Structure of the B DNA cationic shell as revealed by an X-ray diffraction study of CsDNA. Sequence-specific cationic stabilization of B form DNA

Synchrotron radiation diffraction data for phage T2 CsDNA fibres have been used to determine the co-ordinates of the caesium ions in crystalline B form DNA. The R value is 0.16 for an optimized structure. The caesium ions are distributed equally between the narrow and wide grooves of B DNA and are located close to the dyad axes lying between the planes of adjacent base-pairs. On the wide-groove side the cations are separated from the nearest phosphate atoms by a hydration layer one to two water molecules thick. In the narrow groove the cations are directly co-ordinated to the base atoms and, for six out of ten possible DNA stacking types, form chelation complexes: O-2(Pyr)-Cs+-O-2(Pyr), O-2(Pyr)-Cs+-N-3(Pur) or N-3(Pur)-Cs+-N-3(Pur), which stabilize the B conformation. The steric properties of such complexes as estimated for different base sequences and for different ions are consistent with the structural behaviour of double-helical polynucleotides with different base sequences, as experimentally observed.     

Mechanochemical study of conformational transitions and melting of Li-, Na-, K-, and CsDNA fibers in ethanol–water solutions

Highly oriented fibers of Li-, Na-, K-, and CsDNA were prepared with a previously developed wet spinning method. The procedure gave a large number of equivalent fiber bundle samples (reference length, L₀, typically = 12–15 cm) for systematic measurements of the fiber length L in ethanol–water solutions, using a simple mechanochemical set up. The decrease in relative length L/L₀ with increasing ethanol concentration at room temperature gave evidence for the B-A transition centered at 76% (v/v) ethanol for NaDNA fibers and at 80 and 84% ethanol for K- and CsDNA fibers. A smaller decrease in L/L₀ of LiDNA fibers was attributed to the B-C transition centered at 80% ethanol. In a second type of experiment with DNA fibers in ethanol–water solutions, the heat-induced helix–coil transition, or melting, revealed itself in a marked contraction of the DNA fibers. The melting temperature T_m, decreased linearly with increasing ethanol concentration for fibers in the B-DNA ethanol concentration region. In the B-A transition region, Na- and KDNA fibers showed a local maximum in T_m. On further increase of the ethanol concentration, the A-DXA region followed with an even steeper linear decrease in T_m. The dependence on the identity of the counterion is discussed with reference to the model for groove binding of cations in B-DNA developed by Skuratovskii and co-workers and to the results from Raman studies of the interhelical bonds in A-DNA performed by Lindsay and co-workers. An attempt to apply the theory of Chogovadze and Frank-Kamenetskii on DNA melting in the B-A transition region to the curves failed. However, for Na- and KDNA the T_m dependence in and around the A-B transition region could be expressed as a weighted mean value of T_m of A- and B-DNA. On further increase of the ethanol concentration, above 84% ethanol for LiDNA and above about 90% ethanol for Na-, K-, and CsDNA, a drastic change occurred. T_m increased and a few percentages higher ethanol concentrations were found to stabilize the DNA fibers so that they did not melt at all, not even at the upper temperature limit of the experiments (～ 80°C). This is interpreted as being due to the strong aggregation induced by these high ethanol concentrations and to the formation of P-DNA. Many features of the results are compatible with the counterion–water affinity model. In another series of measurements, T_m of DNA fibers in 75% ethanol was measured at various salt concentrations. No salt effect was observed (with the exception of LiDNA at low salt concentrations). This result is supported by calculations within the Poisson–Boltzmann cylindrical cell model. © 1994 John Wiley & Sons, Inc.     

Locating monovalent cations in the grooves of B-DNA

Here we demonstrate that monovalent cations can localize around B-DNA in geometrically regular, sequence-specific sites in oligonucleotide crystals. Positions of monovalent ions were determined from high-resolution X-ray diffraction of DNA crystals grown in the presence of thallium(I) cations (Tl(+)). Tl(+) has previously been shown to be a useful K(+) mimic. Tl(+) positions determined by refinement of model to data are consistent with positions determined using isomorphous F(Tl) - F(K) difference Fouriers and anomalous difference Fouriers. None of the observed Tl(+) sites surrounding CGCGAATTCGCG are fully occupied by Tl(+) ions. The most highly occupied sites, located within the G-tract major groove, have estimated occupancies ranging from 20% to 35%. The occupancies of the minor groove sites are estimated to be around 10%. The Tl(+) positions in general are not in direct proximity to phosphate groups. The A-tract major groove appears devoid of localized cations. The majority of the observed Tl(+) ions interact with a single duplex and so are not engaged in lattice interactions or crystal packing. The locations of the cation sites are dictated by coordination geometry, electronegative potential, avoidance of electropositive amino groups, and cation-pi interactions. It appears that partially dehydrated monovalent cations, hydrated divalent cations, and polyamines compete for a common binding region on the floor of the G-tract major groove.     

