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.     

A compact form of double-stranded RNA in solutions containing poly(ethyleneglycol).

Molecules of single-stranded ribosomal RNA and double-stranded replicative form of phage f2 RNA (dsRNA) adopt a compact form in solutions, containing sufficiently high concentrations of salt (NaCl) and polymer (PEG). However, only in the cases of native dsRNA molecules the compact particles are characterized by a regular internal structure, which accounts for the appearance of an intense positive band in CD spectra. Heating or acidification of PEG-containing solutions of dsRNA leads to the disappearance of the intense positive CD band, which results from the "destruction" of the regular internal structure of compact particles. Comparison of properties of DNA and dsRNA compact particles formed in PEG-containing water-salt solutions suggests the existence of similar mechanisms of compactization of double-stranded polynucleotides.     

A compact from of methylated DNA is solutions containing poly (ethylene glycol).

Some peculiarities of compactization of double-stranded DNA molecules containing methylated nitrogen bases have been studied in water-salt solutions of PEG. It is shown that the methylation of N7-atoms of guanyl residues in original DNA molecules does not prevent the formation of DNA compact particles, but results in a decrease of the amplitude of the negative band in the CD spectrum of compact particles. The influence of N7-guanine methylation on the shape of the CD spectrum being the greater, the lower is the concentration of PEG. The dependence of the negative band amplitude in the CD spectrum on the content of methylated guanyl residues is practically the same for low-molecular weight DNA's from different sources. The observed decrease in the negative band amplitude is interpreted as a result of alterration of guanyl residue orientation relative to the helix axis which leads to diminished optical activity of the "microcrystalline" domains of compact particles. The evidence obtained suggests that changes in the secondary structure of DNA lead to considerable difference between CD spectra of compact particles of methlated DNA and psi-form of DNA. (The changes in the CD spectrum of the DNA compact particles occur also as a result of methylation of C5-atoms of cytosine residues). It is suggested that the negative band in the CD spectrum can be used a criterion for detection of negligible alterations in the DNA secondary structure.     

Compaction of 2-chain DNA as a method of procedure in determining its binding constant with antibiotics]

The binding of antibiotics and dyes with a compact form of DNA produced in water-salt solutions containing polyethylenglycol (PEG) presents a possibility of studying antibiotic interaction with DNA molecules contained in biological objects, such as viruses and chromosomes, since the compact form of DNA reflects some DNA properties in vivo. Possibly the use of the compact and not the "open" or linear form of DNA in chemical reactions will provide data on the efficiency of the compound "action" under conditions close to intracellular ones. The results well be useful in screening substances with "optimal" pharmacological effect. The paper presents a method for determination of the constant of antibiotic or dye binding with DNA and two-chain synthetic polynucleotides in water-salt solutions containing PEG. The method is based on "elimination" of the DNA molecules in the form of compact particles bound in a complex with an antibiotic or a dye. Comparison of the data with the results of estimation of the constants of antibiotic binding with DNA by the routine methods showed close conformity of the binding constants determined by different methods. It was found that the value of the binding constant of the antibiotics studied slightly depended on the structural state of DNA. The value was practically the same for the linear and the compact forms of DNA.     

A comparative X-ray diffraction and circular dichroism study of DNA compact particles formed in water-salt solutions, containing poly(ethylene glycol).

Comparative CD and X-ray diffraction studies of DNA compact particules which were obtained in PEG-containing water-salt solutions, have been carried out. Compact particles, formed from native DNA, produce a psi CD spectrum (characterized by a negative band at lambda-270 nm) and a small-angle X-ray diffraction pattern, which shows two reflections: I at 34-40 A and II at 80-90 A (together with its second-order reflection). Compact particules, formed from DNA molecules with partially disordered secondary structure, do not produce the psi CD spectrum and the reflection I, while the reflection II remains unchanged. It is suggested that the spacing of 34-40 A is associated with a side-by-side packing of DNA fragments in "microcrystallization' regions in compact particules and that such "microcrystallization' accounts for the generation of the psi CD spectrum.     

