Mechanisms of stabilizing nucleosome structure. Study of dissociation of histone octamer from DNA.

The influence of ionic strength on DNA-histone and histone-histone interactions in reconstituted nucleosomes was studied by measuring the parameters of histone tyrosine fluorescence: fluorescence intensity and lambda(max) position. The first parameter is sensitive to histone-DNA interactions. The changes of the second one accrue due to hydrogen bond formation/disruption between tyrosines in the histone H2A-H2B dimer and the (H3-H4)2 tetramer. The simultaneous measurement of these parameters permits the recording of both the dissociation of histone complexes from DNA, as well as changes in histone-histone interactions. As ionic strength is increased, the H2A-H2B histone dimer dissociated first, followed by dissociation of the (H3-H4)2 tetramer [Yager, T.G., McMurray, C.T. and Van Holde, K.E. (1989) Biochemistry 28, 2271-2276]. The H2A-H2B dimer is dissociated in two stages: first, the ionic bonds with DNA were disrupted, followed by the dissociation of the histone dimer from the tetramer. And secondly, the disruption of dimer-tetramer specific H-bonds. It was established that the energy of electrostatic interactions of the histone dimer with DNA within the nucleosome is much less than the energy of interaction of the histone dimer with the tetramer.

Electrostatic contribution to the bending of DNA.

A model is derived that accounts for the short-range electrostatic contribution to the bending of DNA molecule in solution and in complexes with proteins in terms of the non-linear Poisson-Boltzmann equation. We defined that the short-range electrostatic interactions depend on the changes of the polyion surface charge density under deformation, while the long-range interactions depend on the bending-induced changes in distances between each two points along the polyion axis. After an appropriate simplification of the Poisson-Boltzmann equation, the short-range term is calculated separately giving the lower limit for the electrostatic contribution to the DNA persistence length. The result is compared with the theoretical approaches developed earlier [M. Fixman, J. Chem. Phys. 76 (1982) 6346; M. Le Bret, J. Chem. Phys. 76 (1982) 6243] and with the experimental data. The conclusion is made that the results of Fixman-Le Bret, which took into account both types of the electrostatic interactions for a uniformly bent polyion, give the upper limit for the electrostatic persistence length at low ionic strength, and the actual behavior of the DNA persistence length lies between two theoretical limits. Only the short-range term is significant at moderate-to-high ionic strength where our results coincide with the predictions of Fixman-Le Bret. The bending of DNA on the protein surface that is accompanied by an asymmetric neutralization of the DNA charge is also analyzed. In this case, the electrostatic bending energy gives a significant favorite contribution to the total bending energy of DNA. Important implications to the mechanisms of DNA-protein interactions, particularly in the nucleosome particle, are discussed.

[Effect of adenosine triphosphate on the cleavage of H1 histones in nuclei of rat spleen and liver]. / Vliianie adenozintrifosfata na rasshcheplenie gistonov H1 iader selezenki i pecheni krys.

Proteolysis of histones H1b and H1(0) is observed after the incubation of rat spleen nuclei at 37 degrees C during 1 hour. Adenosine triphosphate, inorganic pyrophosphate and nicotinamide adenine dinucleotide decrease the digestion of histone H1b. ATP, PP1 and NAD+, in the case of 3 hours incubation, do not affect proteolysis of H1 histones in rat spleen nuclei. The incubation of rat liver nuclei at 37 degrees C during 1 hour leads to a decrease of the amount of histone H1b and, to much more extent, H1a. In this case ATP, PP1 and NAD+ increase proteolysis of histone H1a but practically do not affect proteolysis of H1b. After 2-hours incubations histone H1a is completely digested but histone H1b is partially preserved: ATP in this case, as well as in spleen nuclei, decreases proteolysis of histone H1b. During the 3 hours incubation, when histones H H1a and H1b are completely digested, partial digestion of histone H3 being observed, ATP does not prevent from proteolysis of histone H1b. A protein appears between the H2A and H4 histones after heating at 37 degrees C in both spleen and liver rat nuclei. Neither ATP nor PP1 and NAD+ affect the amount of this protein. It is suggested that the location of histones H1a and H1b in different chromatin domains determines the digestion of these histones by ATP-dependent proteinases.

