Double helix conformation, groove dimensions and ligand binding potential of a G/C stretch in B-DNA.

The self-complementary DNA fragment CCGGCGCCGG crystallizes in the rhombohedral space group R3 with unit cell parameters a = 54.07 A and c = 44.59 A. The structure has been determined by X-ray diffraction methods at 2.2 A resolution and refined to an R value of 16.7%. In the crystal, the decamer forms B-DNA double helices with characteristic groove dimensions: compared with B-DNA of random sequence, the minor groove is wide and deep and the major groove is rather shallow. Local base pair geometries and stacking patterns are within the range commonly observed in B-DNA crystal structures. The duplex bears no resemblance to A-form DNA as might have been expected for a sequence with only GC base pairs. The shallow major groove permits an unusual crystal packing pattern with several direct intermolecular hydrogen bonds between phosphate oxygens and cytosine amino groups. In addition, decameric duplexes form quasi-infinite double helices in the crystal by end-to-end stacking. The groove geometries and accessibilities of this molecule as observed in the crystal may be important for the mode of binding of both proteins and drug molecules to G/C stretches in DNA.     

DNA associations: packing calculations in A-, B-, and Z-DNA structures.

A detailed theoretical study has been carried out to examine the modes of DNA-DNA interactions on the basis of hard-sphere contact criteria. Two helices of identical structure and length are oriented side-by-side and their relative positions are controlled by translations along and rotations about specific axes. Short atomic contacts between pairs of atoms in the structures are assessed and contact-free configurations are compiled. The computed contact-free arrangements of A, B, and Z double helices are found to be remarkably similar to the packing motifs observed in DNA crystals and stretched fibers. Equally interesting in the study are the broad ranges of sterically acceptable arrangements that preserve the overall packing morphology of neighboring duplexes: Among the most notable morphological features in the helical complexes are extended "super" major and minor grooves which might facilitate the wrapping and packaging of DNA chains in supramolecular assemblies. The hard-sphere computations, however, are insufficient for quantitative interpretation of the packing of DNA helices in the solid state. The results are, nevertheless, a useful starting point for energy based studies as well as relevant to the analysis of long-range interactions in DNA supercoils and cruciforms.     

The effect of crystal packing on oligonucleotide double helix structure

One of the questions that constantly is asked regarding x-ray crystal structure analyses of macromolecules is: To what extent is the observed crystal structure representative of the molecular conformation when free in solution, and to what degree is the structure perturbed by intermolecular crystal forces? This can be assessed with DNA oligomers because of an unusual aspect of crystallization self-complementary oligomers should possess a twofold symmetry axis normal to their helix axis, yet more often than not crystal of such oligomers do not use this internal symmetry. The two ends of the helix are crystallographically distinct though chemically identical. Complexes of DNA oligomers with intercalating drugs such as triostin A tend to use their twofold symmetry when they crystallize, whereas complexes with non-intercalating, groove-binding drugs ignore this symmetry unless the drug molecule is very small. A detailed examination of crystal packing in the dodecamer C-G-C-G-A-A-T-T-C-G-C-G provides an explanation of all of the foregoing behavior in terms of the mechanism of nucleation of DNA or DNA-drug complexes on the surface of a growing crystal. Asymmetry of the ends of the DNA helix is the price that is paid for efficient lateral packing of helices within the crystal. The actual end-for-end variation in standard helix parameters is compared with the experimental noise level as gauged by independent re-refinement of the same oligonucleotide structure where available, and with the observed extent of variation of these same parameters along the helix. Oligomers analyzed are the B-DNA dodecamer C-G-C-G-A-A-T-T-C-G-C-G, the A-DNA octamer G-G-T-A-T-A-C-C, and the phosphorothioate analogue of the B-DNA hexamer G-C-G-C-G-C. End-for-end variation, presumably the result of crystal packing is typically double the experimental noise level, and half the variation in the same parameter along the helix. Analysis of crystal packing in the phosphorothioate hexamer, which uses the same P212121 space group as the dodecamer, shows that the highly unsymmetrical B1 vs. BII backbone conformation probably is to be ascribed to crystal packing forces, and not to the sequence of the hexamer.     