Osmoelastic coupling in biological structures: formation of parallel bundles of actin filaments in a crystalline-like structure caused by osmotic stress

A high molecular weight inert molecule, poly(ethylene glycol) (PEG), or a soluble protein, ovalbumin, causes parallel bundles of actin filaments in a crystalline-like structure under physiological conditions of ionic compositions and pH. The bundle formation depends on the molecular weight of PEG, and a larger molecular weight of PEG can make the bundle at a lower concentration. Actin bundle formation has a discrete dependence on the concentration of PEG. The light scattering following PEG-induced bundle formation increased abruptly at 4.5% (w/w) PEG 6000, while at concentrations less than or equal to 4.0% (w/w) no increase was observed. Labeling actin filaments with heavy meromyosin indicated that the polarity of the filament in the bundle is random. The PEG-induced bundle formation depends on the ionic strength of the solutions and also the concentration of the filament, showing that a higher concentration of PEG was required at lower ionic strength or a lower concentration of the filament. The results described above cannot be explained on the basis of the postulation that the direct binding of PEG molecules to the actin filaments may cause bundle formation. Alternatively, the mechanism can be explained reasonably by the theory of osmoelastic coupling based on preferential exclusion of PEG molecules from the filament surface. High molecular weight molecules such as PEG should be preferentially excluded from the region adjacent to the actin filaments (exclusion layer) by steric hindrance, thereby making imbalance of osmolarity between the bulk and the exclusion layer. This imbalance puts an osmotic stress on the actin filament.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two modes of longe-range orientation of DNA bases realized upon compaction.

Formation of compact particles from linear DNA-anthracycline complexes is accompanied by appearance of intense bands in the CD spectra in the region of absorption of DNA bases (UV-region) and in the region of absorption of anthracycline chromophores (visible region). The intense (positive or negative) bands in the region of anthracycline absorption demonstrate an ordered helical location of anthracycline molecules on the DNA template. This fact, in its turn, is related to formation of the DNA superstructure in PEG-containing water-salt solutions with a long-range orientation of nitrogen bases. Possible types of DNA superstructures and the relation between the local- and the long-range order of bases in the DNA superstructure are discussed.     

A theoretical model of the mechanism of DNA compactization by polycations

DNA compactization in the presence of polycationic ligands was analysed theoretically. The concept is substantiated which states that the formation of polycationic bridges between the DNA regions adjoining in the chain is the principal mechanism of compactization. A phase diagram is plotted for DNA transition into the compact state in the coordinates of solution ionic strength and ligand concentration. The ligand binding with DNA under compactization conditions is shown to be characterized by positive cooperativity. DNA compactization by protamines and histones HI is discussed in terms of the results obtained.     

Correlation between conformation distortion of the DNA sugar-phosphate backbone and high optical activity of its compact form

Different physico-chemical methods (CD, ORD, small-angle X-ray diffraction, etc) were used for investigating the properties of the DNA compact particles formed in PEG-containing water-salt solutions. It has been shown that small-angle reflection, characteristic of the DNA compact particles, changes from 36.8 A (CPEG = 140 mg/ml) to 25 A (CPEG = 300 mg/ml). The maximal optical activity (the intense negative CD-band and optical rotation [alpha] = 60 000 degrees) are inherent properties of the DNA compact particles formed at CPEG 120--180 mg/ml. The high optical activity points to the twist of DNA chromophores through the DNA molecule resulting in a long-rang pitch (P approximately 2000A).Such macroscopic superhelical structure (diameter 40--30 A) is due to conformational distortion of the DNA double-helix with alternating "left" and "right" orientation of chromophoes. Disappearance of conformation distortion is accompanied by disappearance of the high optical activity of the DNA compact particles and results in a small-angle reflection of 25 A. Taking into account the reasons of formation of the optically-active DNA compact particles conditions are suggested to conserve high optical activity at CPEG equal to 400 mg/ml.     