[The effect of thymogen on the chromosome aberration level in a culture of human peripheral blood lymphocytes]. / Vliianie timogena na uroven' khromosomnykh aberratsii v kul'ture limfotstiov perifericheskoi krovi cheloveka.

The influence of thymogene on the frequency of spontaneous and formaldehyde-induced chromosome aberrations in human peripheral blood lymphocytes was detected. High thymogene concentration (1000, 100, and 10 micrograms/ml) significantly decreased the proliferative activity of lymphocytes in culture. Low thymogene concentrations (1.0; 0.1; 0.01; and 0.001 microgram/ml) didn't decrease the mitotic activity of culture and hadn't mutagenic activity. Thymogene concentrations 1.0; 0.1; and 0.001 microgram/ml significantly decreased the frequency of formaldehyde-induced chromosome aberrations in lymphocytes. Possible mechanisms of gene protection by thymogene is discussed.

Translational positioning of nucleosomes on DNA: the role of sequence-dependent isotropic DNA bending stiffness.

A model has been derived that accounts for the nucleosome translational position in terms of the bending free energy that depends on the nearest-neighbor interactions between base-pairs. The available data on the nucleosome positioning on defined DNA sequences in the reconstituted systems have been analyzed. It has been shown that the model allows one to predict the preferred nucleosome translational positioning with an accuracy of about one turn of the double helix. The conclusion is made that the isotropic elastic properties of the DNA molecule are very important for nucleosome translational positioning. The anisotropic flexibility modulates the sequence-dependent preference and defines the precise rotational placement. The analysis points to a possible involvement of DNA bendability in nucleosome structural transitions. To model the nucleosome positioning within the chromatin fiber, the derived algorithm has been applied to random DNA sequences. The nucleosome distribution obtained is close to random, but nucleosomes, according to calculations, are placed on sites with a low value of bending free energy and decreased G+C-content. Relations with other work and some implications are discussed.

[Reaction of fluorescamine with nitrogen bases in nucleotides and single-stranded synthetic and native oligonucleotides]. / Reaktsiia fluoreskamina s azotistnymi osnovaniiami v sostave nukleotidov i odnonitevykh sinteticheskikh i prirodnykh oligonukleotidov.

Reactions of fluorescamine with nucleoside bases have been studied. Fluorescamine reacts with NH2 groups of dA, dG, dC, and the characteristic pyrrolinone spectrum with maximum at 490 nm appears. Fluorescamine does not react with dU and dT. The reaction is pH-dependent, but not linked to protonization of amino groups. pK values for dA, dG, dC are respectively 7.3, 8.4, 8.7. At pH greater than 10 or lower than 3 the fluorescence decreases due to formation of non-fluorescent fluorescamine derivates. The average fluorescence yield of pyrrolinone (the product of reaction of fluorescamine with denatured DNA and single-stranded polymers containing dA) is 2.7-3.5 fold higher as compared to mixture of individual nucleotides. The studied reaction can be used sequencing of nucleic acids.

[Molecular mechanisms of the damaging effect of laser radiation on DNA]. / Molekuliarnye mekhanizmy povrezhdaiushchego deistviia lazernogo izlucheniia na DNK.

The effects of DNA photoscission were studied in the presence of ethidium bromide (EB) and riboflavin (RF) (vitamin B2). Laser irradiation (337 nm) of DNA in the presence of EB and RF induces single- and double-strand scissions in the superhelical form of DNA pBR322 (registered as increasing content of its nicked and linear forms). Possibility of DNA scission by energy absorbed by a nonintercalating chromophore (riboflavin) was demonstrated for the first time. The dependence of DNA photoscission in complex with riboflavin on the energy density in the pulse suggests a nonlinear (two-quantum) character of the photoscission process. It was shown that for riboflavin the quantum yield of DNA photoscission exceeds several times the one for ethidium bromide.