PackHelix: a tool for helix-sheet packing during protein structure prediction

The three‐dimensional structure of a protein is organized around the packing of its secondary structure elements. Predicting the topology and constructing the geometry of structural motifs involving α‐helices and/or β‐strands are therefore key steps for accurate prediction of protein structure. While many efforts have focused on how to pack helices and on how to sample exhaustively the topologies and geometries of multiple strands forming a β‐sheet in a protein, there has been little progress on generating native‐like packings of helices on sheets. We describe a method that can generate the packing of multiple helices on a given β‐sheet for αβα sandwich type protein folds. This method mines the results of a statistical analysis of the conformations of αβ₂ motifs in protein structures to provide input values for the geometric attributes of the packing of a helix on a sheet. It then proceeds with a geometric builder that generates multiple arrangements of the helices on the sheet of interest by sampling through these values and performing consistency checks that guarantee proper loop geometry between the helices and the strands, minimal number of collisions between the helices, and proper formation of a hydrophobic core. The method is implemented as a module of ProteinShop. Our results show that it produces structures that are within 4–6 Å RMSD of the native one, regardless of the number of helices that need to be packed, though this number may increase if the protein has several helices between two consecutive strands in the sequence that pack on the sheet formed by these two strands. Proteins 2011; Published 2011 Wiley‐Liss, Inc.     

Structural rigidity of paranemic crossover and juxtapose DNA nanostructures

Crossover motifs are integral components for designing DNA-based nanostructures and nanomechanical devices due to their enhanced rigidity compared to the normal B-DNA. Although the structural rigidity of the double helix B-DNA has been investigated extensively using both experimental and theoretical tools, to date there is no quantitative information about structural rigidity and the mechanical strength of parallel crossover DNA motifs. We have used fully atomistic molecular dynamics simulations in explicit solvent to get the force-extension curve of parallel DNA nanostructures to characterize their mechanical rigidity. In the presence of monovalent Na⁺ ions, we find that the stretch modulus (γ₁) of the paranemic crossover and its topoisomer JX DNA structure is significantly higher (∼30%) compared to normal B-DNA of the same sequence and length. However, this is in contrast to the original expectation that these motifs are almost twice as rigid compared to the double-stranded B-DNA. When the DNA motif is surrounded by a solvent with Mg²⁺ counterions, we find an enhanced rigidity compared to Na⁺ environment due to the electrostatic screening effects arising from the divalent nature of Mg²⁺ ions. To our knowledge, this is the first direct determination of the mechanical strength of these crossover motifs, which can be useful for the design of suitable DNA for DNA-based nanostructures and nanomechanical devices with improved structural rigidity.     

Groove-backbone interaction in B-DNA. Implication for DNA condensation and recombination

DNA self-fitting is revealed by the study of intermolecular contacts found in the crystal packing of a dodecamer where the helices are locked together by a reciprocal groove-backbone interaction and form a crossed structure. It is proposed that it could be a model for DNA-DNA interaction in several biological processes such as the node of supercoiled DNA and synapsis in recombination. The main topological and symmetrical features of this crossed structure are described and the symmetry-homology relationships are analyzed in the more general case of B-DNA interacting helices. Model-building of Holliday junctions with minimal change from the starting crystal coordinates of the crossed structure leads to at least three different solutions. These various models are compared from the point of view of their symmetry and topology, in the light of their branch migration and resolution properties. In addition, a model for a self-favored reciprocal unwinding mechanism based on the experimentally observed structural alterations, such as the packing-induced opening of G.C base-pairs is proposed. In this model, the phosphate groups of the invading backbone trigger the opening of the base-pairs of the other helix, by pulling cytosine or adenine bases out of the major groove after binding to their amino group.     

Is counterion delocalization responsible for collapse in RNA folding?

