DNA Axis Research Articles

Integration Host Factor (IHF) Dictates the Structure of Polyamine-DNA Condensates: Implications for the Role of IHF in the Compaction of Bacterial Chromatin

Integration host factor (IHF), a nucleoid-associated protein in bacterial cells, is implicated in a number of chromosomal functions including DNA compaction. IHF binds to all duplex DNA with micromolar affinity and at sequence-specific sites with much higher affinity. IHF is known to induce sharp bends in the helical axis of DNA in both modes of binding, but the role of IHF in controlling DNA condensation within bacterial cells has remained undetermined. Here we demonstrate that IHF influences the morphology of DNA condensed by polyamines in vitro. In the absence of IHF, spermidine and spermine condense DNA primarily into toroidal structures, whereas in the presence of IHF, polyamines condense DNA primarily into rodlike structures. Computer simulations of DNA condensation in the absence and presence of IHF binding lend support to our model in which DNA bending proteins, such as IHF and HU, promote the condensation of DNA into rodlike structures by providing the free energy necessary to bend DNA at the ends of linear bundles of condensed DNA. We propose that a common function of IHF and HU in bacterial cells is to facilitate DNA organization in the nucleoid by the introduction of sharp bends in chromosomal DNA.

II. Model Building: An Electrical Theory of Control of Growth and Development in Animals, Prompted by Studies of Exogenous Magnetic Field Effects (Paper I), and Evidence of DNA Current Conduction, In Vitro

A theory of control of cellular proliferation and differentiation in the early development of metazoan systems, postulating a system of electrical controls “parallel” to the processes of molecular biochemistry, is presented. It is argued that the processes of molecular biochemistry alone cannot explain how a developing organism defies a stochastic universe.The demonstration of current flow (charge transfer) along the long axis of DNA through the base-pairs (the “π-way) in vitro raises the question of whether nature may employ such current flows for biological purposes. Such currents might be too small to be accessible to direct measurement in vivo but conduction has been measured in vitro, and the methods might well be extended to living systems. This has not been done because there is no reasonable model which could stimulate experimentation. We suggest several related, but detachable or independent, models for the biological utility of charge transfer, whose scope admittedly outruns current concepts of thinking about organization, growth, and development in eukaryotic, metazoan systems. The ideas are related to explanations proposed to explain the effects demonstrated on tumors and normal tissues described in Article I (this issue).Microscopic and mesoscopic potential fields and currents are well known at sub-cellular, cellular, and organ systems levels. Not only are such phenomena associated with internal cellular membranes in bioenergetics and information flow, but remarkable long-range fields over tissue interfaces and organs appear to play a role in embryonic development (Nuccitelli, ). The origin of the fields remains unclear and is the subject of active investigation. We are proposing that similar processes could play a vital role at a “sub-microscopic level,” at the level of the chromosomes themselves, and could play a role in organizing and directing fundamental processes of growth and development, in parallel with the more discernible fields and currents described.

Force induced DNA melting

When pulled along the axis, double-strand DNA undergoes a large conformational changeand elongates by roughly twice its initial contour length at a pulling force of about 70 pN.The transition to this highly overstretched form of DNA is very cooperative. Applying aforce perpendicular to the DNA axis (unzipping), double-strand DNA can also beseparated into two single-stranded DNA, this being a fundamental process inDNA replication. We study the DNA overstretching and unzipping transitionusing fully atomistic molecular dynamics (MD) simulations and argue that theconformational changes of double-strand DNA associated with either of the abovementioned processes can be viewed as force induced DNA melting. As the force atone end of the DNA is increased the DNA starts melting abruptly/smoothlyabove a critical force depending on the pulling direction. The critical forcefm, at which DNA melts completely decreases as the temperature of the system is increased.The melting force in the case of unzipping is smaller compared to the melting forcewhen the DNA is pulled along the helical axis. In the case of melting throughunzipping, the double-strand separation has jumps which correspond to the differentenergy minima arising due to sequence of different base pairs. The fraction ofWatson–Crick base pair hydrogen bond breaking as a function of force does not showsmooth and continuous behavior and consists of plateaus followed by sharp jumps.

