Structural features of B-DNA dodecamer crystal structures: Influence of crystal packing versus base sequence

We have analyzed the set of inter and intra base pair parameters for each dinucleotide step in single crystal structures of dodecamers, solved at high and medium resolution and all crystallized in P2(1)2(1)2(1) space group. The objective was to identify whether all the structures which have either the Drew-Dickerson (DD) sequence d[CGCGAATTCGCG] with some base modification or related sequence (non-DD), would display the same sequence dependent structural variability about its palindromic sequence, despite the molecule being bent at one end because of similar crystal lattice packing effect. Most of the local doublet parameters for base pairs steps G2-C3 and G10-C11 positions, symmetrically situated about the lateral twofold, were significantly correlated between themselves. In non-DD sequences, significant correlations between these positional parameters were absent. The different range of local step parameter values at each sequence position contributed to the gross feature of smooth helix axis bending in all structures. The base pair parameters in some of the positions, for medium resolution DD sequence, were quite unlike the high-resolution set and encompassed a higher range of values. Twist and slide are the two main parameters that show wider conformational range for the middle region of non-DD sequence structures in comparison to DD sequence structures. On the contrary, the minor and major groove features bear good resemblance between DD and non-DD sequence crystal structure datasets. The sugar-phosphate backbone torsion angles are similar in all structures, in sharp contrast to base pair parameter variation for high and low resolution DD and non-DD sequence structures, consisting of unusual (epsilon =g(-), xi =t) B-II conformation at the 10(th) position of the dodecamer sequence. Thus examining DD and non-DD sequence structures packed in the same crystal lattice arrangement, we infer that inter and intra base pair parameters are as symmetrically equivalent in its value as the symmetry related step for the palindromic DD sequence about lateral two-fold axis. This feature would lead us to agree with the conclusion that DNA conformation is not substantially affected by end-to-end or lateral inter-molecular interaction due to crystal lattice packing effect. Non-DD sequence structures acquire step parameter values which reflect the altered sequence at each of the dodecamer sequence position in the orthorhombic lattice while showing similar gross features of DD sequence structures

Energy Hyperspace for Stacking Interaction in AU/AU Dinucleotide Step: Dispersion-Corrected Density Functional Theory Study

Double helical structures of DNA and RNA are mostly determined by base pair stacking interactions, which give them the base sequence-directed features, such as small roll values for the purine-pyrimidine steps. Earlier attempts to characterize stacking interactions were mostly restricted to calculations on fiber diffraction geometries or optimized structure using ab initio calculations lacking variation in geometry to comment on rather unusual large roll values observed in AU/AU base pair step in crystal structures of RNA double helices. We have generated stacking energy hyperspace by modeling geometries with variations along the important degrees of freedom, roll, and slide, which were chosen via statistical analysis as maximally sequence dependent. Corresponding energy contours were constructed by several quantum chemical methods including dispersion corrections. This analysis established the most suitable methods for stacked base pair systems despite the limitation imparted by number of atom in a base pair step to employ very high level of theory. All the methods predict negative roll value and near-zero slide to be most favorable for the purine-pyrimidine steps, in agreement with Calladine's steric clash based rule. Successive base pairs in RNA are always linked by sugar-phosphate backbone with C3-endo sugars and this demands C1-C1 distance of about 5.4 angstrom along the chains. Consideration of an energy penalty term for deviation of C1-C1 distance from the mean value, to the recent DFT-D functionals, specifically B97X-D appears to predict reliable energy contour for AU/AU step. Such distance-based penalty improves energy contours for the other purine-pyrimidine sequences also. (c) 2013 Wiley Periodicals, Inc. Biopolymers 101: 107-120, 2014.

The structural transition of DNA-Tris(1,10-phenanthroline) cobalt(III) complexes in ethanol-water solution

The interaction of DNA with Tris(1,10-phenanthroline) cobalt(III) was studied by means of atomic force microscopy. Changes in the morphologies of DNA complex in the presence of ethanol may well indicate the crucial role of electrostatic force in causing DNA condensation. With the increase of the concentration of ethanol, electrostatic interaction is enhanced corresponding to a lower dielectric constant. Counterions condense along the sugar phosphate backbone of DNA when e is lowered and the phosphate charge density can thus be neutralized to the level of DNA condensation. Electroanalytical measurement of DNA condensed with Co(phen)(3)(3+) in ethanol solution indicated that intercalating reaction remains existing. According to both the microscopic and spectroscopic results, it can be found that no secondary structure transition occurs upon DNA condensing. B-A conformation transition takes place at more than 60% ethanol solution.