Structure of Escherichia coli uracil DNA glycosylase and its complexes with nonhydrolyzable substrate analogues in solution observed by synchrotron small-angle X-ray scattering

The structure of native and modified uracil DNA glycosylase from E. coli in solution was studied by synchrotron small-angle X-ray scattering. The modified enzyme (6His-uracyl DNA glycosylase) differs from the native one by the presence of an additional N-terminal 11-meric sequence amino acid residues including a block of six His residues. It was found that the conformations of these enzymes in solution at moderate ionic strength (60 mM NaCI) substantially differ in spite of minimal differences in the amino acid sequences and functional activity. The structure of native uracil DNA glycosylase in solution is close to that in crystal, showing a tendency for association. The interaction of this enzyme with nonhydrolyzable analogues of DNA ligands causes a partial dissociation of associates and a compactization of protein structure. At the same time, 6His-uracyl DNA glycosylase has a compact structure essentially different from the crystal one. A decrease in the ionic strength of solution results in a partial disruption of compact structure of the modified protein, without changes in its functional activity.     

Monovalent cations affect the free solution mobility of DNA by perturbing the hydrogen-bonded structure of water

The free solution mobilities of single- and double-stranded DNA molecules of various molecular weights have been measured by capillary electrophoresis in solutions of constant ionic strength containing a common anion and fifteen different monovalent cations. In solutions with the same ionic composition, the mobilities of different DNA molecules can vary by up to 20%, depending on molecular weight, the number of strands, and the presence or absence of A-tracts, runs of four or more contiguous adenine residues. Importantly, the mobilities observed for the same DNA sample can vary by up to 40% in solutions containing different cations. The mobility differences observed for the same DNA in solutions containing different cations cannot be rationalized by differences in the anhydrous radii or intrinsic conductivities of the various cations, or by the sequence-dependent binding of certain cations to A-tracts. Instead, the observed mobilities are linearly correlated with the average number of water-water hydrogen bonds that are present in solutions containing different cations. The mobilities are also correlated with the viscosity B coefficients of the various cations and with the rotational correlation times frictional coefficients observed for water molecules in solutions containing different cations. Hence, monovalent cations modify the free solution mobility of DNA primarily by perturbing the hydrogen-bonded structure of water, affecting the friction experienced by the migrating DNA molecules during electrophoresis.     

Liquid-crystalline dispersions formed by the complexes of linear double-stranded DNA molecules with dendrimers

The specific features of liquid-crystalline dispersions formed by double-stranded DNA molecules interacting with polypropylenimine dendrimers of five generations (G1—G5) in aqueous saline solutions of various ionic strengths were studied. It was demonstrated that the binding of dendrimer molecules to DNA led to the formation of dispersions independently of solution ionic strength and dendrimer structure. By the example of a generation 4 dendrimer, it was shown that the shape of dispersion particles of the (DNA-dendrimer G4) complex were close to a sphere with a diameter of 300–400 nm. The boundary conditions (ionic strength of solution and molecular mass of dendrimer) for the formation of optically active (cholesteric) and optically inactive (DNA-dendrimer) dispersions were determined by circular dichroism spectroscopy. The dispersions formed by dendrimers G1–G3 and G5 were optically inactive. Dendrimers G4 formed liquid-crystalline dispersions of two types. Cholesteric liquid-crystalline dispersions were formed in high ionic strength solutions (μ > 0.4), whereas the dispersions formed in low and intermediate ionic strength solutions (μ < 0.4) lacked an intense negative band in their circular dichroism spectra. The effect of molecular crowding on both the (DNA-dendrimer G4) binding efficiency and the pattern of spatial packing of the (DNA-dendrimer G4) complexes in the liquid-crystalline dispersion particles was demonstrated. The factors determining the structural polymorphism of the liquid-crystalline dispersions of (DNA-dendrimer) complexes are postulated.     