Sequence-Dependent Bending of DNA and Phasing of Nucleosomes

Abstract

Conformational analysis has revealed anisotropic flexibility of the B-DNA double helix: it bends most easily into the grooves, being the most rigid when bent in a perpendicular direction. This result implies that DNA in a nucleosome is curved by means of relatively sharp bends (“mini-kinks”) which are directed into the major and minor grooves alternatively and separated by 5–6 base pairs. The “mini-kink” model proved to be in keeping with the x-ray structure of the B-DNA dodecamer resolved later, which exhibits two “annealed kinks”, also directed into the grooves.

The anisotropy of B DNA is sequence-dependent: the pyrimidine-purine dimers (YR) favor bending into the minor groove, and the purine-pyrimidine dinucleotides (RY), into the minor one. The RR and YY dimers appear to be the most rigid dinucleotides. Thus, a DNA fragment consisting of the interchanging oligopurine and oligopyrmidine blocks 5–6 base pairs long should manifest a spectacular curvature in solution.

Similarly, a nucleotide sequence containing the RY and YR dimers separated by a half-pitch of the double helix is the most suitable for wrapping around the nucleosomal core. Analysis of the numerous examples demonstrating the specific alignment of nucleosomes on DNA confirms this concept. So, the sequence-dependent “mechanical” properties of the double helix influence the spatial arrangement of DNA in chromatin.     

The structure of DAPI bound to DNA

The structure of the DNA fluorochrome 4'-6-diamidine-2-phenyl indole (DAPI) bound to the synthetic B-DNA oligonucleotide C-G-C-G-A-A-T-T-C-G-C-G has been solved by single crystal x-ray diffraction methods, at a resolution of 2.4 A. The structure is nearly isomorphous with that of the native DNA molecule alone. With one DAPI and 25 waters per DNA double helix, the residual error is 21.5% for the 2428 reflections above the 2-sigma level. DAPI inserts itself edgewise into the narrow minor groove, displacing the ordered spine of hydration. DAPI and a single water molecule together span the four AT base pairs at the center of the duplex. The indole nitrogen forms a bifurcated hydrogen bond with the thymine O2 atoms of the two central base pairs, as with netropsin and Hoechst 33258. The preference of all three of these drugs for AT regions of B-DNA is a consequence of three factors: (1) The intrinsically narrower minor groove in AT regions than in GC regions of B-DNA, leading to a snug fit of the flat aromatic drug rings between the walls of the groove. (2) The more negative electrostatic potential within the minor groove in AT regions, attributable in part to the absence of electropositive-NH2 groups along the floor of the groove, and (3) The steric advantage of the absence of those same guanine-NH2 groups, thus permitting the drug molecule to sink deeper into the groove. Groove width and electrostatic factors are regional, and define the relative receptiveness of a section of DNA since they operate over several contiguous base pairs. The steric factor is local, varying from one base pair to the next, and hence is the means of fine-tuning sequence specificity.     

On the hydration of DNA. II. Base composition dependence of the net hydration of DNA

The dependence of the net hydration of DNA on its base composition has been measured by density gradient ultracentrifugation of three DNA's in a series of cesium and lithium salt solutions of different water activities. Extrapolation to zero water activity showed the dependence of the partial specific volume on base composition to be very small for CsDNA and aero for LiDNA. At least 99% of the dependence of buoyant density on base composition can be accounted for on the basis of a differential hydration, with a mole of adenine–thymine pairs binding about 2 moles more water than a mole of guanine–cytosine pairs in CsCl.     

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.     