[ATP-dependent structural changes in chromatin]. / ATP-zavisimye strukturnye izmeneniia khromatina.

Incubation (1 hour, 37 degrees C) of nuclei and chromatin from guinea pig spleen (but not from thymus or the liver) resulted in a proteolysis of H1 and H2A histones and accumulation of specific hydrolytic products. Sodium dodecyl sulfate gel electrophoresis revealed a decline in H1 and H2A and the appearance of new protein bands under histone H10 and between H2A and H4. ATP (10 mM) (but not cAMP or PPi) added to the incubation mixture prevented the H1 and H2A digestion and accumulation of the aforementioned products. The ATP, which protects the histones in the nucleus and chromatin from proteinases, promoted the cleavage of cytosolic low molecular weight proteins. The mechanisms of ATP-dependent chromatin structural rearrangements determining the resistance of nuclear proteins to proteolysis are discussed.

[The possible role of endogenous nucleases in the structural organization of chromatin]. / Vozmozhnaia rol' éndogennykh nukleaz v strukturnoi organizatsii khromatina.

Two fractions of rat liver nuclei with different buoyant density have been obtained. The electrophoretic analysis of the oligonucleosome patterns of DNA out of nuclei of these two fractions revealed different levels of activity in endonucleases. In case of inhibition during the extraction of activity in Ca, Mg-dependent endonucleases, the average size of high polymeric DNA is larger for nuclei with bigger buoyant density (fraction I) than for nuclei with smaller ones (fraction II). This finding is evidence of in situ existence of two pools of liver nuclei with different endogenic nuclease activities. In nuclear chromatin fraction I DNA is torsionally stressed; in fraction II it is relaxed that correlates with larger activity of endonucleases and smaller buoyant density of this fraction. A hypothesis on a possible role of endonucleases in chromatin structure organization has been put forward. According to this hypothesis a modulation of activity in nuclear endonucleases can determine different packaging and activity of chromatin from different pools of cellular nuclei.

[A rapid method of preparing the (H3-H4-H2A-H2b)(2) histone octamer in large quantities]. / Bystryi metod polucheniia oktamera (H3-H4-H2A-H2B)(2) gistonov v bol'shikh kolichestvakh.

A simple and fast method for isolation of large amounts of the histone octamer (H2A-H2B-H3-H4)2 is proposed. This method is based on chromatin adsorption by hydroxyapatite with subsequent extraction of the histone octamer with 50 mM sodium-phosphate buffer containing 4 M NaCl pH 8.0. It was shown that the properties of the histone octamer isolated by this extractive procedure are identical with those of the histone octamer obtained by elution on a Sephadex G-100 column. The histone tetramer (H3-H4)2 and dimer (H2A-H2B) were obtained after gel filtration on Sephadex G-100 in 50 mM sodium-acetate (pH 5.6).

[The effect of DNA supercoiling DNA on nucleosome structure]. / Vliianie sverkhspiralizatsii DNK na strukturu nukleosom.

The circular DNA which contains nucleosomes and additional supercoils has been considered theoretically. The different possible effect of increased negative supercoiling on the nucleosome structure have been studied. According to the model proposed all supercoils in the nucleosome-containing circular DNA are realized as torsional deformations of the double helix. The free energy of both supercoiling (torsional deformations) and nucleosome stabilization have been taken into consideration to obtain the equation for free energy of nucleosome-containing circular DNA. The analysis of this equation and the experimental data by Garner et al. (II Psoc. Natl. Acad. Sci. USA. 1987. P. 2620-2623) about the maximum amount of supercoiling obtained by DNA-topoisomerase II treatment of nucleosome-containing pBR322 plasmid has been performed. It has been shown that two possibilities are consistent with both the equation and experimental data. These are: (1) the increased supercoiling induces the torsional strains not only in linker regions but also in nucleosome DNA and thus supercoiling causes an instability on nucleosome structure; (2) increased supercoiling induces a structural change of nucleosome which is accompanied by nucleosome DNA unwinding and its transition into form with approximately 11 base pairs per turn of double helix. It has been evaluated that in the first case the average torsional rigidity of nucleosome DNA should be approximately 2.5 times as much and in the second case--much more than the rigidity of naked DNA. Both types of nucleosome structural changes may cause its transition to a potentially active state for transcription. It is suggested that increased supercoiling can be a switch mechanism of chromatin activation.

