Azimuthal frustration and bundling in columnar DNA aggregates

The interaction between two stiff parallel DNA molecules is discussed using linear Debye-Hückel screening theory with and without inclusion of the dielectric discontinuity at the DNA surface, taking into account the helical symmetry of DNA. The pair potential furthermore includes the amount and distribution of counterions adsorbed on the DNA surface. The interaction does not only depend on the interaxial separation of two DNA molecules, but also on their azimuthal orientation. The optimal mutual azimuthal angle is a function of the DNA-DNA interaxial separation, which leads to azimuthal frustrations in an aggregate. On the basis of the pair potential, the positional and orientational order in columnar B-DNA assemblies in solution is investigated. Phase diagrams are calculated using lattice sums supplemented with the entropic contributions of the counterions in solution. A variety of positionally and azimuthally ordered phases and bundling transitions is predicted, which strongly depend on the counterion adsorption patterns.     

DNA length-dependent cooperative interactions in the binding of Ku to DNA

Despite its central role in the nonhomologous DNA end joining process, we still have an incomplete picture of the interaction between Ku and DNA. Here we describe both kinetic (surface plasmon resonance or SPR) and equilibrium (electrophoretic mobility shift assay or EMSA) studies of Ku binding to linear double-stranded DNA. Ku interaction with 1-site DNA is noncooperative, as expected. Electrophoretic mobility shift assays indicate cooperativity in the binding of Ku molecules to DNA long enough for two Ku molecules to bind (2-site DNA). For the kinetic studies, we use surface plasmon resonance in which one end of the DNA molecules is linked to a surface while the other end is free to interact with Ku. We find that one Ku molecule dissociates from 1-site DNA with simple Langmuir (i.e., independent) kinetics. However, two Ku molecules associate and dissociate from 2-site DNA with a time course that cannot be described as a simple Langmuir interaction. On 3- and 4-site DNA, EMSA and SPR studies do not reveal any cooperativity, suggesting that the middle Ku does not exhibit cooperative interaction with the two Ku molecules bound at the DNA ends. These results indicate that Ku molecules can demonstrate cooperative interaction, and this is influenced by their positions along the DNA.     

Preliminary morphological and X-ray diffraction studies of the crystals of the DNA cetyltrimethylammonium salt.

Double-stranded DNA molecules (molecular weight 2.5 X 10(5) - 5 X 10(5) daltons) have been crystallized from water-salt solutions as cetyltrimethylammonium salts (CTA-DNA). Variation of crystallization conditions results in a production of different types of CTA-DNA crystals: spherulits, dendrites, needle-shaped and faceted rhombic crystals, the latter beeing up to 0.3 mm on a side. X-ray diffraction data indicate that DNA molecules in the crystals form a hexagonal lattice which parameters vary slightly with the morphological type of the crystal. Comparison of the melting curves of the DNA preparation before and after crystallization suggests that DNA molecules are partially fractionated in the course of crystallization. Crystals of the CTA-DNA-proflavine complex have also been obtained.     

DNA conformation and its changes upon binding transcription factors

Pyrimidine-purine steps are flexible and can roll around the major groove. This feature is used to follow the protein surface and thereby create a particular superstructure of DNA. The interaction of the two molecules can be understood in terms of a close fitting of the two surfaces, in particular, how the DNA surface adapts to the protein surface. For analysing the fitting the widths of two DNA grooves are useful parameters.Much progress has been made since Schrödinger predicted “a gene to be an aperiodic solid (or a crystal)” (29) and we hope that our review may contribute in a small way.     

Distribution functions, describing the binding of extended ligands with DNA molecules. Possible use for cases of DNA condensation

Due to noncooperative binding of ligands to DNA molecules, DNA molecules are in equilibrium with different numbers of adsorbed ligands. This equilibrium for a given concentration of the free ligand in the solution is characterized by the distribution function, which describes the probability of revealing the DNA molecule with a definite number of adsorbed ligands. If polycations act as ligands, DNA molecules with the number of ligands sufficient for neutralizing the charges on phosphates may undergo a phase transition. One example of this transition is the formation of liquid-crystalline dispersions during the binding of DNA to chitosan. We analyzed the binding of chitosan to DNA on the assumption that this binding is due to equilibrium adsorption. At a definite concentration of chitosan in solution, DNA molecules are in equilibrium with different numbers of adsorbed molecules of chitosan. If the number of adsorbed ligands exceeds some critical value, the DNA molecule covered with chitosan becomes capable of interacting with other DNA molecules. As a result of this interaction (attraction), liquid-crystalline dispersions can form. Equations describing the dependence of the concentration of DNA molecules on the concentration of the ligand in solution were derived. It was shown that, at given parameters of the model, it is possible to describe experimental data characterizing the formation of cholesteric liquid-crystalline dispersions. The analysis of the data makes it possible to reconstitute both the size of the binding site occupied by chitosan on the DNA and the energy of interaction of chitosan with DNA.     