Distinct functional domains of the Salmonella enterica WbaP transferase that is involved in the initiation reaction for synthesis of the O antigen subunit

WbaP is a membrane enzyme that initiates O antigen synthesis in Salmonella enterica by catalysing the transfer of galactose 1-phosphate (Gal-1-P) onto undecaprenyl phosphate (Und-P). WbaP possesses at least three predicted structural domains: an N-terminal region containing four transmembrane helices, a large central periplasmic loop, and a C-terminal domain containing the last transmembrane helix and a large cytoplasmic tail. In this work, we investigated the contribution of each region to WbaP function by constructing a series of mutant WbaP proteins and using them to complement O antigen synthesis in DeltawbaP mutants of S. enterica serovars Typhi and Typhimurium. Truncated forms of WbaP lacking the periplasmic loop exhibited altered chain-length distributions in O antigen polymerization, suggesting that this central domain is involved in modulating the chain-length distribution of the O polysaccharide. The N-terminal and periplasmic domains were dispensable for complementation of O antigen synthesis in vivo, suggesting that the C-terminal domain carries the sugar-phosphate transferase activity. However, despite the fact that they complemented the synthesis of O antigen in the DeltawbaP mutant in vivo, membrane extracts containing WbaP derivatives without the N-terminal domain failed to transfer radioactive Gal from UDP-Gal into a lipid-rich fraction. These results suggest that the N-terminal region of WbaP, which contains four transmembrane domains, is essential for the insertion or stability of the protein in the bacterial membrane. We propose that the domain structure of WbaP enables this protein not only to function in the transfer of Gal-1-P to Und-P but also to establish critical interactions with additional proteins required for the correct assembly of O antigen in S. enterica.

The application of gas liquid chromatography to the simultaneous analysis of sugars and sugar phosphates in biological samples /|nby David J. LeBlanc. -- 260 St. Catharines [Ont. : s. n.],

A study was undertaken' to determine the applicability of gas liquid chromatography to the simultaneous analysis of sugars and sugar phosphates from biological samples. A new method of silylation involving dimethylsulfoxide, hexamethyldisilazane, trimethylchlorosilane and cyclohexane (1:0.2:0.1:1) which rapidly silylated sugars and sugar phosphates was developed. Subsequent chromatography on a 5% SE-52 column gave good resolution of the sugar and sugar phosphate samples. Sugar phosphates decomposed during chromatography and were lost at the 7 x 10-3 ~mole level. Acidic ethanol extraction of yeast samples revealed background contamination from the yeast sample, the culture medium and the silylation reagents which would further limit the level of detection obtainable with the glc for sugars in biological samples to the 3 x 10-4 ~mole level.

Le motif d’empaquetage le long du sillon: une nouvelle entité structurale récurrente dans les ARN ribosomiques