Although energetic and phylogenetic methods have been very successful for prediction of nucleic acid secondary structures, arrangement of these secondary structure elements into tertiary structure has remained a difficult problem. Here we explore the packing arrangements of DNA, RNA, and DNA/RNA hybrid molecules in crystals. In the conventional view, the highly charged double helix will be pushed toward isolation by favorable solvation effects; interactions with other like-charged stacks would be strongly disfavored. Contrary to this expectation, we find that most of the cases analyzed ( approximately 80%) exhibit specific, preferential packing between elements of secondary structure, which falls into three categories: (i) interlocking of major grooves of two helices, (ii) side-by-side parallel packing of helices, and (iii) placement of the ribose-phosphate backbone ridge of one helix into the major groove of another. The preponderance of parallel packing motifs is especially surprising. This category is expected to be maximally disfavored by charge repulsion. Instead, it comprises in excess of 50% of all packing interactions in crystals of A-form RNA and has also been observed in crystal structures of large RNA molecules. To explain this puzzle, we introduce a novel model for RNA folding. A simple calculation suggests that the entropy gained by a cloud of condensed cations surrounding the helices more than offsets the Coulombic repulsion of parallel arrangements. We propose that these condensed counterions are responsible for entropy-driven RNA collapse, analogous to the role of the hydrophobic effect in protein folding.     

Differential stability of DNA crossovers in solution mediated by divalent cations

The assembly of DNA duplexes into higher-order structures plays a major role in many vital cellular functions such as recombination, chromatin packaging and gene regulation. However, little is currently known about the molecular structure and stability of direct DNA–DNA interactions that are required for such functions. In nature, DNA helices minimize electrostatic repulsion between double helices in several ways. Within crystals, B-DNA forms either right-handed crossovers by groove–backbone interaction or left-handed crossovers by groove–groove juxtaposition. We evaluated the stability of such crossovers at various ionic concentrations using large-scale atomistic molecular dynamics simulations. Our results show that right-handed DNA crossovers are thermodynamically stable in solution in the presence of divalent cations. Attractive forces at short-range stabilize such crossover structures with inter-axial separation of helices less than 20 Å. Right-handed crossovers, however, dissociate swiftly in the presence of monovalent ions only. Surprisingly, left-handed crossovers, assembled by sequence-independent juxtaposition of the helices, appear unstable even at the highest concentration of Mg²⁺studied here. Our study provides new molecular insights into chiral association of DNA duplexes and highlights the unique role divalent cations play in differential stabilization of crossover structures. These results may serve as a rational basis to understand the role DNA crossovers play in biological processes.     

C-DNA may facilitate homologous DNA pairing

Recombination-independent homologous pairing represents a prominent yet largely enigmatic feature of chromosome biology. As suggested by studies in the fungus Neurospora crassa, this process may be based on the direct pairing of homologous DNA molecules. Theoretical search for the DNA structures consistent with those genetic results has led to an all-atom model in which the B-DNA conformation of the paired double helices is strongly shifted toward C-DNA. Coincidentally, C-DNA also features a very shallow major groove that could permit initial homologous contacts without atom–atom clashes. The hereby conjectured role of C-DNA in homologous pairing should encourage the efforts to discover its biological functions and may also clarify the mechanism of recombination-independent recognition of DNA homology.     

Mediation of the A/B-DNA helix transition by G-tracts in the crystal structure of duplex CATGGGCCCATG

The crystal structure of the DNA dodecamer duplex CATGGGCCCATG lies on a structural continuum along the transition between A- and B-DNA. The dodecamer possesses the normal vector plot and inclination values typical of B-DNA, but has the crystal packing, helical twist, groove width, sugar pucker, slide and x-displacement values typical of A-DNA. The structure shows highly ordered water structures, such as a double spine of water molecules against each side of the major groove, stabilizing the GC base pairs in an A-like conformation. The different hydration of GC and AT base pairs provides a physical basis for solvent-dependent facilitation of the A↔B helix transition by GC base pairs. Crystal structures of CATGGGCCCATG and other A/B-DNA intermediates support a ‘slide first, roll later’ mechanism for the B→A helix transition. In the distribution of helical parameters in protein–DNA crystal structures, GpG base steps show A-like properties, reflecting their innate predisposition for the A conformation.     