Diffusion Constant of a Nonspecifically Bound Protein Undergoing Curvilinear Motion along DNA

The mechanism of a protein's diffusion along a DNA segment is a subject of much current interest because of the involvement of this diffusion in numerous biological processes, including the recognition of DNA sequences and chemical modifications of DNA. In this work we present a theoretical derivation of the diffusion coefficient of a nonspecifically bound protein, assuming that the protein follows a helical track along the DNA. It is shown that, for protein-sized molecules, the principal contribution to the total translational friction comes from the curvilinear motion along the helix, and this contribution is given by 6pietaRR(oc)(2) + 8pietaR(3), where R is the protein radius, ROC is the distance of separation between the center of mass of the protein and the helical axis of DNA, and eta is the viscosity of the medium. The translational diffusion of the protein along the helical track of DNA is thus predicted to have a nearly R(-3) size dependence, not the R(-1) dependence characterizing simple translational diffusion. It is shown that this expression gives rather good estimates of the translational diffusion coefficient measured in single molecule experiments.

Cylindrical Projection of Electrostatic Potential and Image Analysis Tools for Damaged DNA: The Substitution of Thymine with Thymine Glycol

Changes of electrostatic potential around the DNA molecule resulting from chemical modifications of nucleotides may play a role in enzymatic recognition of damaged sites. Effects of chemical modifications of nucleotides on the structure of DNA have been characterized through electronic structure computations. Quantum mechanical structural optimizations of fragments of five pairs of nucleotides with thymine or thymine glycol were performed at the density functional level of theory with a B3LYP exchange-correlation functional and 6-31G(d,p) basis sets. The electrostatic potential (EP) around DNA fragments was projected on a cylindrical surface around the double helix. The 2D maps of EP of intact and damaged DNA fragments were compared using image analysis methods to identify and measure modifications of the EP that result from the occurrence of thymine glycol. It was found that distortions of phosphate groups and displacements of the accompanying countercations by up to approximately 0.5 angstroms along the axis of DNA are clearly reflected in the EP maps. Modifications of the EP in the major groove of DNA near the damaged site are also reported.

Dynamic Basis for One-Dimensional DNA Scanning by the Mismatch Repair Complex Msh2-Msh6

The ability of proteins to locate specific sites or structures among a vast excess of nonspecific DNA is a fundamental theme in biology. Yet the basic principles that govern these mechanisms remain poorly understood. For example, mismatch repair proteins must scan millions of base pairs to find rare biosynthetic errors, and they then must probe the surrounding region to identify the strand discrimination signals necessary to distinguish the parental and daughter strands. To determine how these proteins might function we used single-molecule optical microscopy to answer the following question: how does the mismatch repair complex Msh2-Msh6 interrogate undamaged DNA? Here we show that Msh2-Msh6 slides along DNA via one-dimensional diffusion. These findings indicate that interactions between Msh2-Msh6 and DNA are dominated by lateral movement of the protein along the helical axis and have implications for how MutS family members travel along DNA at different stages of the repair reaction.

DNA sequence-specific ligands: XIII. New dimeric Hoechst 33258 molecules, inhibitors of HIV-1 integrase in vitro

Dimeric Hoechst 33258 molecules [dimeric bisbenzimidazoles (DBBIs)] that, upon binding, occupy one turn of the B form of DNA in the narrow groove were constructed by computer simulation. Three fluorescent DBBIs were synthesized; they consist of two bisbenzimidazole units tail-to-tail linked to phenolic hydroxy groups via penta- or heptamethylene or tri(ethylene glycol) spacers and have terminal positively charged N.N-dimethylaminopropyl carboxamide groups in the molecule. The absorption spectra of the DBBIs in the presence of different DNA concentrations showed a hypochromic effect and a small shift of the absorption band to longer wavelengths, which indicated the formation of a complex with DNA. The presence of an isobestic point in the spectrum indicates the formation of one type of DBBI-DNA complexes. The interaction of DBBIs with DNA was studied by CD using a cholesteric liquid-crystalline dispersion (CLD) of DNA. The appearance of a positive band in the absorption region of ligand chromophores in the CD spectrum of the DNA CLD indicates the formation of a DBBI-DNA complex in which ligand chromophores are arranged at an angle close to 54 degrees relative to the helix axis of DNA, which suggests the localization of the DBBI in the narrow groove of DNA. All the DBBIs were found to be in vitro inhibitors of HIV-1 DNA integrase in the 3'-processing reaction, and, of the three DBBIs, two dimers inhibit HIV-1 integrase even in submicromolar concentrations.