La plupart des molécules d’ARN doivent se replier en structure tertiaire complexe afin d’accomplir leurs fonctions biologiques. Cependant, les déterminants d’une chaîne de polynucléotides qui sont nécessaires à son repliement et à ses interactions avec d’autres éléments sont essentiellement inconnus. L’établissement des relations structure-fonction dans les grandes molécules d’ARN passe inévitablement par l’analyse de chaque élément de leur structure de façon individuelle et en contexte avec d’autres éléments. À l’image d’une construction d’immeuble, une structure d’ARN est composée d’unités répétitives assemblées de façon spécifique. Les motifs récurrents d’ARN sont des arrangements de nucléotides retrouvés à différents endroits d’une structure tertiaire et possèdent des conformations identiques ou très similaires. Ainsi, une des étapes nécessaires à la compréhension de la structure et de la fonction des molécules d’ARN consiste à identifier de façon systématique les motifs récurrents et d’en effectuer une analyse comparative afin d’établir la séquence consensus. L’analyse de tous les cas d’empaquetage de doubles hélices dans la structure du ribosome a permis l’identification d’un nouvel arrangement nommé motif d’empaquetage le long du sillon (AGPM) (along-groove packing motif). Ce motif est retrouvé à 14 endroits dans la structure du ribosome de même qu’entre l’ARN ribosomique 23S et les molécules d’ARN de transfert liées aux sites ribosomaux P et E. Le motif se forme par l’empaquetage de deux doubles hélices via leur sillon mineur. Le squelette sucre-phosphate d’une hélice voyage le long du sillon mineur de l’autre hélice et vice versa. Dans chacune des hélices, la région de contact comprend quatre paires de bases. L’empaquetage le plus serré est retrouvé au centre de l’arrangement où l’on retrouve souvent une paire de bases GU dans une hélice interagissant avec une paire de bases Watson-Crick (WC) dans l’autre hélice. Même si la présence des paires de bases centrales GU versus WC au centre du motif augmente sa stabilité, d’autres alternatives existent pour différents représentants du motif. L’analyse comparative de trois librairies combinatoires de gènes d’AGPM, où les paires de bases centrales ont été variées de manière complètement aléatoire, a montré que le contexte structural influence l’étendue de la variabilité des séquences de nucléotides formant les paires de bases centrales. Le fait que l’identité des paires de bases centrales puisse varier suggérait la présence d’autres déterminants responsables au maintien de l’intégrité du motif. L’analyse de tous les contacts entre les hélices a révélé qu’en dehors du centre du motif, les interactions entre les squelettes sucre-phosphate s’effectuent via trois contacts ribose-ribose. Pour chacun de ces contacts, les riboses des nucléotides qui interagissent ensemble doivent adopter des positions particulières afin d’éviter qu’ils entrent en collision. Nous montrons que la position de ces riboses est modulée par des conformations spécifiques des paires de bases auxquelles ils appartiennent. Finalement, un autre motif récurrent identifié à l’intérieur même de la structure de trois cas d’AGPM a été nommé « adenosine-wedge ». Son analyse a révélé que ce dernier est lui-même composé d’un autre arrangement, nommé motif triangle-NAG (NAG-triangle). Nous montrons que le motif « adenosine-wedge » représente un arrangement complexe d’ARN composé de quatre éléments répétitifs, c’est-à-dire des motifs AGPM, « hook-turn », « A-minor » et triangle-NAG. Ceci illustre clairement l’arrangement hiérarchique des structures d’ARN qui peut aussi être observé pour d’autres motifs d’ARN. D’un point de vue plus global, mes résultats enrichissent notre compréhension générale du rôle des différents types d’interactions tertiaires dans la formation des molécules d’ARN complexes.

Molecular Dynamics Simulation of a PNA·DNA·PNA Triple Helix in Aqueous Solution

Molecular dynamics simulations have been used to explore the conformational flexibility of a PNA·DNA·PNA triple helix in aqueous solution. Three 1.05 ns trajectories starting from different but reasonable conformations have been generated and analyzed in detail. All three trajectories converge within about 300 ps to produce stable and very similar conformational ensembles, which resemble the crystal structure conformation in many details. However, in contrast to the crystal structure, there is a tendency for the direct hydrogen-bonds observed between the amide hydrogens of the Hoogsteen-binding PNA strand and the phosphate oxygens of the DNA strand to be replaced by water-mediated hydrogen bonds, which also involve pyrimidine O2 atoms. This structural transition does not appear to weaken the triplex structure but alters groove widths and so may relate to the potential for recognition of such structures by other ligands (small molecules or proteins). Energetic analysis leads us to conclude that the reason that the hybrid PNA/DNA triplex has quite different helical characteristics from the all-DNA triplex is not because the additional flexibility imparted by the replacement of sugar−phosphate by PNA backbones allows motions to improve base-stacking but rather that base-stacking interactions are very similar in both types of triplex and the driving force comes from weak but definate conformational preferences of the PNA strands.

Probing molecular changes induced in DNA by reactive oxygen species with monoclonal antibodies

Antibodies reactive with native double stranded DNA are characteristic of the chronic inflammatory disease systemic lupus erythematosus. Native DNA is however, a poor immunogen and the mechanism of anti-DNA antibody production is incompletely understood. Modification of DNA can increase its immunogenicity and in inflammatory disease states reactive oxygen species produced from phagocytic cells have been shown to thus modify DNA. In this study, monoclonal antibodies produced spontaneously by two mice strains with lupus-like disease were used in a competition ELISA to monitor changes to DNA induced by reactive oxygen species. Different procedures for reactive oxygen species generation were found to cause distinct and characteristic changes to DNA involving modifications of base residues, the sugar-phosphate backbone and the gross conformational structure of double-stranded DNA. In view of this, it may be possible to use these antibodies further to probe DNA and infer the source and nature of the reactive oxygen species it has been exposed to, particularly in vivo.