Electron microscopic study of compactization of individual DNA molecules in films during the interaction with histone H1]

The protein-free method was applied for the investigation of histone H1 DNA complexes formation. The main advantage of this method is the possibility to get intramolecular compact structures at interaction of individual spread molecules of DNA with histone H1. It was shown that in the presence of 0.2-5 micrograms/ml of histone H1 in hypophase there are three types of structures on electronmicroscopic preparations: fibres of non-compacted DNA, compact fibres with twisted strands of duplex DNA and compacted rod-like and circular structures where separate fibres of duplex DNA could not be distinguished. The study of compact structures morphology allows to conclude that they are formed by side-by-side association of DNA fibres, as it takes place in the case of triple rings formation at the compactization of circular DNA due to trivaline binding. At increasing ionic strength there is a tendency for transition from second type structures to the third type structures. The latter can be explained by transition from non-cooperative to cooperative binding of histone H1 to DNA.     

DNA complexes with synthetic oligopeptides: a model for studying the regularity of DNA compactization in vivo

The electron microscopic data on compactization of DNA at interaction with the synthetic oligopeptides having the trend of beta-structures formation in solutions are summarized. The new types of intramolecular and intermolecular compact structures are described in brief. Sequence of compactization process steps is discussed, the models of DNA packaging in the structures are presented. On the basis of the data presented the general principles of arrangement of the described compact structures are formulated, the mechanisms are proposed for formation of different types of compact particles on the final stage of the process of DNA condensation. Some processes of the genetic material compactization in vivo are discussed in which the proposed mechanisms for compact structures formation may have realization.     

Description of the binding of chitosan to DNA at different ionic strengths in terms of the theories of ion condensation and adsorption

The binding of chitosan molecules to DNA in solutions of different ionic strength has been studied. The data were analyzed in terms of the model of ion condensation and the thermodynamic theory of the binding of protracted ligands to DNA. Combining these approaches made it possible to estimate the sterical and energetic characteristics of chitosan-DNA binding and establish the dependence of the chitosan-DNA binding constant on the ionic strength of solution.     

Probing the electrostatic shielding of DNA with capillary electrophoresis

The free solution mobility of a 20-bp double-stranded DNA oligomer has been measured in diethylmalonate (DM) and Tris-acetate buffers, with and without added NaCl or TrisCl. DM buffers have the advantage that the buffering ion is anionic, so the cation composition in the solution can be varied at will. The results indicate that the free solution mobility of DNA decreases linearly with the logarithm of ionic strength when the ionic strength is increased by increasing the buffer concentration. The mobility also decreases linearly with the logarithm of ionic strength when NaCl is added to NaDM buffer or TrisCl is added to TrisDM buffer. Nonlinear effects are observed if the counterion in the added salt differs from the counterion in the buffer. The dependence of the mobility on ionic strength cannot be predicted using the Henry, Debye-Hückel-Onsager, or Pitts equations for electrophoresis. However, the mobilities observed in all buffer and buffer/salt solutions can be predicted within approximately 20% by the Manning equation for electrophoresis, using no adjustable parameters. The results suggest that the electrostatic shielding of DNA is determined not only by the relative concentrations of the various ions in the solution, but also by their equivalent conductivities.     

Intramolecular compactization of circular DNA in interaction with dansyl hydrazide trivaline leading to a formation of a new type of structure--triple rings

Compactization of supercoiled circular plasmid pBR322 caused by interaction with synthetic oligopeptide dansyl hydrazide trivaline capable of beta-structure formation was studied by electron microscopy. The results show that at rising input peptide concentration circular DNA molecules undergo intramolecular structural transition with the formation of compact ring structures. The compact ring structures are formed by the fiber having the thickness of 60 A. The analysis of morphology of intermediate structures and the contour length measurements enable us to conclude that 60 A-fiber contains three lying side-by-side and interwound double-stranded DNA segments. Thus, the compact ring structures are addressed to as triple rings. The triple ring have one special point, where the triple region ends are locked by a duplex DNA segment. The mechanisms responsible for the triple ring formation may be of importance for DNA and chromatin compactization processes in vivo.     

