Simulations of the solvent structure for macromolecules. I. Solvation of B-DNA double helix at T = 300 K

Monte Carlo simulations are reported for a system of 447 water molecules enclosing a B-DNA double-helix fragment with 12 base pairs and the corresponding sugar and phosphate units. From a detailed analysis on the interaction energies and probability distributions (at a simulated temperature of 300 K), the water molecules can be partitioned into clusters strongly interacting with (1) the phosphates, (2) the sugars, (3) the sugars and the bases, and (4) the base pairs. In addition, transgroove and interphosphate filament of hydrogen-bonded water molecules have been detected. From simulations performed with variable numbers of water molecules, a theoretical isotherm has been obtained, with the characteristic sigmoidal shape, known from absorption–desorption experiments on related systems. The expected main features for the structure of water molecules solvating B-DNA with Na⁺ counterions are briefly discussed at the end of the paper.     

Theoretical Study of the Sequence Selectivity of Isolexins,Isohelical DNA Groove Binding Ligands. Proposal for the GC Minor Groove Specific Compounds

Abstract

Atheoretical study is presented of complex formation between DNA fragments of different base sequences and isolexins, “isohelical base reading polymers”, formed of heteroaromatic pentagonal rings joined by appropriate linkers. Extensive computations are performed for the isolexin composed of the furan-pyrrole-furan sequence. They involve charged ligands with propioamidinium groups at both ends as well eis neutral molecules with terminal methyl, carbonyl and amino groups. Two different groups (C=O and NH) are used as linkers between the base reading moieties. The role of these elements on the binding preference of the ligands has been examined. The results show that the mere possibility of formation of hydrogen bonds between a ligand and the nucleic acid bases is not sufficient to ensure its binding specificity which is determined largely by the interplay of electrostatic factors. Thus the dicationic isolexins uniformly prefer AT sequences. For the neutral isolexins the nature of the groups forming the linkers is a major factor in defining the specificity, although these groups do not participate directly in the interaction with DNA The C=O linkers favour binding to AT sequence while the N-H linkers permit preferential binding to the GAG sequence. Finally, for the first time in theoretical computations, a ligand is proposed which should bind preferentially to the minor groove of GC sequences: this ligand is a neutral isolexin composed of three furan rings linked by two N-H groups. This ligand is considered as an improvable prototype. Altogether the results presented open the path for the designing of minor groove ligands specific for any desirable DNA base sequence.     

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.     

An absolute method for the determination of the persistence length of native DNA from electron micrographs.

Information on spatial correlation in the tangent direction along electron microscope images of filamentous molecule is shown to be obtainable by the analysis of statistical fluctuations in curvature, yielding an absolute measure of the persistence parameter a_micro. The relationship of a_micro, a local, microscopic parameter, to the persistence length introduced by Kratky and Porod is discussed. The hypotheses underlying the assumed theoretical model concern (1) the shape of the angle distribution, assumed to be Gaussian; (2) the passage from a three- to a two-dimensional situation, which is supposed to occur by deformation of the flexible chain in a manner that preserves the memory of the spatial correlation in orientation (except for the blocking of one degree of freedom); and (3) the adsorption conditions, which should meet the equilibrium requirement as closely as possible. The analytical method has been checked on computer simulated “Gaussian” molecules: the study of the simulated sample was essential in solving the problems connected with minimum statistics requirements and the effect of the reading error. Experimental images obtained for T2 DNA fragments at different ionic strengths by Kleinschmidt's adsorption technique have been analyzed by means of an automatic flying spot digitizer, the “Precision Encoder and Pattern Recognition.” The results show that adsorbed molecules do in fact “remember” the rigidity they possessed in solution and that the Gaussian hypothesis is well verified. Consequently, the slopes of log cosθ (l) or θ² (l) may be used indifferently in the estimate of a_micro. The dependence of this parameter on ionic strength in the range explored shows the expected behavior.     

Size distribution and maturation of newly replicated DNA through the S and G2 phases of physarum polycephalum

The size distribution of newly made DNA and the dynamics of size maturation of progeny DNA molecules were studied in the synchronous S and G2 phases of Physarum polycephalum. Pulse labeling of DNA and analysis of the products on alkaline sucrose gradients showed that synthesis of primary replication units (which will also be referred to as “Okazaki” fragments) occurred throughout the S period. Pulse and pulse-chase experiments revealed a distinct pattern of size maturation. An apparently linear increase in molecular weight of progeny DNA molecules during the first hour of the S phase occurred at a rate of approximately 4–5 × 10⁵ daltons per min at 26°C, corresponding to the joining of 6–8 Okazaki fragments. The resulting 35–45S (1.1–2.2 × 10⁷ daltons) DNA molecules may correspond to the Physarum “replicon.” The further size increases of the newly made DNA appear to occur in steps, possibly reflecting a clustering of isochronous replicons along the chromatide. These observations are discussed with regard to mechanisms of DNA replication and size maturation.     