The mechanical strength of phosphates under friction-induced cross-linking

Purpose: In the present study, we consider mechanical properties of phosphate glasses under high temperatureinduced and under friction-induced cross-linking, which enhance the modulus of elasticity. Design/methodology/approach: Two nanomechanical properties are evaluated, the first parameter is the modulus of elasticity (E) (or Young's modulus) and the second parameter is the hardness (H). Zinc meta-, pyro - and orthophosphates were recognized as amorphous-colloidal nanoparticles were synthesized under laboratory conditions and showed antiwear properties in engine oil. Findings: Young's modulus of the phosphate glasses formed under high temperature was in the 60-89 GPa range. For phosphate tribofilm formed under friction hardness and the Young's modulus were in the range of 2-10 GPa and 40-215 GPa, respectively. The degree of cross-linking during friction is provided by internal pressure of about 600 MPa and temperature close to 1000°C enhancing mechanical properties by factor of 3 (see Fig 1). Research limitations/implications: The addition of iron or aluminum ions to phosphate glasses under high temperature - and friction-induced amorphization of zinc metaphosphate and pyrophosphate tends to provide more cross-linking and mechanically stronger structures. Iron and aluminum (FeO4 or AlO4 units), incorporated into phosphate structure as network formers, contribute to the anion network bonding by converting the P=O bonds into bridging oxygen. Future work should consider on development of new of materials prepared by solgel processes, eg., zinc (II)-silicic acid. Originality/value: This paper analyses the friction pressure-induced and temperature–induced the two factors lead phosphate tribofilm glasses to chemically advanced glass structures, which may enhance the wear inhibition. Adding the coordinating ions alters the pressure at which cross-linking occurs and increases the antiwear properties of the surface material significantly.

NUPARM and NUCGEN: software for analysis and generation of sequence dependent nucleic acid structures

Software packages NUPARM and NUCGEN, are described, which can be used to understand sequence directed structural variations in nucleic acids, by analysis and generation of non-uniform structures. A set of local inter basepair parameters (viz. tilt, roll, twist, shift, slide and rise) have been defined, which use geometry and coordinates of two successive basepairs only and can be used to generate polymeric structures with varying geometries for each of the 16 possible dinucleotide steps. Intra basepair parameters, propeller, buckle, opening and the C6...C8 distance can also be varied, if required, while the sugar phosphate backbone atoms are fixed in some standard conformation ill each of the nucleotides. NUPARM can be used to analyse both DNA and RNA structures, with single as well as double stranded helices. The NUCGEN software generates double helical models with the backbone fixed in B-form DNA, but with appropriate modifications in the input data, it can also generate A-form DNA ar rd RNA duplex structures.

The effect of tungsten additive on the surface characteristics of amorphous Ni-P alloy

The variation of surface compositions on amorphous Ni80.4W1.5P18.1 alloy by O-2 oxidation and H-2 reduction treatments have been studied by XPS, UPS and ISS. It shows that addition of tungsten in the amorphous Ni-P alloy leads to dramatic changes of the relating component distributions in the surface layers before and after these treatments. Oxidation of a Ni80.4W1.5P18.1 amorphous alloy in 1 bar of oxygen at 513 K caused a significant segregation of nickel in different oxide states at the surface. The subsequent reduction of the oxidized surface with I bar hydrogen at 553 K resulted in only a small portion of Ni and P being reduced into elemental states, while most of them was found to combine to form a kind of nickel phosphate compound. On the other hand, under the same conditions, the oxidation and reduction of a Ni80P20 alloy gave rise to metallic Ni and elemental P as the predominate species on the alloy surface. The addition of W in the amorphous alloy might act as nuclei for a favorable formation of the phosphate structure which was proposed to be an active species for hydrogen-relating catalytic reactions. (C) 1999 Elsevier Science B.V. All rights reserved.

Conformational Dynamics of the Human Propeller Telomeric DNA Quadruplex on a Microsecond Time Scale

The human telomeric DNA sequence with four repeats can fold into a parallel-stranded propeller-type topology. NMR structures solved under molecular crowding experiments correlate with the crystal structures found with crystal-packing interactions that are effectively equivalent to molecular crowding. This topology has been used for rationalization of ligand design and occurs experimentally in a number of complexes with a diversity of ligands, at least in the crystalline state. While G-quartet stems have been well characterised, the interactions of the TTA loop with the G-quartets are much less defined. To better understand the conformational variability and structural dynamics of the propeller-type topology, we performed molecular dynamics simulations in explicit solvent up to 1.5 µs. The analysis provides a detailed atomistic account of the dynamic nature of the TTA loops highlighting their interactions with the G-quartets including formation of an A:A base pair, triad, pentad and hexad. The results present a threshold in quadruplex simulations, with regards to understanding the flexible nature of the sugar-phosphate backbone in formation of unusual architecture within the topology. Furthermore, this study stresses the importance of simulation time in sampling conformational space for this topology.