Comparison of the solution and crystal conformations of (G + C)-rich fragments of DNA.

DNA fragments crystallize in an unpredictable manner, and relationships between their crystal and solution conformations still are not known. We have studied, using circular dichroism spectroscopy, solution conformations of (G + C)-rich DNA fragments, the crystal structures of which were solved in the laboratory of one of the present authors. In aqueous trifluorethanol (TFE) solutions, all of the examined oligonucleotides adopted the same type of double helix as in the crystal. Specifically, the dodecamer d(CCCCCGCGGGGG) crystalized as A-DNA and isomerized into A-DNA at high TFE concentrations. On the other hand, the hexamer d(CCGCGG) crystallized in Z-form containing tilted base pairs, and high TFE concentrations cooperatively transformed it into the same Z-form as adopted by the RNA hexamer r(CGCGCG), although d(CCGCGG) could isomerize into Z-DNA in the NaCl + NiCl2) aqueous solution. The fragments crystallizing as B-DNA remained B-DNA, regardless of the solution conditions, unless they denatured or aggregated. Effects on the oligonucleotide conformation of 2-methyl-2,4-pentanediol and other crystallization agents were also studied. 2-Methyl-2,4-pentanediol induced the same conformational transitions as TFE but, in addition, caused an oligonucleotide condensation that was also promoted by the other crystallization agents. The present results indicate that the crystal double helices of DNA are stable in aqueous TFE rather than aqueous solution.     

Determination of handedness of DNA double helices from NOE difference spectra: the structure of poly(dG-dC).poly(dG-dC) in low salt

Combined use of shielding constant computations, measurements of chemical shifts and NOE studies reveal that poly(dG-dC).(poly)dG-dC) in low salt solutions exist as a right-handed B-DNA double helix described by Gupta, Dhingra, Sarma, Sarma, Rajagopalan and Sasisekharan, J. Biomole. Str. Dyn. 1. 395, 1983. We present a simple and direct method to determine the handedness of DNA double helices from NOE difference spectra. This method takes advantage of the NOE between base protons and the H2'H2" sugar protons; and in the difference NOE spectra in the H2'H2" region the signatures of the right and left-handed helices become imprinted.     

GU receptors of double helices mediate tRNA movement in the ribosome

A new RNA structural motif consisting of two double helices closely packed via minor grooves is found in many places in the ribosome structure. The packing requires that a GU base pair in one helix be packed against a Watson-Crick pair in the other helix. Two such motifs mediate the interaction of the P- and E-tRNA with the large ribosomal subunit. Analysis of the particular positions of these two motifs in view of the available data on occupancy of tRNA-binding sites and structural changes in the ribosome during the elongation cycle suggests a distinct role for each motif in tRNA translocation.     

Crystallographic study of one turn of G/C-rich B-DNA

The DNA decamer d(CCAGGCCTGG) has been studied by X-ray crystallography. At a nominal resolution of 1.6 A, the structure was refined to R = 16.9% using stereochemical restraints. The oligodeoxyribonucleotide forms a straight B-DNA double helix with crystallographic dyad symmetry and ten base-pairs per turn. In the crystal lattice, DNA fragments stack end-to-end along the c-axis to form continuous double helices. The overall helical structure and, notably, the groove dimensions of the decamer are more similar to standard, fiber diffraction-determined B-DNA than A-tract DNA. A unique stacking geometry is observed at the CA/TG base-pair step, where an increased rotation about the helix axis and a sliding motion of the base-pairs along their long axes leads to a superposition of the base rings with neighboring carbonyl and amino functions. Three-center (bifurcated) hydrogen bonds are possible at the CC/GG base-pair steps of the decamer. In their common sequence elements, d(CCAGGCCTGG) and the related G.A mismatch decamer d(CCAAGATTGG) show very similar three-dimensional structures, except that d(CCAGGCCTGG) appears to have a less regularly hydrated minor groove. The paucity of minor groove hydration in the center of the decamer may be a general feature of G/C-rich DNA and explain its relative instability in the B-form of DNA.     