AHNAK interacts with the DNA ligase IV-XRCC4 complex and stimulates DNA ligase IV-mediated double-stranded ligation

AHNAK is a high molecular weight protein that is under-expressed in several radiosensitive neuroblastoma cell lines. Using immunoaffinity purification or purified proteins, we show that AHNAK interacts specifically with the DNA ligase IV-XRCC4 complex, a complex that functions in DNA non-homologous end-joining. Furthermore, AHNAK and the DNA ligase IV-XRCC4 complex co-immunoprecipitate demonstrating an in vivo interaction. This interaction is specific and is not observed with other DNA ligases nor with other components of the DNA non-homologous end-joining machinery. We characterised AHNAK as a protein that stimulates the double-stranded (DS) ligation activity of DNA ligase IV-XRCC4. We show that AHNAK has weak DNA-binding activity and forms a stable complex with the DNA ligase IV-XRCC4 complex on DNA. AHNAK is also able to link two DNA molecules to a similar extent to that previously reported for Ku. Together, these findings demonstrate new activities for AHNAK, and raise the possibility that it may function to modulate DNA non-homologous end-joining.     

Translocation of Escherichia coli recA protein from a single-stranded tail to contiguous duplex DNA

Duplex DNA with a contiguous single-stranded tail was nearly as effective as single-stranded DNA in acting as a cofactor for the ATPase activity of recA protein at neutral pH and concentrations of MgCl2 that support homologous pairing. The ATP hydrolysis reached a steady state rate that was proportional to the length of the duplex DNA attached to a short 5' single-stranded tail after a lag. Separation of the single-stranded tail from most of the duplex portion of the molecule by restriction enzyme cleavage led to a gradual decline in ATP hydrolysis. Measurement of the rate of hydrolysis as a function of DNA concentration for both tailed duplex DNA and single-stranded DNA cofactors indicated that the binding site size of recA protein on a duplex DNA lattice, about 4 base pairs, is similar to that on a single-stranded DNA lattice, about four nucleotides. The length of the lag phase preceding steady state hydrolysis depended on the DNA concentration, length of the duplex region, and the polarity of the single-stranded tail, but was comparatively independent of tail length for tails over 70 nucleotides in length. The lag was 5-10 times longer for 3' than for 5' single-stranded tailed duplex DNA molecules, whereas the steady state rates of hydrolysis were lower. These observations show that, after nucleation of a recA protein complex on the single-stranded tail, the protein samples the entire duplex region via an interaction that is labile and not strongly polarized.     

DNA binding by dihydrofolate reductase from Lactobacillus casei

The protein-dependent retention of double-stranded DNA molecules on nitrocellulose filters has been used to show that pure dihydrofolate reductase from Lactobacillus casei has affinity for DNA. Dihydrofolate reductase will bind to end-labeled linear double-stranded DNA and to DNA in supercoiled form. Coenzymes and certain inhibitors do not affect the affinity of the protein to DNA, indicating that the DNA-binding region of the protein is distinct from the binding sites for these molecules. Comparison of the retention on filters by dihydrofolate reductase of two plasmid DNAs, differing only in a 3000-base pair insert containing the L. casei gene for dihydrofolate reductase, showed that in the presence of this DNA region lower concentrations of the protein were required to give significant retention; it is possible that a specific DNA-protein interaction underlies this effect. This presents the possibility of studying the interaction with DNA of a protein for which a crystal structure and considerable nuclear magnetic resonance data are already available.     

Interaction of alkaline-earth metal ions with calf thymus DNA. Volume and compressibility effects in diluted aqueous solutions

The binding of Mg2+, Ca2+, Sr2+ and Ba2+ ions to calf thymus DNA in solutions has been investigated by ultrasonic and densimetric techniques. The obtained parameters, the apparent molar volume, phiV, and the apparent molar adiabatic compressibility, phiK(S), are very sensitive to hydration of investigated molecules. The interaction between the cations and DNA is accompanied by overlapping their hydration shells and consequently releasing the water molecules from hydration shells to bulk state. The change in the hydration is reflected in the measured parameters, phiV and phiK(S). The magnitude of these hydration changes is determined by the position of the cation relative to DNA atomic groups involved in the binding, and thus can characterize the structure of cation-DNA complexes. The values of the dehydration effects of the binding, deltaphiV and deltaphiK(S), correspond to two direct or higher number of indirect contacts between calf thymus DNA and the cations.     