[Primary structure of the basic nuclear protein from spermatozoa of the mollusk Illex argentinus and its comparison with the structure of sperm proteins in other animals]. / Pervichnaia struktura osnovnogo iadernogo belka iz spermatozoidov golovonogogo molliuska Illex argentinus i sravnenie ee so strukturami spermal'nykh belkov drugikh zhivotnykh.

The spermatic protein of chromatin I2 of squid Illex argentinus was separated by HPLC into two components I2-1 and I2-2. Amino acid sequences of the major portion of protein I2-1 (52 residues) and the N-terminal sequence of protein I2-2 (21 residues) were determined. Arginines in protein I2-1 are arranged in clusters typical of protamines; the first cluster is in the N-terminus, the longest heterogeneous basic cluster is in the central part of the protein chain, the C-terminal part of the molecule contains two clusters of three hydroxyamino acids each. The N-terminal sequences of illexins I2-1 and I2-2 (1-14 residues) are highly homologous. Homologous regions were found in illexin I2-1, tunnin of tuna fish and avian gallin thus defining the notion of proteins of an intermediate type from mollusc spermatozoa chromatin exemplified by the squid protamine-like protein.

[A theoretical model of the mechanism of DNA compactization by polycations]. / Teoreticheskoe rassmotrenie mekhanizmov kompaktizatsii DNK polikationami.

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.

Displacement of histones by sperm-specific proteins at different stages of spermatogenesis of squid.

Changes in the composition of the chromatin basic proteins during spermatogenesis of the squid Illex argentinus were studied. The core histones of I. argentinus slightly differ from those of calf thymus in the subfractional composition of histones H2A and H2B. A similar amino acid composition is revealed in the histones H1 of the squid I. argentinus and calf thymus. Histone H1 of the squid has a lower molecular mass and a special subfractional composition as compared to those of calf thymus, grass carp and carp studied formerly [Kadura et al. (1983) Comp. Biochem. Physiol. 743, 343-350]. Neither the fractional nor subfractional composition of histones changes during spermatogenesis. The two new proteins were revealed in the chromatin composition of squid testes and spermatozoa illexines I1 and I2. Illexine I2 is composed of two subfractions I2-1 and I2-2. Illexine I2 shows a high content of arginine (75 mol/100 mol). Serine (10 mol/100 mol), histidine (3,2 mol/100 mol) and tyrosine residues (2,9 mol/100 mol) are also present. Illexine I1 shows the presence of arginine (45,6 mol/100 mol), lysine (7.6 mol/100 mol), serine (11.4 mol/100 mol), hystidine (2.3 mol/100 mol) and tyrosine residues (2.8 mol/100 mol). Molecular masses of illexines I2 and I1 are approximately 7 kDa and 9 kDa respectively. It is supposed that during spermatogenesis the histones are displaced in two-stage order: histones----I1----I2.

Rearrangements of chromatin structure during spermatogenesis of squid.