Hydrogen and hydration of DNA and RNA oligonucleotides

In this paper, hydrogen bonding interaction and hydration in crystal structures of both DNA and RNA oligonucleotides are discussed. Their roles in the formation and stabilization of oligonucleotides have been covered. Details of the Watson-Crick base pairs G.C and A.U in DNA and RNA are illustrated. The geometry of the wobble (mismatched) G.U base pairs and the cis and almost trans conformations of the mismatched U.U base pairs in RNA is described. The difference in hydration of the Watson-Crick base pairs G.C, A.U and the wobble G.U in different sequences of codon-anticodon interaction in double helical molecules are indicative of the effect of hydration. The hydration patterns of the phosphate, the 2'-hydroxyl groups, the water bridges linking the phosphate group, N7 (purine) and N4 of Cs or O4 of Us in the major groove, the water bridges between the 2'-hydroxyl group and N3 (purine) and O2 (pyrimidine) in the minor groove are discussed.     

Intermediates in genetic recombination of bacteriophage T7 DNA. Biological activity and the roles of gene 3 and gene 5

When Escherichia coli cells were infected with ³²P- and 5-bromodeoxyuridine-labeled T7 bacteriophage defective in genes 1.3, 2.3, 4 and 5, doubly branched T7 DNA molecules with “H” or “X”-like configurations were found in the half-heavy density fractions. Physical study showed that they are dimeric molecules composed of two parental DNA molecules (Tsujimoto & Ogawa, 1977a). The transfection assay of these molecules revealed that they were infective. Genetic analysis of progeny in infective centers obtained by transfection of dimeric molecules formed by infection of genetically marked T7 phage showed that these dimeric molecules were genetically biparental.To elucidate the roles of the products of gene 3 (endonuclease I) and gene 5 (DNA polymerase) of phage T7 in the recombination process, the ³²P/BrdUrd hybrid DNA molecules which were formed in the infected cells in the presence of these gene products were isolated, and their structures were analyzed. The presence of T7 DNA polymerase seems to stimulate and/or stabilize the interaction of parental DNAs. At an early stage of infection few dimeric molecules were formed in the absence of T7 DNA polymerase, whereas a significant number of doubly branched molecules were formed in its presence. With increasing incubation time, the multiply branched DNA molecules with a high sedimentation velocity accumulated.In contrast to the accumulation of multiply branched molecules in phage with mutations in genes 2, 3 and 4, almost all of the ³²P/BrdUrd hybrid DNA formed in phage with mutations in genes 2 and 4 were monomeric linear molecules. Shear fragmentation of monomeric linear ³²P/BrdUrd-labeled DNA shifted the density of [³²P]DNA to almost fully light density. It was also found that approximately 50% of [³²P]DNA was linked covalently to BrdUrd-labeled DNA. These linear monomer DNA molecules had infectivity and some of those formed by infection of genetically marked parents yielded recombinant phages. Therefore the gene 3 product seems to process the branched intermediates to linear recombinant molecules by trimming the branches.     

Letter: The isolation and properties of the specific binding sites for Escherichia coli RNA polymerase on T4 and T7 bacteriophage DNAs.

RNA polymerase of Escherichia coli was allowed to bind to labeled T4 or T7 bacteriophage DNA. The unbound and “weakly” bound polymerase molecules were removed by adding an excess of poly(I) which has a high affinity for the enzyme (Bautz et al., 1972). After the unbound DNA regions were digested with pancreatic DNAase and snake venom phosphodiesterase, the “protected” DNA-RNA polymerase complexes were isolated by Sephadex G200 column chromatography. The protected DNA sites were then isolated by phenol extraction and hydroxylapatite chromatography. Studies of the DNA recognition regions led to the following conclusions. (1) No binding is observed in the absence of the sigma subunit or at low temperatures. (2) The amount of protection ranges from 0·18% to 0·24% of T4 DNA and from 0·25% to 0·34% of T7 DNA. In the absence of poly(I), higher protections are observed and the protected regions display heterogeneity in size and secondary structure. (3) The protected regions are double-stranded, as shown by hydroxylapatite chromatography, base composition analysis, and thermal chromatography. (4) The length of the protected regions comprise about 50 to 55 nucleotide pairs, as suggested by end-group analysis, sucrose density-gradient centrifugation, and polyacrylamide gel electrophoresis. (5) The results suggest the interaction of dimeric polymerase molecules at these sites. On the basis of DNA sizes, there are 7 to 9 such sites on T4 DNA and 2 to 3 on T7 DNA. (6) The protected regions are high in (A + T): 68% for T4 and 62% for T7 DNA. (7) Thermal chromatograms reflect these base compositions and suggest the homogeneity of these regions with respect to size and base composition.     

