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 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.     

Structural organization of kinetoplast DNA and its compactization in a model system in vitro

Studies on compactization and decompactization of the genome are of great importance for elucidation of structural mechanisms taking part in the regulation of gene activity. Kinetoplast DNA (kpDNA) is a convenient model for studies of compactization processes. KpDNA represents unique structure ("network"), consisting of catenated circular molecules of two types: minicircles (900 b.p.) and maxicircles (40 000 b.p.). The compactization process of kpDNA in vitro caused by interaction with synthetic peptide-dansylhydraside trivaline was studied. It was shown that at the initial stages the hairpins are observed on minicircles as if triple rings are being organized. The formation of hairpin is probably favoured by the presence in the minicircles of bent DNA, a specific nucleotide sequence causing rigid bending of the DNA helix. The hairpin does not make contact with the neighbouring DNA segment to form a triple ring, because the sizes of minicircles are too small. The minicircles compactization is finished with a complete collapse of the minicircles with the formation of rod-like structures. The catenation causes branching of rod-like structures. As a result of their intermolecular interaction, the branched rod-like structures become thicker. The process is completed with formation of the compact network, its diameter being 3-6 times smaller compared to the initial one.     

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.     

Intracellular localization of XCAP-E and pEg7 condensins in normal mitosis and after the treatment inducing artificial changes in structural organization of mitotic chromosomes

Function of condensin subunits XCAP-E and pEg7 (XCAP-D2) in the formation and maintaining of special organization of mitotic chromosomes has been studied in Xenopus laevis cells (XL-2). The experimental conditions involved blocking chromosomes being in the condensed state in cells treated by cytostatics, or during their reversible artificial decondensation. The latter was induced by incubation of living cells in hypotonic medium. In extensively mollen chromosomes, XCAP-E and pEg7, remained associated with axial regions of chromosomes. In contrast, upon adaptation of cells to hypotonic conditions and recondensation of chromosomes to nearly initial state, both proteins dissociated from chromosomes into the cytoplasm. In K-mitotic cells, after a 3-6 h treatment with nocodazole or taxol, considerable dissociation of XCAP-E and pEg7 from chromosomes was observed without significant changes in overall level of chromosome compactization. Taken together the data suggested that condensins play no important role in maintaining mitotic chromosomes being in condensed state. Rather, it seems probable that mitotic function of condensins may be associated either with the formation of the higher order chromosome structure, and/or segregation of sister chromatids, the processes being tightly linked with chromosome compactization. This paper is in memory of Professor Katherine Le Guellec of Rennes-1 University, who left us in June 2001. Professor Le Guellec initiated this work in Rennes and offered all the possible help that this work be continued in Moscow University. Let the memory of Katherine, a great scientist and sympathetic friend, live for ever in ours hearts.     

Oversupercoiling and compactization of supercoiled DNA

Supercoiled DNA pGEMEX with length of 3993 nucleotides was immobilized on the different substrates (freshly cleaved mica, standard aminomica and modified aminomica) and visualized by atomic force microscopy. Plectonomically supercoiled DNA molecules as well as molecules with extremely high level of compactization (i.e. molecules with considerably higher supercoiled density values in comparing with experimentally measured and theoretically investigated ones) were visualized on modified aminomica. At the further increasing of the compactization level an axis length of oversupercoiled molecules was decreased from approximately 390 nm to approximately 140 nm and formation of minitoroids of approximately 50 nm diameter and molecules in sphere conformation were observed. Model of possible conformational transitions of supercoiled DNA was proposed basing on the analysis of captured AFM images at the increasing of supercoiling density.     

DNA damage in cells exposed to ionizing radiation

Ionizing radiation induces variety of structural lesions in DNA of irradiated organisms. Their formation depends largely on the degree of cell oxygenation, the level of endogenous antioxidants, on DNA-protein complexes and compactization of DNA in the chromatin and activity of DNA repair systems. All ionizing radiation-induced DNA lesions can arbitrarily be divided into two groups. Group 1 includes singly damaged sites (single-sites): base modification, single-strand breaks, alkaline-labile sites (including a basic sites). Group 2 contains: locally multiply damaged sites (clustered lesions), double-strand breaks, intermolecular cross-links. The yields of lesions of group 2 increases with high linear energy transfer of radiation and these lesions play a dominant role in the radiation death, formation of chromosome and gene mutations, cell transformation.     

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.     

Structure of chromatin from the pigeon brain. I. Composition, electrophoretic motility and circular dichroism spectra of nucleosome particles

Composition and structural properties of pigeon brain hemisphere neuron chromatin have been studied. In these cells nucleosomal DNA repeat length is about 165 nucleotide pairs. The content of H1 and H2A histones was found to decrease by 18 and 30% respectively in comparison with the chromatin possessing the normal quantity of those histones. At the same time the content of protein uH2A (A-24), being the conjugate of H2A histone and ubiquitine, is increased. Mononucleosomes isolated from neuron chromatin was found to have relatively low electrophoretic mobility in polyacrylamide gel taking into account the size of their DNA fragment. Circular dichroism spectra of nucleosome particles show that the neuron mononucleosomes are more unfolded than the rat thymus ones. Data obtained allow to suggest that the short DNA repeat and accumulation of protein uH2A in neurons are the factors influencing the compactization of neuron chromatin.     

Effect of heparin on DNA

It was found that heparin is capable of increasing the sedimentation coefficient of DNA over alkaline and neutral sucrose density gradients as well as of reducing the intrinsic viscosity of native DNA. Irreversible compactization of DNA induced by heparin apparently by means of intramolecular aggregation of DNA with involvement of residual protein underlies the above-described phenomena. The influence of heparin on DNA is potentiated in more concentrated solutions. This should be taken into account in the assay of DNA-containing systems in the presence of heparin.     