Simulations of the B-DNA molecular dynamics of d(CGCGAATTCGCG)2 and d(GCGCGCGCGC)2: an analysis of the role of initial geometry and a comparison of united and all-atom models

Molecular dynamics simulations on the sequence d(CGCGAATTCGCG)2 have been carried out using both united atom and all-atom representations, and starting the simulations both from a regular repeating B-DNA structure and from the x-ray single crystal B-DNA structure. An all-atom B-DNA simulation on the sequence d(GCGCGCGCGC)2 has also been carried out, in order to compare it with a previous united atom simulation. The helix repeats, H-bonding, sugar pucker profiles, and average torsional angles are all in the range observed in crystallographic and nmr studies for B-DNA helices. In some of the sequences, there is a significant bend in the DNA helices. The individual helix repeats, with focus on 3'CpG5' and 3'GpC5' units, show the opposite helix repeat to that suggested by Calladine's rules.     

Modelling and refinement of the crystal structure of nucleoprotamine from Gibbula divaricata

The molecular structure of nucleoprotamine from Gibbula divaricata and its packing in oriented fibers has been modelled both to fit the X-ray diffraction pattern and to avoid steric compression. The representative model consists of 51 poly (dinucleotide) B-DNA helices with 51 poly(hexapeptide) chains associated with the major grooves. The prevailing peptide conformation is beta. The four arginine residues present are hydrogen-bonded to DNA phosphates while neutral peptides protrude into the minor grooves of neighboring nucleoprotamine molecules which are packed 2.61 nm apart in a screw-disordered, quasi-hexagonal lattice. This model reconciles a number of earlier, apparently conflicting experimental results and explains the remarkable stability of nucleoprotamines.     

Structural prediction of A- and B-DNA duplexes based on coordinates of the phosphorus atoms.

The sequence-dependent structure of DNA double helices was studied extensively during the past 10 years. How the backbone structure correlates with the base structure in a duplex conformation is still an important yet open question. Using a set of reduced coordinates and a least-squares fitting procedure, we have developed a method to predict structures for B-DNA duplexes based on coordinates of the phosphorus atoms. This method can be used to predict all-atom structures for both bent and straight molecules. We estimated the accuracies of the predictions by studying a set of 10 oligonucleotides with their structures available from the Protein Data Bank. We used this method to construct a modeled structure for the bacteriophage lambda cro operator for which the phosphorus coordinates were known from 3.5-angstrum resolution crystal data (4CRO).     

Geometry of the DNA strands within the RecA nucleofilament: role in homologous recombination

Homologous recombination consists of exchanging DNA strands of identical or almost identical sequence. This process is important for both DNA repair and DNA segregation. In prokaryotes, it involves the formation of long helical filaments of the RecA protein on DNA. These filaments incorporate double-stranded DNA from the cell's genetic material, recognize sequence homology and promote strand exchange between the two DNA segments. DNA processing by these nucleofilaments is characterized by large amplitude deformations of the double helix, which is stretched by 50% and unwound by 40% with respect to B-DNA. In this article, information concerning the structure and interactions of the RecA, DNA and ATP molecules involved in DNA strand exchange is gathered and analyzed to present a view of their possible arrangement within the filament, their behavior during strand exchange and during ATP hydrolysis, the mechanism of RecA-promoted DNA deformation and the role of DNA deformation in the process of homologous recombination. In particular, the unusual characteristics of DNA within the RecA filament are compared to the DNA deformations locally induced by architectural proteins which bind in the DNA minor groove. The possible role and location of two flexible loops of RecA are discussed.