Nucleic acid binding properties of Escherichia coli ribosomal protein S1. I. Structure and interactions of binding site I.

Escherichia coli ribosomal protein S1 plays a central role in initiation of protein synthesis, perhaps via participation in the binding of messenger RNA to the ribosome. S1 protein has two nucleic acid binding sites with very different properties: site I binds either single-stranded DNA or RNA, while site II binds single-stranded RNA only (Draper et al., 1977). The nucleic acid binding properties of these sites have been explored using the quenching of intrinsic protein fluorescence which results from binding of oligo- and polynucleotides, and are reported in this and the accompanying paper (Draper &; von Hippel, 1978).Site I has been studied primarily using DNA oligomers and polymers, and has been found to have the following properties. (1) The intrinsic binding constant (K) of site I for poly(dA) and poly(dC) is ~3 × 10⁶m^?1 at 0.12 m-Na⁺, and the site size (n, the number of nucleotide residues covered per S1 bound) is 5.1 ± 1.0 residues. (2) Binding of site I to polynucleotides is non-co-operative. (3) The K value for binding of S1 to single-stranded polynucleotides is ~10³ larger than K for binding to double-stranded polynucleotides, meaning that S1 (via site I) is a potential “melting” or “double-helix destabilizing” protein. (4) The dependence of log K on log [Na⁺] is linear, and analysis of the data according to Record et al. (1976) shows that two basic residues in site I form charge-charge interactions with two DNA phosphates. In addition, a major part of the binding free energy of site I with the nucleic acid chain appears to involve non-electrostatic interactions. (5) Oligonucleotides bound in site II somewhat weaken the binding affinity of site I. (6) Binding affin is virtually independent of base and sugar composition of the nucleic acid ligand; in fact, the total absence of the base appears to have little effect on the binding, since the association constant for 2′-deoxyribose 5′-phosphate is approximately the same as that for dAMP or dCMP. (7) Two molecules of d(ApA) can bind to site I, suggesting the presence of two “subsites” within site I. (8) Iodide quenching experiments with S1-oligonucleotide complexes show differential exposure of tryptophans in and near the subsites of site I, depending upon whether neither, one, or both subsites are complexed with an oligonucleotide.     

Effect of gold nanoparticle on stability of the DNA molecule: A study of molecular dynamics simulation

An understanding of the mechanism of DNA interactions with gold nanoparticles is useful in today medicine applications. We have performed a molecular dynamics simulation on a B-DNA duplex (CCTCAGGCCTCC) in the vicinity of a gold nanoparticle with a truncated octahedron structure composed of 201 gold atoms (diameter ～1.8 nm) to investigate gold nanoparticle (GNP) effects on the stability of DNA. During simulation, the nanoparticle is closed to DNA and phosphate groups direct the particles into the major grooves of the DNA molecule. Because of peeling and untwisting states that are occur at end of DNA, the nucleotide base lies flat on the surface of GNP. The configuration entropy is estimated using the covariance matrix of atom-positional fluctuations for different bases. The results show that when a gold nanoparticle has interaction with DNA, entropy increases. The results of conformational energy and the hydrogen bond numbers for DNA indicated that DNA becomes unstable in the vicinity of a gold nanoparticle. The radial distribution function was calculated for water hydrogen–phosphate oxygen pairs. Almost for all nucleotide, the presence of a nanoparticle around DNA caused water molecules to be released from the DNA duplex and cations were close to the DNA.     