Molecular dynamics simulations of A. T-rich oligomers: sequence-specific binding of Na+ in the minor groove of B-DNA

Molecular dynamics simulations have been employed to probe the sequence-specific binding of sodium ions to the minor groove of B-DNA of three A. T-rich oligomers having identical compositions but different orders of the base pairs: C(AT)(4)G, CA(4)T(4)G, and CT(4)A(4)G. Recent experimental investigations, either in crystals or in solution, have shown that monovalent cations bind to DNA in a sequence-specific mode, preferentially in the narrow minor groove regions of uninterrupted sequences of four or more adenines (A-tracts), replacing a water molecule of the ordered hydration structure, the hydration spine. Following this evidence, it has been hypothesized that in A-tracts these events may be responsible for structural peculiarities such as a narrow minor groove and a curvature of the helix axis. The present simulations confirm a sequence specificity of the binding of sodium ions: Na(+) intrusions in the first layer of hydration of the minor groove, with long residence times, up to approximately 3 ns, are observed only in the minor groove of A-tracts but not in the alternating sequence. The effects of these intrusions on the structure of DNA depend on the ion coordination: when the ion replaces a water molecule of the spine, the minor groove becomes narrower. Ion intrusions may also disrupt the hydration spine modifying the oligomer structure to a large extent. However, in no case intrusions were observed to locally bend the axis toward the minor groove. The simulations also show that ions may reside for long time periods in the second layer of hydration, particularly in the wider regions of the groove, often leading to an opening of the groove.     

Crystalline aggregation of a proteolytic fragment of the major head protein of bacteriophage T4.

An analysis has been made of the composition and structure of the two types of sheets assembled from material from dissociated bacteriophage T2 (Poglazov &; Mesyhanzhinov, 1967) and T4 capsids. Serological techniques have been used to show that both types of sheet are assembled from proteolytic fragment of P23¹, the major capsid constituent. The two types of sheets have been found to interconvert depending on the concentration of Mg²⁺ ions in the buffer. Computer modelling experiments show that the “hexagonal” and “rectangular” morphologies observed in the negative stain are due to in-register and staggered associations, respectively, of a single basic hexagonal lattice. Analysis by polyacrylamide gel electrophoresis of samples of sheets and dissociated capsids, together with previous results from immune electron microscopy (Kistler et al., 1978), suggest that hexamers of the proteolytic fragment are derived conservatively from capsomers of the phage head.The value of this proteolytic P23 fragment has been twofold: (1) it has proved to be a useful peptide in the ongoing primary sequence determination of P23 and (2) antibodies raised against it have been employed to follow the fate of P23 antigenic sites during various steps of phage capsid maturation (Kistler et al., 1978).     

Effect of discrete distribution of ions on B- and Z-DNA: a theoretical investigation

Using an iterative approach, we have placed monovalent (“solvated”) and divalent (both solvated and “unsolvated”) ions around a 20 base pair sequence, (dC-dG)₁₀, in standard B and ZI conformations. The molecule with its attendant ions in the various conformations is subjected to to energy minimization using the program AMBER. In the presence of solvated cations (both monovalent as well as divalent) the B form is more stable than the Z form. However, direct binding with the unsolvated divalent cations makes the Z form more stable. Groove-binding provides some insight into the facility with which the B to Z transition occurs with higher charged cations. In the presence of unsolvated divalent cations, the Z form binds more charges at the groove through more ligands, compared to the B form. The orientation around the CpG phosphates in the minor groove of the Z form is found ideal for ion binding. Detailed molecular models for the ion binding have been developed. In general, phosphate groups dominate the ion binding. Large perturbations are seen mostly in the angles that control the phosphate orientation.     

Conformational transitions of the phosphodiester backbone in native DNA: two-dimensional magic-angle-spinning 31P-NMR of DNA fibers.

Solid-state 31P-NMR is used to investigate the orientation of the phosphodiester backbone in NaDNA-, LiDNA-, MgDNA-, and NaDNA-netropsin fibers. The results for A- and B-DNA agree with previous interpretations. We verify that the binding of netropsin to NaDNA stabilizes the B form, and find that in NaDNA, most of the phosphate groups adopt a conformation typical of the A form, although there are minor components with phosphate orientations close to the B form. For LiDNA and MgDNA samples, on the other hand, we find phosphate conformations that are in variance with previous models. These samples display x-ray diffraction patterns that correspond to C-DNA. However, we find two distinct phosphate orientations in these samples, one resembling that in B-DNA, and one displaying a twist of the PO4 groups about the O3-P-O4 bisectors. The latter conformation is not in accordance with previous models of C-DNA structure.     

An ordered precipitate of polyadenylic acid formed by freezing at acidic pH: comparison of X-ray diffraction and other properties of the precipitate with those of fibers or direct acid-precipitates

Poly A was found to precipitate upon freezing acidic solutions at p^H values where it is normally soluble; this precipitate tends to have the form of small thin plates of irregular outline (“plates”). The X-ray diffraction pattern and solubility properties of the “plates” were compared with those of poly A precipitated solely by exposure to lower p^H values, and with fibers drawn from acidic solution. There is considerable molecular order in each of these three types of preparation. In all cases, the diffraction patterns are consistent with the presence of the double-stranded helical structure proposed by Rich, Davies, Crick, and Watson (J. Mol. Biol., 3 , 71 (1961)) based on fiber diffraction data. The diffraction pattern from the “plates” is compared in detail with that of the fibers, and is shown to be in accord with a packing scheme having the chain axis of the molecular structure confined to the plane of the “plate,” but oriented randomly in that plane.     