How to Switch the Direction of Photoinduced Charge Injection into DNA?

Photoinduced hole injection by an acridine derivative X + within the complex 5'-TG-2TX+TG2T-3/ has been studied using quantum chemical calculations. The probability of charge shift from the excited state of the intercalated chromophore, (X + )*, to the guanine acceptors, G -2 and G 2 , is computed for 24 different conformations of the complex generated by rotation of the chromophore about the DNA axis. Because the free energies and the reorganization energies for the charge shift in both direction are very similar, the probability of G -2 or G 2 oxidation is determined by the ratio of squared electronic couplings between (X + )* and the diabatic states with a hole on the guanine bases. To estimate the electronic couplings we employ the multistate generalized Mulliken-Hush method and electronic adiabatic states of the system calculated within the multiconfigurational INDO/S approach. We show that the rate of the charge shift reaction critically depends on the chromophore position and that there are well-separated regions in the conformational space, where the hole charge is mainly injected either into G -2 or into G 2 or is unlikely. Thus, the direction of the hole injection can be switched by rotation of the intercalated chromophore and the photocleavage efficiency of the acridine derivative and related species can be modulated by the conformational changes.

Terminal Twist-Induced Writhe of DNA with Intrinsic Curvature

Supercoiling of a closed circular DNA rod may result from an application of terminal twist to the DNA rod by cutting the rod, rotating one of the cut faces as the other being fixed and then sealing the cut. According to White's formula, DNA supercoiling is probably accompanied by a writhe of the DNA axis. Deduced from the elastic rod model for DNA structure, an intrinsically straight closed circular DNA rod does not writhe as subject to a terminal twist, until the number of rotation exceeds a rod-dependent threshold. By contrast, a closed circular DNA rod with intrinsic curvature writhes instantly as subject to a terminal twist. This noteworthy character in fact belongs to many intrinsically curved DNA rods. By solving the dynamic equations, the linearization of the Euler-Lagrange equations governing intrinsically curved DNA rods, this paper shows that almost every clamped-end intrinsically curved DNA rod writhes instantly when subject to a terminal twist (clamped-end DNA rods include closed circular DNA rods and topological domains of open DNA rods). In terms of physical quantities, the exceptions are identified with points in R(6) whose projections onto R(5) (through ignoring the total energy density of a rod) form a subset of a quadratic hypersurface. This paper also suggests that the terminal twist induced writhe is due to the elasticity and the clamped-end boundary conditions of the DNA rods.

Structural Basis for p53 Binding-induced DNA Bending

Specific p53 binding-induced DNA bending has important biological implications such as transcription activation. However, the detailed structures of the bent DNA and the p53-DNA complex are still unavailable, hampering our understanding of the mechanism for p53-induced DNA bending and its consequent biological significance. To gain insight into the p53 binding-induced DNA bending, we performed molecular dynamics simulations on DNA segments with the consensus sequence for p53-specific binding, half site DNA-p53 complexes, and full site DNA-p53 complexes. We show that each DNA-bound p53 core domain caused a local DNA conformational change within the quarter site; upon the binding of the p53 dimer, there was an apparent DNA bending at the center of the half site; when bound with two p53 dimers, the full site DNAs with two different sequences bent 20 and 35 degrees, respectively. These results are in agreement with experimental observations. Our simulations demonstrate that the two p53 dimers favored a staggered conformation in which they make favorable interactions at the interface. This dimer-dimer interface organization necessitated conformational changes in the DNA, leading to the bending at the center of the full site, which in turn is dependent on the DNA sequence. Overall, our results provide the detailed atomic model for the DNA-p53 tetramer complex and delineate the roles of DNA-p53, p53 dimer-dimer interactions, and DNA sequence in specific p53 binding-induced DNA conformational changes.