Repair of abasic sites in DNA

Repair of both normal and reduced AP sites is activated by AP endonuclease, which recognizes and cleaves a phosphodiester bond 5' to the AP site. For a short period of time an incised AP site is occupied by poly(ADP-ribose) polymerase and then DNA polymerase beta adds one nucleotide into the repair gap and simultaneously removes the 5'-sugar phosphate. Finally, the DNA ligase III/XRCC1 complex accomplishes repair by sealing disrupted DNA ends. However, long-patch BER pathway, which is involved in the removal of reduced abasic sites, requires further DNA synthesis resulting in strand displacement and the generation of a damage-containing flap that is later removed by the flap endonuclease. Strand-displacement DNA synthesis is accomplished by DNA polymerase delta/epsilon and DNA ligase I restores DNA integrity. DNA synthesis by DNA polymerase delta/epsilon is dependent on proliferating cell nuclear antigen, which also stimulates the DNA ligase I and flap endonuclease. These repair events are supported by multiple protein-protein interactions. (C) 2003 Elsevier B.V. All rights reserved.

Conducão eletrônica e propriedades termodinâmicas da molécula de DNA

In this thesis, we study the thermo-electronic properties of the DNA molecule. For this purpose, we used three types of models with the DNA, all assuming a at geometry (2D), each built by a sequence of quasiperiodic (Fibonacci and / or Rudin-Shapiro) and a sequence of natural DNA, part of the human chromosome Ch22. The first two models have two types of components that are the nitrogenous bases (guanine G, cytosine C, adenine A and thymine T) and a cluster sugar-phosphate (SP), while the third has only the nitrogenous bases. In the first model we calculate the density of states using the formalism of Dyson and transmittance for the time independent Schr odinger equation . In the second model we used the renormalizationprocedure for the profile of the transmittance and consequently the I (current) versus V (voltage). In the third model we calculate the density of states formalism by Dean and used the results together with the Fermi-Dirac statistics for the chemical potential and the quantum specific heat. Finally, we compare the physical properties found for the quasi-periodic sequences and those that use a portion of the genomic DNA sequence (Ch22).

During the initiation of fermentation overexpression of hexokinase PII in yeast transiently causes a similar deregulation of glycolysis as deletion of Tps1

In the yeast Saccharomyces cerevisiae a novel control exerted by TPS1 (=GGS1=FDP1=BYP1=CIF1=GLC6=TSS1)-encoded trehalose-6-phosphate synthase, is essential for restriction of glucose influx into glycolysis apparently by inhibiting hexokinase activity in vivo. We show that up to 50-fold overexpression of hexokinase does not noticeably affect growth on glucose or fructose in wild-type cells. However, it causes higher levels of glucose-6-phosphate, fructose-6-phosphate and also faster accumulation of fructose-1,6-bisphosphate during the initiation of fermentation. The levels of ATP and Pi correlated inversely with the higher sugar phosphate levels. In the first minutes after glucose addition, the metabolite pattern observed was intermediate between those of the tps1Δ mutant and tile wild-type strain. Apparently, during the start-up of fermentation hexokinase is more rate-limiting in the first section of glycolysis than phosphofructokinase. We have developed a method to measure the free intracellular glucose level which is based on the simultaneous addition of D-glucose and an equal concentration of radiolabelled L-glucose. Since the latter is not transported, the free intracellular glucose level can be calculated as the difference between the total B-glucose measured (intracellular + periplasmic/extracellular) and the total L-glucose measured (periplasmic/extracellular). The intracellular glucose level rose in 5 min after addition of 100 mM-glucose to 0.5-2 mM in the wild-type strain, ± 10 mm in a hxk1Δ hxk2Δ glk1Δ and 2-3 mM in a tps1Δ strain. In the strains overexpressing hexokinase PII the level of free intracellular glucose was not reduced. Overexpression of hexokinase PII never produced a strong effect on the rate of ethanol production and glucose consumption. Our results show that overexpression of hexokinase does not cause the same phenotype as deletion of Tps1. However, it mimics it transiently during the initiation of fermentation. Afterwards, the Tps1-dependent control system is apparently able to restrict Properly up to 50-fold higher hexokinase activity.