The Interaction Between the Novel Intercalator Diethidium Cation and B-Form DNA: a Theoretical Study

Abstract

This research is an effort to further understand the physicochemical interaction between the novel drug molecule diethidium (2,7-diamino 9-[2,7 diamino 10-nN- phenanthridium] 10- nN- phenanthridium) and its biological receptor DNA. The ultimate goal is the elucidation of this novel class of drugs as potential pharmaceutical agents. Understanding the physico- chemical properties of this drug as well as the mechanism by which it interacts with DNA should ultimately allow the rational design of novel anti-cancer or anti-viral drugs.

A novel binding structure for the diethidium cation to B-form DNA is herein described. Molecular modeling on the complex formed between diethidium and a dodecamer of double-stranded B-form DNA, CGCGAATTCGCG, has shown that this complex is indeed fully capable of participating in the formation of a stable intercalation site. It was expected that diethidium would have a mechanism of intercalation significantly different from other classical intercalators because a) Its structure, that of two perpendicular planes, each known to have excellent intercalation properties, is novel b) The linker region length is zero c) The tilt between the two planes of the drug matches the geometry of the space available to this drug in the major groove.

We have studied the complex formed when diethidium enters the central site of the B-DNA dodecamer through the major groove. The complex forms several classes of intercalation structures, which are all stable and vary from “partially intercalate” to “fully intercalated”. Multiple minimizations show the drug to be very mobile within the intercalation site. Further, some structures show organization and concomitant stiffening of the DNA above the intercalation site, with a disorganization and disruption of the regular B-DNA structure immediately below the intercalation site. This particular phenomena may be expected to lead to significantly different physicochemical properties for the diethidium complex with respect to other known intercalators, because this sort of vectorial difference in structure above and below the site of intercalation is unknown in existing intercalators, as far as the authors are aware. In addition, we expect the mechanism of interaction between drug and DNA to be described by “direct ligand transfer”, wherein the drug is transferred from duplex DNA to duplex DNA without re-entering the solvent.¹

This work is the first instance known to the authors of a novel drug entity that was deduced solely by mathematical reasoning ² and described subsequently by computational methods. Evidence that diethidium should interact with its target site DNA differently from other known intercalators is strong.     

Localization of chromatin proteins within DNA grooves by methylation of chromatin with dimethyl sulphate

The use of the comparative modification with ³H-dimethyl sulphate (DMS) of free DNA and DNA in different complexes is proposed to evaluate the shielding of the minor and major grooves of the DNA double helix and to determine the presence of single-stranded DNA in the complexes.Glucosyl groups in DNA of T6 phage protect, as expected, the major groove, and actinomycin d in its complex with DNA shields the minor groove against methylation with DMS.The data obtained suggest that histones and protamine in reconstituted nucleohistone and nucleoprotamine are allocated within partly the major groove leaving the minor groove open, while polylysine does not seem to be buried within either of the grooves, and cations of cetyltrimethylammonium lie within the minor groove of DNA.     

Crystal structures of A-DNA duplexes

All crystal structures of A-DNA duplexes exhibit a typical crystal packing, with the termini of one molecule abutting the shallow grooves of symmetry related neighbors, while all other forms (B, Z, and RNA) tend to form infinitely stacked helices. The A-DNA arrangement leads to the formation of shallow groove base multiples that have implications for the structure of DNA in compacted states. The characteristic packing leaves big solvent channels, which can be sometimes occupied by B-DNA duplexes. Comparisons of the structures of the same oligomer crystallizing in two different space groups and of different sequences crystallizing in the same space group show that the lattice forces dominate the A-DNA conformation in the crystals, complicating the effort to elucidate the influence of the base sequence on the structures. Nevertheless, in both alternating and nonalternating fragments some sequence effects can still be uncovered. Furthermore, several studies have started to define the minimal sequence changes or chemical modifications that can interconvert the oligomers between different double-helical conformers (A-, B-, and Z-form). Overall, it is seen that the rigid nucleotide principle applies to the oligomeric fragments. Besides the structures of the naked DNAs, their interactions with water, polyamines, and metal ions have attracted considerable attention. There are conserved patterns in the hydration, involving both the grooves and the backbone, which are different from those of B-DNA or Z-DNA. Overall, A-DNA seems to be more economically hydrated than B-DNA, particularly around the sugar-phosphate backbone. Spermine was found to be able to bind exclusively to either of the grooves or to the phosphate groups of the backbone, or exhibit a mixed binding mode. The located metal cations prefer binding to guanine bases and phosphate groups. The only mispairs investigated in A-DNA are the wobble pairs, yielding structural insight into their effects on helix stabilities and hydration. G · T wobble pairs have been determined in various sequence contexts, where they differentially affect the conformations and stableness of the duplexes. The structure of a G · ^m5C base pair, which surprisingly also adopted the wobble conformation, suggests that a similar geometry may transiently exist for G · C pairs. These results from the crystalline state will be compared to the solution state and discussed in relation to their relevance in biology. © 1997 John Wiley & Sons, Inc. Biopoly 44: 45–63, 1997     