Exploring the potential of nuclear and mitochondrial sequencing data generated through genome‐skimming for plant phylogenetics: A case study from a clade of neotropical lianas

Few botanical studies have explored the potential of nuclear ribosomal DNA (nrDNA) and mitochondrial DNA (mtDNA) data obtained through genome skimming for phylogeny reconstruction. Here, we analyzed the phylogenetic information included in the nrDNA and mtDNA of 44 species of the “Adenocalymma‐Neojobertia” clade (Bignoniaceae). To deal with intraindividual polymorphisms within the nrDNA, different coding schemes were explored through the analyses of four datasets: (i) “nrDNA contig,” with base call following the majority rule; (ii) “nrDNA ambiguous,” with ambiguous base calls; (iii) “nrDNA informative,” with ambiguities converted to multistate characters; and, (iv) “mitochondrial,” with 39 mitochondrial genes. Combined analyses using the nrDNA and mtDNA data and previously published “plastid” datasets were also conducted. Trees were obtained using Maximum Likelihood and Bayesian criteria. The congruence among genomes was assessed. The nrDNA datasets were shown to be highly polymorphic within individuals, while the “mitochondrial” dataset was the least informative, with 0.36% of informative bases within the ingroup. The topologies inferred using the nrDNA and mtDNA datasets were broadly congruent with the tree derived from the analyses of the “plastid” dataset. The topological differences recovered were generally poorly supported. The topology that resulted from the analyses of the “combined” dataset largely resembles the “plastid” tree. These results highlight limitations of nuclear ribosomal DNA and mitochondrial genes for phylogeny reconstruction obtained through genome skimming and the need to include more data from both genomes. The different topologies observed among genomes also highlight the importance of exploring data from various genomes in plant phylogenetics.     

Collective motion in DNA and its role in drug intercalation

The effects of collective motion in DNA as reflected by resonance coupling among its intact segments have been discussed for both linear and circular DNA molecules. The results indicate that due to the effects of this kind of internal collective motion, the energy will be at times highly concentrated at some spots. As a result of the overfocus of energy, the stress built up along the direction of hydrogen bonds between complementary base pairs will be dramatically increased, rupturing a series of consecutive hydrogen bonds simultaneously and resulting in a suddenly free jerk, such that the DNA molecule will undergo a local “quake.” The “hole” formed by this kind of quake-like motion will be large enough for bulky drugs to gain entrance and intercalate into DNA. Even for smaller drugs, this local quake-like motion can also provide a significant mode of entry for intercalation. Energy minimizations carried out for DNA–drug complexes indicate that, for most drugs, a distortion or disruption of 2 to 4 base pairs occurs at the intercalation site in DNA molecules.     

Parallel Double Helices of DNA. Conformational Analysis of Regular Helices with the Second Order Symmetry Axis

Abstract

We have performed a conformational analysis of DNA double helices with parallel directed backbone strands connected with the second order symmetry axis being at the same time the helix axis. The calculations were made for homopolymers poly(dA) · poly(dA), poly(dC) · poly(dC), poly(dG) poly(dG), and poly(dT) · poly(dT). All possible variants of hydrogen bonding of base pairs of the same name were studied for each polymer. The maps of backbone chain geometrical existence were constructed. Conformational and helical parameters corresponding to local minima of conformational energy of “parallel” DNA helices, calculated at atom-atom approximation, were determined. The dependence of conformational energy on the base pair and on the hydrogen bond type was analysed. Two major conformational advantageous for “parallel” DNA's do not depend much on the hydrogen-bonded base pair type were indicated. One of them coincided with the conformational region typical for “antiparallel” DNA in particular for the B-form DNA Conformational energy of “parallel” DNA depends on the base pair type and for the most part is similar to the conformational energy of “antiparallel” B-DNA.     

Calorimetry of DNA-dye interactions in aqueous solution: I. Proflavine and ethidium bromide

The results of an investigation on the interaction of proflavine and of ethidium bromide with DNA (calf thymus) in dilute aqueous solution are reported. The binding of the two dyes by DNA has been studied by means of microcalorimetric and of equilibrium dialysis measurements. Data on the thermodynamics of dimerization of both proflavine and ethidium bromide in aqueous solution obtained on the basis of spectroscopic and/or calorimetric experiments are also reported.The enthalpy data show that dye-dimerization and dye “strong” interaction with DNA are energetically favourable and quite similar while only in the latter case the phenomenon is also entropy driven. This is taken as further evidence in support of the concept that “strong” interaction-of both proflavine and ethidium bromide with DNA means dye molecules intercalation into the native, double helical structure of the biopolymer.     

Amplification as a Rectification Mechanism for the Redundant rRNA Genes

IN eukaryotic cells multiple copies of ribosomal RNA (rRNA) genes are clustered in one or a few nucleolar organizers per haploid genome. These “redundant” or “reiterated” genes have all been shown to be, within the limits of available experimental techniques, identical within a given organism and species, producing homogeneous populations of rRNA molecules. On the other hand, rRNAs from organisms of different taxonomic position display evidence of considerable evolutionary change from the point of view of size, base sequence (revealed by RNA-DNA hybridization) and base composition^1,2.     

