameter variation for repressor-operator interactions are considered.     

Twisted hyperboloid (strophoid) as a model of β-barrels in proteins

Using a least-squares fitting procedure, polypeptide backbones of one parallel and seven antiparallel β-barrels were approximated with various curved surfaces. Although the hyperboloid gave better approximations to all the β-barrel backbones than the ellipsoid, elliptical cylinder or catenoid, the best approximations were obtained with a novel surface, a twisted hyperboloid (strophoid). The root-mean-square errors between individual β-barrels and the fitted strophoid surfaces ranged from 0.75 Å to 1.64 Å. The parameters which determine the strophoid surface allow groups of β-barrel shapes to be defined according to their barrel twists (i.e. angles subtended by directions of the long axis of cross-section at the top and the bottom of the barrel), course of elliptical cross-sections (either monotonically increasing along the barrel axis, as in cones, or having a middle “waist”, as in hyperboloids), and types of backbone curvatures (either convex or concave). The curvatures at individual points of strophoid surface are local, variable quantities related to the local helicity (coil) of the polypeptide backbone, in contrast to values of β-sheet twist (i.e. dihedral angles subtended by adjacent β-strands) known to be virtually identical in all the β-sheets. The variability found in parameters such as barrel shapes and curvatures suggests that simple models (isotropically stressed surfaces, principle of minimal surface tension) proposed in the past to account for β-barrel shapes are not sufficient. Rather, the complex nature of best-fit theoretical surfaces points to an important role played by a local variability of the forces involved.     

Strict co-linearity of genetic and protein folding domains in an intragenically duplicated rat lens γ-crystallin gene

Two complementary DNA clones pRLγ-2 and pRLγ-3 of different rat lens γ-crystallin messenger RNAs have been used to identify γ-crystallin gene sequences in rat genomic DNA. Subsequently, the DNA present in the 18,000 to 20,000 bases region of the EcoRI digest, giving rise to a strong doublet hybridization signal, was cloned in λ phage Charon-4A. One of the clones, λRCHγ-3, carrying an insert of 17,500 bases has been characterized in detail. From analysis at the restriction enzyme level with 5′-, “middle” and 3′-specific subprobes of pRLγ-3 it could be deduced that λRCHγ-3 contains only one γ-crystallin gene. The coding sequences of this gene are interrupted by intronic DNA. The primary structure of this gene and its flanking regions have been established by sequencing the relevant regions of a subclone of λRCHγ-3, designated pRCHγ-3.1. The sequence data show that the γ-crystallin gene extends over 2700 bases of rat genomic DNA. The gene is split by two introns, one of 87 base-pairs after the third translation codon and a large one of 1880 base-pairs after codon 84. The mosaic structure of the gene is strictly co-linear with the structure of the γ-crystallin polypeptide in that the large intron is positioned in a region which specifies the so-called “connecting peptide” and which links the two highly symmetrical and homologous protein domains. Although expected from the cDNA and protein sequence no introns were observed between the coding regions in the DNA specifying the two homologous folding motifs present in each protein domain. The relevance of this phenomenon in terms of the evolution of the mature γ-crystallin gene is discussed.     

Molecular Dynamics Simulation Study of DNA Triplex Formed by Mixed Sequences in Solution

Abstract

The unrestrained molecular dynamics simulation of the triple helical DNA with mix sequences d(GACTGGTGAC)?d(CTGACCACTG)*d (GACTGGTGAC), using the particle mesh Ewald sum, is presented here. The Ewald summation method effectively eliminates the usual “cut-off” of the long—range interactions and allowed us to evaluate the full effect of the electrostatic forces. The AMBER5.0 force field has been used during the simulation in solvent. The MD results support a dynamically stable model of DNA triplex over the entire length of the trajectory. The duplex structure assumes the conformation, which is very close to B-DNA. In mixed sequences the purine bases occurs in both strand of DNA duplex. The bases of third strand do not favor the Hoogsteen or/and reverse Hoogsteen type of Hydrogen bonding but they form hydrogen bonds with the bases of both the strand of DNA duplex. The orientation of the third strand is parallel to one of the strand of duplex and all nucleotides (C, A, G & T) show isomorphic behavior with respect to the DNA duplex. The conformation of all the three strands is almost same except few exceptions. Due to interaction of third strand the conformational change in the duplex structure and a finite amount of displacement in the W-C base pairs have been observed. The conformational variation of the back bone torsion angles and helicoidal parameters, groove widths have been discussed. The sequence—dependent effects on local conformation, helicoidal and morphological structure, width of the grooves of DNA helix may have important implication for understanding the functional energetics and specificity of interactions of DNA and its triplexes with proteins, pharmaceutical agents and other legends.     

Configurational effects on conformational properties of cyclic nucleotides. I. Theoretical calculations of conformer preferences in α-nucleoside 3′,5′ cyclic monophosphates

A comparative study has been made of the configurational effects on the conformational properties of α- and β-anomers of purine and pyrimidine nucleoside 3′,5′,-cyclic monophosphates and their 2′-arabino epimers. Correlation between orientation of the base and the 2′-hydroxyl group have been studied theoretically using the PCILO (Perturbative Configuration Interaction using Localized Orbitals) method. The effect of change in ribose puckering on the base-hydroxyl interaction has also been studied. The result show that steric repulsions and stabilizing effects of intramolecular hydrogen bonding between the base and the 2′-hydroxyl (OH) group are of major importance in determining configurations of α-anomers and 2′-arabino-β-epimers. For example, hydrogen bonding between the 2′-hydroxyl group and polar centers on the base ring is clearly implicated as a determinant of syn-anti preferences of the purine (adenine) or pyrimidine (uracil) bases in α-nucleoside 3′,5′-cyclic monophosphates. Moreover, barrier heights for interconversion between conformers are sensitive to ribose pucker and 2′-OH orientations. The result clearly show that a change in ribose-ring pucker plays an essential role in relieving repulsive interaction between the base and the 2′-hydroxyl group. Thus a C2′-exo-C3′-endo (₂T³) pucker is favored for α-anomers in contrast with the C4′-exo-C3′-endo (₄T³) from found in β-compounds.     

Identification of new polymorphic positions in rDNA sequences of the “intermediate” Fasciola forms

Fascioliasis is a foodborne zoonotic disease generally caused by the parasitic flukes Fasciola gigantica and Fasciola hepatica in class Trematoda. An “intermediate” Fasciola forms between F. gigantica and F. hepatica has been shown to exist. However, the relationships among F. gigantica, F. hepatica, and “intermediate” Fasciola forms remain unclear. In this study, we found five new polymorphic positions in 18S and 28S rDNAs sequences of “intermediate” Fasciola forms. According to the high-throughput sequencing results, all known 16 polymorphic positions of “intermediate” Fasciola forms show a clear and consistent tendency for F. gigantica or F. hepatica, and the percentages of the most frequently occurring bases were different in specimens. In the three ITS sequence fragments, hybrid-type base combinations of the polymorphic positions were detected, and the percentages of the most frequent base combinations were different in specimens too. In addition, interestingly, the newly detected ITS-802 position was not a traditional polymorphic position in “intermediate” Fasciola forms, and the bases in ITS-802 position are not same as the allele bases of F. gigantica or F. hepatica. Our results will be helpful to investigations into the molecular taxonomy, population genetics, and ecology of F. gigantica, F. hepatica, and “intermediate” Fasciola forms.