Labelling of histone H5 and its interaction with DNA. 2. Cooperative binding of histone H5 to DNA as probed by steady-state fluorescence and diffusion-enhanced energy transfer

The interaction of histone H5 labelled with fluorescein isothiocyanate (FITC) with DNA has been studied by fluorescence titration, and diffusion-enhanced fluorescence energy transfer (DEFET) measurements with Tb(III) lanthanide chelates as donors. Analysis of the binding data by the model of Schwarz and Watanabe (J.Mol.Biol. 163, 467-484 (1983)) yielded a mean stoichiometry of 60 nucleotides per H5 molecule, independently of ionic strength, in the range of 3 to 300 mM NaCl, at very low DNA concentration (6 microM in mononucleotide). It ensues an approximate electroneutrality of the saturated complexes. Histone H5 molecules appeared to be clustered along the DNA lattice in clusters containing on average 3 to 4 H5 molecules separated by about 79 base pairs, at mid-saturation of the binding sites. The interaction process was found highly cooperative but the cooperativity parameter was also insensitive to ionic strength in the above range. DEFET experiments indicated an important decrease of accessibility of the FITC label to the TbHED3A and TbEDTA- chelates with ionic strength in the 0 to 100 mM NaCl range. In the presence of DNA, H5 appears already folded at low ionic strength so that the FITC probe is also not accessible to the donor chelate. The present study constitutes an indispensable preliminary step to further studies on the localization of histone H5 in condensed chromatin structures.     

G.T wobble base-pairing in Z-DNA at 1.0 A atomic resolution: the crystal structure of d(CGCGTG).

The DNA oligomer d(CGCGTG) crystallizes as a Z-DNA double helix containing two guanine-thymine base pair mismatches of the wobble type. The crystal diffracts to 1 A resolution and the structure has been solved and refined. At this resolution, a large amount of information is revealed about the organization of the water molecules in the lattice generally and more specifically around the wobble base pairs. By comparing this structure with the analogous high resolution structure of d(CGCGCG) we can visualize the structural changes as well as the reorganization of the solvent molecules associated with wobble base pairing. There is only a small distortion of the Z-DNA backbone resulting from introduction of the GT mismatched base pairs. The water molecules cluster around the wobble base pair taking up all of the hydrogen bonding capabilities of the bases due to wobble pairing. These bridging water molecules serve to stabilize the base-base interaction and, thus, may be generally important for base mispairing either in DNA or in RNA molecules.     

Construction of DNA Hemicatenanes from Two Small Circular DNA Molecules

DNA hemicatenanes, one of the simplest possible junctions between two double stranded DNA molecules, have frequently been mentioned in the literature for their possible function in DNA replication, recombination, repair, and organization in chromosomes. They have been little studied experimentally, however, due to the lack of an appropriate method for their preparation. Here we have designed a method to build hemicatenanes from two small circular DNA molecules. The method involves, first, the assembly of two linear single strands and their circularization to form a catenane of two single stranded circles, and, second, the addition and base-pairing of the two single stranded circles complementary to the first ones, followed by their annealing using DNA topoisomerase I. The product was purified by gel electrophoresis and characterized. The arrangement of strands was as expected for a hemicatenane and clearly distinct from a full catenane. In addition, each circle was unwound by an average of half a double helical turn, also in excellent agreement with the structure of a hemicatenane. It was also observed that hemicatenanes are quickly destabilized by a single cut on either of the two strands passing inside the junction, strongly suggesting that DNA strands are able to slide easily inside the hemicatenane. This method should make it possible to study the biochemical properties of hemicatenanes and to test some of the hypotheses that have been proposed about their function, including a possible role for this structure in the organization of complex genomes in loops and chromosomal domains.     