A compact form of DNA in solution. III. Influence of the ion composition of the solution on the compactization process of double-stranded DNA in the presence of peg]

The data showing the features of the DNA compactization process in PEG-containing solutions of chlorides of different alkaline metals (LiCl, KCl, RbCl and CsCl) and an ammonium salt (CH3-(CH2)17-N-(CH3)3Br) are presented. The data indicate that the formation of a compact form of the double-stranded DNA in PEG-containing water-salt solutions depends not only on the PEG concentration and ionic strength but on tha cation nature as well. The compactization occurs most easily in the presence of Na+-ions. This indicates a specific character of interaction between Na+-ions and DNA phosphate groups which may be due to an optimum structural fit between the hydrated Na+-ions and orientation of the phosphate groups in the DNA molecule. The nature of forces involved in the processes of the intramolecular compactization and intermolecular aggregation of double-stranded DNA molecules in water-salt solution is discussed. The difference between the effect of Na+ and that of K+-ions on the compactization process at the ionic strengths close to physiological values makes it possible to suggest that the changes of the tertiary structure of double-stranded DNA which accompany its function in vivo may take place under conditions of a decreased water activity at the expense of relatively slight changes in ion composition of the water surrounding DNA.     

Control by cell interaction of phosphate uptake in 3T3 cells.

Phosphate uptake by monolayers of 3T3 cell decreases when the cultures enter the stationary phase, even when incubated in fresh medium containing 10% serum. However, SV 3T3 cultures retain a high rate of phosphate uptake when the cells reach saturation densities.We have observed that 3T3 cells grown to stationary phase in monolayers and then trypsinized and incubated in suspension, display an increase in phosphate uptake when the cell concentration is decreased from 10⁶ cells/ml to 10⁵ cells/ml. Where the cell concentration is further reduced from 10⁵ cells/ml to 2.5 × 10⁴ cells/ml there is no further increase in the rate of phosphate uptake. We observed, on the contrary, a small decrease.The “concentration effect” (the decrease of phosphate uptake when the cell concentration increases from 10⁵ to 10⁶ cells/ml) is larger when cells originate from a culture in stationary phase than when they originate from a culture in log phase.The “concentration effect” may be observed 10 min after cell incubation but is larger after a lag time of 40 min incubation.Differences in the “concentration effect” may be noted between 3T3 and SV 3T3 cells. In SV 3T3 cells no significant variations of phosphate uptake were observed when the cell concentration was changed. Thus, differences between phosphate uptake in 3T3 and SV 3T3 cells are large when cells are incubated at high concentrations or at high densities and small when they are incubated at low concentrations or at low densities.The “concentration effect” in 3T3 cells supports the assumption that interactions between cells cause the decrease of phosphate metabolism in dense culture. Diffusion of an inhibitor into the medium remains the more plausible explanation of the data.     

Hysteresis and partial irreversibility of denaturation of DNA as a means of investigating the topology of base distribution constraints: application to a yeast rho- (petite) mitochondrial DNA

Low salt concentrations prevent reassociation of separated single strands of DNA, but not the renaturation of partially melted molecules. Rewinding, however, may be delayed (hysteresis) and/or incomplete (partial irreversibility). Long-range fluctuations in base compositioncould account for these observations: (a) the “zippering-up” of a denatured (G + C)-rich section may have to await that of one of its neighbouring (A + T)-rich sections, hence a temperature lag in rewinding; (b) the removal of intramolecular heterogeneities in base composition by fragmentation will give rise to a dispersal of strand-separation temperatures. Conversely, it is shown how a considerable amount of information about the topology of base distribution constraints could be derived from these phenomena.Some yeast ρ^? (petite) mitochondrial DNAs, the melting of which is quasidiscontinuous, provide an excellent opportunity for testing the applicability of this new approach to denaturation mapping. Alternating partial denaturation and renaturation with a low rate of temperature change were followed by high-frequency recording of absorbance at 260 nm. A typical experiment (counterion concentration 0.015 m-Na⁺) carried out on a low-complexity (length of repetitive unit about 3000 base-pairs) ρ^? DNA is reported in full detail. Analysis of the data disclosed the existence of two relatively (G + C)-rich clusters separated by long homogeneous stretches of high (A + T) content.The rewinding of ρ^? DNAs is a discontinuous process. Both equilibrium and non-equilibrium melting processes were observed. Hysteresis in rewinding, which is restricted to the melting range, increases discontinuously with the extent of unwinding reached prior to cooling. Results are shown to be fully consistent with a model that presupposes that nucleation does not play any part in the renaturation process. They are briefly discussed further in the light of current concepts in the theory of helix-coil transitions of DNA.