The state of a toroid-like DNA globule: the persistent macromolecule in a low-molecular solvent

Dimensions of a toroidal globule appearing at the compactization of DNA macro-molecule in a poor low-molecular solvent are estimated. Diagram of DNA molecule state was analysed in variables: contour length-concentration of the precipitating agent. Possible existence of the tricritical point in the joint of the regions of coil, isotropic globular and liquid crystalline globular states was suggested.     

Live-cell imaging microscopy and quantitative analysis of Ca2+-dependent effects of neurotransmitters on DNA in snail neurons

The effects of serotonin and glutamate on DNA spatial fluorescent pattern after preliminary application of nifedipine and verapamil (inhibitors of L-type of potential-regulated Ca²⁺ channels) or caffeine (ryanodine receptors agonist) or BAPTA AM (membrane-permeable Ca²⁺ buffer) were in vivo studied in L-RPl1 neurons in isolated snail CNS preparation stained with SYTO16 AM. It was found out that preliminary applied BAPTA AM, caffeine, nifedipine or verapamil suppressed the neurotransmitter-induced changes in the number and size of spots in the neuronal nucleus of edible snail. At the same time by 30–120 min after an application of neurotransmitters in the presence of nifedipine, verapamil or BAPTA AM but not caffeine a pronounced increase in the number of large spots is produced, that probably reflects an activation of DNA compactization and chromatin condensation for this period. Obtained data suggest the presence of two consequent phases of DNA transformation evoked by neurotransmitters: (1) — extracellular Ca²⁺ dependent phase of chromatin decondensation and DNA decompactization (duration: about 1–2 h) and (2) subsequent (in 30 min after application of transmitters) extracellular Ca²⁺ independent caffeine-susceptible phase within which DNA compactization, chromatin condensation and the restoration of the initial DNA state occur.     

A replication enhancer of viral origin increases the mitotic stability of yeast transformants]

It is shown in this paper that a DNA fragment of Hepatitis B virus possessing structural features of yeast replication enhancer increases the mitotic stability of yeast transformants containing hybrid plasmids of episomal and replicative types. The mitotic stability of transformants with plasmid of the replicative type and with the replication enhancer increases only in [cir+] cells. Comparison of primary sequences of HBV DNA of different subtypes revealed that only DNA has unique structural features of the yeast enhancer of replication.     

The role of Ca ions in restoration of the structure of interphase and mitotic chromosomes in PK living cells after hypotonic stress.

The dynamics of mitotic chromosome and interphase chromatin recondensation in living PK cells during their adaptation to hypotonic medium was studied. The recondensation process was found to be slowed down by the modification of plasma membrane with low concentrations of glutaraldehyde, while osmotic reactions of glutaraldehyde-treated cells remain unchanged. The effect of glutaraldehyde can be rapidly reversed by the addition of Ca(2+)-ionophore A23187. Intracellular Ca(2+)measurements show that the adaptation to hypotonic shock is accompanied by restoration of free Ca concentration, whereas the delay of chromatin condensation in glutaraldehyde-treated cells is paralleled by the decrease of Ca level. The mechanisms implying the role of low concentration of Ca(2+)in chromatin compactization in vivo are discussed.     

Electron microscopic study of DNA compactization under the effect of putrescine]

DNA-putrescine complexes were studied by electron-microscopy with the use of protein-free method. The latter gives the opportunity to investigate the interaction of DNA molecules spread on the surface layer of hypophase and the polyamine molecules in the thick layer of hypophase. Polyamine concentration varied from 5 x 10(-4) mM to 5 x 10(-1) mM. Under the low concentration of putrescine the complexes are represented by agglomerations of kinked knobbed fibres 10 to 20 nm thick, consisting of several fibres of duplex DNA. Upon increasing of putrescine concentration from 5 x 10(-4) to 1.5 x 10(-1) mM, the fibres become more thick (up to 25 nm), highly twisted and have the appearance of cylinders. Very often in the composition of complexes, it is possible to encounter the circular structures, which were formed at the expense of intermolecular interaction of different parts of the complex. The circular structures can serve as "embryos" of toroids of different sizes, that is of different degree of saturation with DNA and putrescine. At the concentration of putrescine 5 x 10(-1) mM the complexes have the appearance of toroids and structures on the basis of toroids, cylinders. The scheme of possible transitions of fibres of various thickness is proposed. The regularities of the compactization process, stimulated by polyamines, don't depend on the degree of compactization (the thickness of compacting fibre), that is they are similar for duplex DNA and for the fibres 25 nm thick, consisting of dozens of DNA molecules.     

Multidomain structure of fibrinogen and its transformations

Generalized concepts of some structural peculiarities of fibrinogen, its transformation into fibrin and assembly have been considered on the basis of author's and published data. The role of local conformational changes in different areas of fibrinogen molecule and of separate reaction centers in formation of single- and double-stranded rod-like equilibrium fibrin oligomers and flexible branched copolymers of fibrinogen with fibrin E fragment has been considered. The mechanism of compactization has been discussed.     

Effect of the pH of the medium on DNA conformation

The hydrodynamical and optical properties of DNA were investigated in the wide-range of pH by the methods of streaming birefringence, viscometry and spectrophotometry for the different ionic strengths of environment. The measurements of the intrinsic viscosity as a function of pH allow us to determine the compactization of protonated DNA without the destruction of double-helical conformation. This transition is accompanied by a decrease in the optical anisotropy of DNA and the coefficient of molar extinction E260 (P). The increase of volume and persistence length of DNA was observed in the alkaline range of pH. Analyses of experimental data lead to an assumption that the predominant cause of these effects is the change of flexibility of DNA as a result of ionization of its bases. The data obtained were compared with those for polycationic molecules.