Electron microscopic and physico-chemical studies of DNA complexes with synthetic oligopeptides: binding specificity and DNA compact structures

Binding to DNA of two synthetic peptides, Val-Thr-Thr-Val-Val-NH-NH-Dns and Thr-Val-Thr-Lys-Val-Gly-Thr-Lsy-Val-Gly-Thr-Val-Val-NH-NH-Dns (where Dns is a residue of 5-dimethylaminonaphthalene-1-sulfonic acid), has been studied by circular dichroism, electron microscopy and fluorescence methods. It has been found that these two peptides can self-associate in aqueous solution as follows from the fact that concentration-dependent changes are observed in the UV absorbance and fluorescence spectra. The two peptides can bind to DNA both in self-associated and monomeric forms. The pentapeptide in the beta-associated form binds more strongly to poly(dG).poly(dC) than to poly[d(A-C)].poly[d(G-T)] and poly(dA).poly(dT) whereas the tridecapeptide exhibits an opposite order of preferences binding more strongly to poly[d(A-C)].poly[d(G-T)] and poly(dA).poly(dT) than to poly(dG).poly(dC). Binding is a cooperative process which is accompanied by the DNA compaction at peptide/DNA base pair ratios greater than 1. At the initial stage of the compaction process, the coalescence of DNA segments covered by bound peptide molecules leads to the formation of DNA loops stabilized by the interaction between peptide molecules bound to different DNA segments. Further increase in the peptide/DNA ratio leads to the formation of rod-like structures each consisting of two or more double-stranded DNA segments. The final stage of the compaction process involves folding of fibrillar macromolecular complexes into a globular structure containing only one DNA molecule.     

Binding of Hoechst 33258 and its Derivatives to DNA

Abstract

In the present work, we employed UV-VIS spectroscopy, fluorescence methods, and circular dichroism spectroscopy (CD) to study the interaction of dye Hoechst 33258, Hoechst 33342, and their derivatives to poly[d(AT)]·poly[d(AT)], poly(dA)·poly(dT), and DNA dodecamer with the sequence 5′-CGTATATATACG-3′. We identified three types of complexes formed by Hoechst 33258, Hoechst 33342, and methylproamine with DNA, corresponding to the binding of each drug in monomer, dimer, and tetramer forms. In a dimer complex, two dye molecules are sandwiched in the same place of the minor DNA groove. Our data show that Hoechst 33258, Hoechst 33342, and methylproamine also form complexes of the third type that reflects binding of dye associates (probably tetramers) to DNA. Substitution of a hydrogen atom in the ortho position of the phenyl ring by a methyl group has a little effect on binding of monomers to DNA. However it reduces strength of binding of tetramers to DNA. In contrast, a Hoechst derivative containing the ortho-isopropyl group in the phenyl ring exhibits a low affinity to poly(dA)·poly(dT) and poly[d(AT)]·poly[d(AT)] and binds to DNA only in the monomer form. This can be attributed to a sterical hindrance caused by the ortho-isopropyl group for side-by-side accommodation of two dye molecules in the minor groove. Our experiments show that mode of binding of Hoechst 33258 derivatives and their affinity for DNA depend on substituents in the ortho position of the phenyl ring of the dye molecule. A statistical mechanical treatment of binding of Hoechst 33258 and its derivatives to a polynucleotide lattice is described and used for determination of binding parameters of Hoechst 33258 and its derivatives to poly[d(AT)]·poly[d(AT)] and poly(dA)·poly(dT).     

沙吸附DNA的分布及转化活性

尽管自然环境中存在着大量的DNase,大分子体外DNA在土等生态环境中存在并保持转化活性。实验表明DNA被沙土吸附之后会被沙土保护而产生对DNase的抗性。然而对沙中DNA的检测多是通过PCR或者探针杂交的方法来进行,采用转化方法进行来检测的却鲜有报道。为了研究DNA释放进沙之后的分布及转化活性,建立了流过式微型离心沙柱。将20μLpUC18置于1.0g直径介于1.2~2.5mm之间的沙中,1.5mL0.1mol/LCaCl2洗脱之后再用200μLTEN缓冲液提取。结果表明吸附于沙中的DNA经提取后仍然具有生物转化活性,并且与洗脱液、提取液具有不同的转化活性和对DNase抗性,说明DNA与沙粒的结合不只是一种简单的附着,而是3种复杂的复合体结构。相对于没有沙保护而只能在室温下保留7d转化活性的DNA,在被沙吸附保护之后的DNA可以保存转化活性达35d以上。研究结果还表明在存在感受态细胞的情况下,生物释放出来的DNA并非直接与沙粒结合,而是优先与感受态细胞结合。推测认为沙吸附DNA的转化活性同DNA的构象及同感受态细胞接触的机会相关,而与DNA的浓度不直接相关。这些结果为研究环境中存在的DNA库及其与混居的微生物间的相互作用,水平基因转移及其生态效应提供了有价值的信息。     

Alignment-free prediction of mycobacterial DNA promoters based on pseudo-folding lattice network or star-graph topological indices

The importance of the promoter sequences in the function regulation of several important mycobacterial pathogens creates the necessity to design simple and fast theoretical models that can predict them. This work proposes two DNA promoter QSAR models based on pseudo-folding lattice network (LN) and star-graphs (SG) topological indices. In addition, a comparative study with the previous RNA electrostatic parameters of thermodynamically-driven secondary structure folding representations has been carried out. The best model of this work was obtained with only two LN stochastic electrostatic potentials and it is characterized by accuracy, selectivity and specificity of 90.87%, 82.96% and 92.95%, respectively. In addition, we pointed out the SG result dependence on the DNA sequence codification and we proposed a QSAR model based on codons and only three SG spectral moments.     