One-Dimensional Thermotropic Dilatation Area of Lipid Headgroups within Lamellar Lipid/DNA Complexes

Using simultaneous synchrotron small- and wide-angle X-ray diffraction (SWAXD), we investigated the thermotropic behavior of a cationic lipid mixture of DOTAP-DOPC (1,2-dioleoyl-3-trimethylammonium-propane-dioleoylphosphatidylcholine) liposomes complexed with calf thymus DNA. The DOTAP-DOPC/DNA complex reacts to temperature change by a bilayer compression normal to its surface and an expansion of the DNA in the plane of the rod lattice. By applying two independent recently developed models, we show here for the first time that the thermotropic dilatation area of lipid headgroups within the complexes is not isotropic but occurs parallel to the 1D DNA lattice (i.e., along the direction perpendicular to the DNA axis). Our results shed light on the role of spatial dimensionality in the DNA packing density within lamellar lipoplexes and provide experimental evidence that the interaction between DNA molecules confined between lipid bilayers can be regarded as a 1D problem.

Geometric Properties of Covalently Bonded DNA on Single-Crystalline Diamond

Diamond is a promising candidate for bioapplications. Properties of hybridized DNA arrays on single-crystalline diamond are studied on a microscopic level by atomic force microscopy (AFM) in buffer solutions. Compact DNA layers in a thickness of 76 A are resolved by optimizing phase and height contrast in AFM. The height shows some long-range (30 nm) undulations of +/-5 A due to tip and DNA interactions. The axis of double helix DNA is oriented at about 36 degrees with respect to the diamond surface. DNA molecules can be removed by contact-mode AFM with forces >45 nN, indicating stronger DNA bonding than on gold substrates.

Salerno's Model of DNA Re-Analysed: Could Breather Solitons have Biological Significance?

We investigate the sequence-dependent behaviour of localised excitations in a toy, nonlinear model of DNA base-pair opening originally proposed by Salerno. Specifically we ask whether "breather" solitons could play a role in the facilitated location of promoters by RNA polymerase (RNAP). In an effective potential formalism, we find excellent correlation between potential minima and Escherichia coli promoter recognition sites in the T7 bacteriophage genome. Evidence for a similar relationship between phage promoters and downstream coding regions is found and alternative reasons for links between AT richness and transcriptionally-significant sites are discussed. Consideration of the soliton energy of translocation provides a novel dynamical picture of sliding: steep potential gradients correspond to deterministic motion, while "flat" regions, corresponding to homogeneous AT or GC content, are governed by random, thermal motion. Finally we demonstrate an interesting equivalence between planar, breather solitons and the helical motion of a sliding protein "particle" about a bent DNA axis.

Molecular Orientation of Porphyrins Accompanying the Formation of Chain-Aligned DNA Films

Porphyrin-containing DNA solid films with several binding orientations were successfully prepared by drying the aqueous solution of porphyrins and DNA in a magnetic field. By the measurement of linear dichroism absorption spectra from three identical spatial directions, the molecular orientations of porphyrins in chain-aligned DNA films were evaluated. Tetra(N-methylpyridinium-4-yl)-porphyrin was found to be bound at the surface of DNA chains like a patch. This orientation is different from the intercalative or groove binding manners observed in solutions. In contrast, tetra(4-sulfonatophenyl)-porphyrin exhibited an orientation perpendicular to the DNA axis, which may be attributed to H-aggregation of porphyrins along the direction parallel to DNA chains. Heme-protein with coiled coil backbone was also aligned along the DNA chains, orienting ferric protoporphyrin parallel to the magnetic field. The major effect for these molecular orientations would be the molecular packing of the rod-disk or the rod-rod systems.

Spectrometric and voltammetric investigation of interaction of neutral red with calf thymus DNA: pH effect

The interaction of neutral red (NR) with calf thymus DNA (CT DNA) was investigated by spectrometric (UV-vis, circular dichroism and fluorescence) and voltammetric techniques. It was shown that the interaction of NR with DNA depended on the values of R ( R is defined as the ratio of the concentration of NR to that of CT DNA) and pH of the solution. NR intercalated into CT DNA base pairs at lower R value ( R < 2.4) and following by NR aggregating along the helical surface of DNA at higher R value ( R > 2.4) in pH 6.0 solution. Interestingly, we found that at lower R value, NR intercalated into CT DNA with its long axis perpendicular or parallel to the dyad axis of DNA in the solution of pH 6.0. While in pH 7.0 solution, NR bound with CT DNA through intercalation and electrostatic interactions. The electrochemical inactive complexes, NR-2CT DNA, 3NR-CT DNA, and NR-CT DNA were formed when NR interacted with nucleic acids in pH 6.0 and 7.2 solutions, respectively. The corresponding intrinsic binding constants for these complexes were obtained by UV-vis and fluorescence spectrometric methods, respectively. The CD spectra showed that the conformation of CT DNA was converted from right-handed B-DNA to left-handed Z-DNA due to the aggregating of NR along the surface of DNA in pH 6.0 solution, whereas a conversion from B-DNA to C-DNA was induced due to the interaction of DNA with NR in pH 7.2 solution. Finally, two binding modes of NR with CT DNA in aqueous with different values of pH were shown in the scheme.