Influence of divalent magnesium ion on DNA: molecular dynamics simulation studies

A large amount of experimental evidence is available on the effect of magnesium ions on the structure and stability of DNA double helix. Less is known, however, on how these ions affect the stability and dynamics of the molecule. The static time average pictures from X-ray structures or the quantum chemical energy minimized structures lack understanding of the dynamic DNA–ion interaction. The present work addresses these questions by molecular dynamics simulation studies on two DNA duplexes and their interaction with magnesium ions. Results show typical B-DNA character with occasional excursions to deviated states. We detected expected stability of the duplexes in terms of backbone conformations and base pair parameter by the CHARMM-27 force field. Ion environment analysis shows that Mg²⁺ retains the coordination sphere throughout the simulation with a preference for major groove over minor. An extensive analysis of the influence of the Mg²⁺ ion shows no evidence of the popular predictions of groove width narrowing by dipositive metal ion. The major groove atoms show higher occupancy and residence time compared to minor groove for magnesium, where no such distinction is found for the charge neutralizing Na⁺ ions. The determining factor of Mg²⁺ ion’s choice in DNA binding site evolves as the steric hindrance faced by the bulky hexahydrated cation where wider major groove gets the preference. We have shown that in case of binding of Mg²⁺ to DNA non electrostatic contributions play a major role.

An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:5     

The lambda head-tail joining reaction: purification, properties and structure of biologically active heads and tails

Procedures were developed to obtain biologically active lambda heads and tails at high purity with 20 to 40% recovery. Free heads, free tails and phage particles differ markedly in stability. Phage are stable in solutions containing Mg²⁺ but tails are not. The protein subunits which form the shaft of the tail dissociate in the presence of Mg²⁺ and form multisubunit spherical structures. EDTA protects free tails against inactivation but disrupts heads and phage particles. The four carbon diamine, putrescine, stabilizes heads against inactivation; the three and five carbon diamines are less effective. Electron micrographs reveal a new “knob” structure at the distal end of the tail fiber of phage and of free tails. Tails released from EDTA-disrupted phage possess a “head-tail connector”, a structure not present on the tail before its joining with a head.     

pH Changes Associated with Iron-Stress Response

When Fe-inefficient T3238fer and Fe-efficient T3238FER tomatoes were supplied iron, and nitrogen as nitrate, they increased the pH of the nutrient culture. When they were supplied nitrogen as ammonium, they decreased the pH. When Fe supply was limited, Fe-stress response developed in T3238FER that opposed the usual nitrate response and decreased, rather than increased, the pH. A “reductant” which reduced Fe³⁺ to Fe²⁺ was released from the roots of these plants and lowered the pH; and there was a tremendous increase in the uptake of Fe. T3238fer did not produce “reductant” in response to Fe-stress; the pH increased, and the plants developed Fe-deficiency when nitrogen was supplied as nitrate. Nitrogen nutrition and iron-stress response are important factors associated with iron chlorosis in plants. Release of hydrogen ions from roots of Fe-stressed plants is caused by more than response to imbalanced uptake of cations and anions.     

X-ray diffraction study of the interaction of the amino terminal half-molecule of histone H4 with duplex DNA

The interaction of the amino terminal half-molecule of histone H4 with duplex DNA has been studied by fiber x-ray diffraction. Changes induced in the diffraction pattern of B-DNA by the presence of the bound peptide have been Fourier-analyzed and the results presented in terms of a deweighted radial projection of the electron density. We conclude that the peptide binds on the major groove side of the sugar–phosphate chain.