Enzymatic cleavage of superhelical DNA in a liquid crystal state]

Superhelical pBR322 DNA molecules form liquid-crystalline dispersions in water-salt solutions containing poly(ethyleneglycol). The formation of the liquid-crystalline dispersions from superhelical DNA molecules results in the appearance of two sites inside the DNA molecules that are split by Micrococcal nuclease. The first site of digestion does not differ from the standard site split by this enzyme in water-salt solutions, whereas the second one represents a new site specific only for the DNA molecules forming liquid-crystalline dispersions. Splitting of the DNA molecule through the first site is accompanied by formation of its linear form; splitting of a new site results in the formation of two linear DNA fragments with molecular masses equal to half of the initial DNA molecules. Enzyme digestion of superhelical DNA molecules forming liquid-crystalline dispersions induces a reformation of the "nonspecific" space organization of dispersions to the cholesteric one. A hypothetic model for packing of the superhelical DNA molecules inside liquid-crystalline dispersions and its transformation under enzyme action is suggested.     

Re-entrant cholesteric phase in DNA liquid-crystalline dispersion particles

In this research, we observe and rationalize theoretically the transition from hexagonal to cholesteric packing of double-stranded (ds) DNA in dispersion particles. The samples were obtained by phase exclusion of linear ds DNA molecules from water-salt solutions of poly(ethylene glycol)—PEG—with concentrations ranging from 120 mg ml^?1 to 300 mg ml^?1. In the range of PEG concentrations from 120 mg ml^?1 to 220 mg ml^?1 at room temperature, we find ds DNA molecule packing, typical of classical cholesterics. The corresponding parameters for dispersion particles obtained at concentrations greater than 220 mg ml^?1 indicate hexagonal packing of the ds DNA molecules. However, slightly counter-intuitively, the cholesteric-like packing reappears upon the heating of dispersions with hexagonal packing of ds DNA molecules. This transition occurs when the PEG concentration is larger than 220 mg ml^?1. The obtained new cholesteric structure differs from the classical cholesterics observed in the PEG concentration range 120–220 mg ml^?1 (hence, the term ‘re-entrant’). Our conclusions are based on the measurements of circular dichroism spectra, X-ray scattering curves and textures of liquid-crystalline phases. We propose a qualitative (similar to the Lindemann criterion for melting of conventional crystals) explanation of this phenomenon in terms of partial melting of so-called quasinematic layers formed by the DNA molecules. The quasinematic layers change their spatial orientation as a result of the competition between the osmotic pressure of the solvent (favoring dense, unidirectional alignment of ds DNA molecules) and twist Frank orientation energy of adjacent layers (favoring cholesteric-like molecular packing).     

Interaction of topotecan--a DNA topoisomerase I inhibitor--with dual-stranded polydeoxyribonucleotides. II. Formation of a complex containing several DNA molecules in the presence of topotecan]

This study is a continuation of a series of papers dealing with topotecan interaction with double-stranded polydeoxyribonucleotides. We showed earlier that topotecan molecules form dimers in solution at concentration above 10(-5) (per base pair). Topotecan interaction with calf thymus DNA in solutions of low ionic strength was studied by fluorescence, circular dichroism, and linear flow dichroism. The data obtained indicate that topotecan forms two types of complex with DNA, DNA molecules combining with each other during formation of one of these complexes. The association constant of two topotecan-filled DNA molecules with each other was estimated at 10(4) M-1 (per base pair) in 1 mM sodium cacodylate buffer, pH 6.8, at 20 degrees C. A possibility of modulation of DNA topoisomerase I activity by topotecan due to complexation with several sites of a supercoiled DNA molecule is discussed.