Orientation of Dye Molecules in DNA-Based Films with Chain Alignment and Judgment of Their DNA-Binding Modes

Novel composite films of chain-oriented DNA, which contain the DNA-binding dyes aligned in specific orientation, were successfully prepared by drying the solution under a horizontal magnetic field. Most of the dye-DNA composite films showed linear dichroism, as revealed by polarized ultraviolet-visible (UV-vis) spectroscopy. The intercalators, ethidium bromide and acridine orange, were fixed in chain-oriented DNA films in a similar binding manner as in solutions. Also, Hoechst 33258 and 4',6-diamidino-2-phenylindole were found to be aligned along the minor groove, even in the solid films. Thus, our new method of preparing dye-DNA composite films with chain orientation is useful for aligning small molecules, and it will provide views of the novel anisotropic materials expected in various application fields. We used this method to prepare composite DNA films with newly designed original compounds. Seven of nine dyes were judged to bind obviously to DNA as intercalators by polarized UV-vis spectroscopy. The DNA-binding manners were further analyzed by fluorescence anisotropy measurements. On the basis of the curves for the rotational angle dependence of the anisotropy, we were able to estimate the angles between the transition-dipole moments of dyes and the aligned chain axis of DNA. Interestingly, two original compounds were found to be in the tilted forms with regard to the plane of base pairs. We emphasize here that the method using aligned dye-DNA films is very convenient for identifying the binding modes of the compounds for double-stranded DNA.

The nucleolus

The nucleolus

Charge Transport Mechanism in Native Deoxyribonucleic Acid

The frequency dependent electrical conductivity below 1 MHz was studied on wet-spun, macroscopically oriented, calf thymus DNA bulk samples. Temperature dependence of the quasistatic electrical conductivity measured perpendicular to the long DNA axis is discussed within the framework of activated variable range hopping mechanism. It is shown that the frequency dependence of the electrical conductivity in the frequency range of approximately 10-3 Hz–1015 Hz agrees well with predictions of the hole hopping mechanism. Temperature dependence of the electrical conductivity at various frequencies exhibits a crossover at lower temperatures to almost temperature independent mechanism.

Axis Curvature and Ligand Induced Bending in the CAP-DNA Oligomers

The origin of DNA axis curvature in complexes of the catabolite activator protein with DNA is studied using multiple molecular dynamics (MD) simulations of the free and protein-bound forms of the DNA. The results are compared to available solution and crystal structure data. The MD simulations reproduce the experimentally observed bend in DNA and indicate that ∼40% of the bending observed in the complex is intrinsic to the DNA sequence, whereas ∼60% is induced on protein binding. The MD provides a model for the dynamical structure of the DNA free in solution and for ligand-induced bending.

Topological insulators inhibit diffusion of transcription‐induced positive supercoils in the chromosome of Escherichia coli

The double helical nature of DNA implies that progression of transcription machinery that cannot rotate easily around the DNA axis creates waves of positive supercoils ahead of it and negative supercoils behind it. Using topological reporters that detect local variations in DNA supercoiling, we have characterized the diffusion of transcription-induced (TI) positive supercoils in plasmids or in the chromosome of wild type Escherichia coli cells. Transcription-induced positive supercoils were able to diffuse and affect local supercoiling several kilobases away from the site of origin. By testing the effect of various DNA sequences, these reporters enabled us to identify elements that impede supercoil diffusion, i.e. behave as topological insulators. All the elements tested correspond to DNA gyrase catalytic targets. These results correlate the ability of a DNA sequence to be cleaved by DNA gyrase with topological insulator activity. Implications of the asymmetry in supercoil diffusion for the control of DNA topology are discussed.