Targeting DNA with novel diphenylcarbazoles

Double-stranded DNA is a therapeutic target for a variety of anticancer and antimicrobial drugs. Noncovalent interactions of small molecules with DNA usually occur via intercalation of planar compounds between adjacent base pairs or minor-groove recognition by extended crescent-shaped ligands. However, the dynamic and flexibility of the DNA platform provide a variety of conformations that can be targeted by structurally diverse compounds. Here, we propose a novel DNA-binding template for construction of new therapeutic candidates. Four bisphenylcarbazole derivatives, derived from the combined molecular architectures of known antitumor bisphenylbenzimidazoles and anti-infectious dicationic carbazoles, have been designed, and their interaction with DNA has been studied by a combination of biochemical and biophysical methods. The substitutions of the bisphenylcarbazole core with two terminal dimethylaminoalkoxy side chains strongly promote the interaction with DNA, to prevent the heat denaturation of the double helix. The deletion or the replacement of the dimethylamino-terminal groups with hydroxyl groups strongly decreased DNA interaction, and the addition of a third cationic side chain on the carbazole nitrogen reinforced the affinity of the compound for DNA. Although the bi- and tridentate molecules both derive from well-characterized DNA minor-groove binders, the analysis of their binding mode by means of circular and linear dichroism methods suggests that these compounds form intercalation complexes with DNA. Negative-reduced dichroism signals were recorded in the presence of natural DNA and synthetic AT and GC polynucleotides. The intercalation hypothesis was validated by unwinding experiments using topoisomerase I. Prominent gel shifts were observed with the di- and trisubstituted bisphenylcarbazoles but not with the uncharged analogues. These observations, together with the documented stacking properties of such molecules (components for liquid crystals), prompted us to investigate their binding to the human telomeric DNA sequence by means of biosensor surface plasmon resonance. Under conditions favorable to G4 formation, the title compounds showed only a modest interaction with the telomeric quadruplex sequence, comparable to that measured with a double-stranded oligonucleotide. Their sequence preference was explored by DNase I footprinting experiments from which we identified a composite set of binding sequences comprising short AT stretches and a few other mixed AT/GC blocks with no special AT character. The variety of the binding sequences possibly reflects the coexistence of distinct positioning of the chromophore in the intercalation sites. The bisphenylcarbazole unit represents an original pharmacophore for DNA recognition. Its branched structure, with two or three arms suitable to introduce a structural diversity, provides an interesting scaffold to built molecules susceptible to discriminate between the different conformations of nucleic acids.     

The interaction of multiply charged intercalating heterocycles with DNA

The interaction with DNA of two aromatic nitrogen heterocycles, 1 and 2 , which at pH 6 have two positive charges on their ring systems and two cationic side chains, have been determined. A third similar compound, 3 , with a single side chain and reduced ring charge, was analyzed as a control. Viscometric titrations with sonicated DNA indicated that all three compounds bind to DNA by intercalation. Spectrophotometric binding studies as a function of ionic strength indicated that both 1 and 2 bind to DNA as tetracations at pH 6. These are the first examples of intercalators with two charges directly on the intercalating ring system. Dissociation kinetics experiments as a function of ionic strength confirmed that 1 and 2 bind to DNA as tetracations. Compound 1 has a G · C base-pair binding preference, 2 seems to prefer binding to alternating pyrimidine–purine sequences regardless of the composition, and 3 has no significant binding specificity.     

Optimization of conditions for the in vitro formation of hybrid DNA molecules by DNA ligase.

Conditions for the ligation with T4 induced DNA ligase of two DNA molecules via their complementary sticky ends have been established which lead preferentially to the formation of hybrid molecules. This is demonstrated with two combinations of parent molecules varying greatly in their relative molecular weights. In one case the intact hybrid molecule could be directly isolated. In addition a DNA dependent quantitative electrophoretic assay for DNA ligase activity is described which does not need a radioactively labeled substrate. The ligation procedure has been shown to be useful in molecular cloning experiments.