A stepwise replacement of somatic histones on sperm-specific proteins (we have termed them illexines I1 and I2) is found to occur during spermatogenesis of squid Illex argentinus [Kadura, S.N. and Khrapunov, S.N. (1988) Eur. J. Biochem. 175, 603-607]. The chromatin from nuclei of squid immature testes has a nucleosomal DNA repeat which corresponds to the nucleosomal repeat of calf thymus chromatin (195 +/- 5 bp). As spermatozoa become mature and illexine I2 accumulates in the chromatin, the nucleosomal structure of the latter disappears and chromatin compacting takes place. The chromatin DNA from squid spermatozoa is highly resistant to micrococcal nuclease action. Spectrophotometry and spectrofluorimetry were to establish that neither illexine I1 nor illexine I2 forms a globular structure in solution under any conditions studied. Illexine I2 (approx. 7 kDa) shows a high affinity to DNA and remains bound to it under conditions when complexes of illexine I1 (approx. 9 kDa) and salmine (approx. 4.5 kDa) with DNA completely dissociate. This fact, allowing for a similar content (about 75%) of arginine in illexine I2 and salmine, suggests high clustering of arginine residues in the composition of illexine I2. It is suggested that the initial stage of histone substitution with illexine I1, which has a more moderate affinity to DNA than illexine I2, prepares chromatin for the formation of a highly packed structure by illexine I2 during squid spermatogenesis.

[Binding of positive charged ligands to DNA. The effect of ionic strength, charge and size of the ligand]. / Sviazyvanie polozhitel'no zariazhennykh ligandov s DNK. Vliianie ionnoi sily, zariada i razmera liganda.

The electrostatic interaction of extended cationic ligands with DNA has been considered on the basis of the analytical solution of a simplified Poisson--Boltzmann equation for the charged polyion cylinder. The control numerical solution of rigorous Poisson--Boltzmann equation shows that the assumption about the absence of coions in the vicinity of the highly charged polyion cylinder does not significantly influence the accuracy of solution and DNA electrostatic free energy evaluation. It was found that the basic contribution to the free energy of electrostatic ligand-DNA interaction is the mixing entropy change due to release of counterions from the vicinity of DNA. The equation for the dependence of the ligand to DNA binding constant K upon ionic strength c has been derived without introduction of any empirical parameters. This equation is consistent with the experimental data and can be used for the determination of a number of ligand--DNA ionic contacts in a wide range of salt concentrations. The main consequences of Manning and Record et al. theories can be considered as limiting cases of the theory presented. In particular the equation d(lnK)/d(lnc) = -0.88 N by Record et al. has a restricted range of application and it can be used only for a relative approximate estimation of the number of electrostatic bonds in ligand-DNA complexes. The analysis of electrostatic interaction of DNA with ligands which neutralize only part of phosphate groups in the binding site of DNA was also performed.(ABSTRACT TRUNCATED AT 250 WORDS)

[Analysis of the heterogeneity of chromatin proteins fractionated on hydroxyapatite]. / Analiz geterogennosti belkov khromatina, fraktsionirovannykh s pomoshch'iu gidroksiapatita.

The protein composition of the liver chromatin has been studied by two techniques for fractionation of histones. The "lability" fraction of histones H2A-H2B is revealed. In these fractions histones H2B have many modified forms and they are not included into octamer (H3, H4, H2A, H2B)2. Young animals rather than old ones have much quantitative subfractions of histone H2B. The "lability" fraction of histones H2A-H2B is stated to be very significant in the activated and repressed chromatin structure.

[3 types of sperm proteins in eukaryotes]. / Tri tipa spermal'nykh belkov éukariot.

Basic spermal proteins of various species of hydrobionts attributed to Pisces and Cephalopoda are studied. It is established that chromatin of nine species referring to two Cypriniformes families includes the somatic histones. Histone H1 of Cypriniformes is attributed to the lysine-rich type histones and contains 35% mol. of lysine and 0.7% mol. of tyrosine. Chromatin of 14 species of fish referring to nine families of the percoid fish superorder includes protamines similar to salmin, a typical protamine of salmon. The amino acidic analysis of protamine from the sandre sperma has shown that it contains 59% mol. of arginine and no tyrosine. Chromatin of three species from squid superorder referring to Cephalopoda includes gametones -- proteins differing from histones and protamines both in the electrophoretic mobility and amino acidic composition (75% mol. of arginine, 3% mol. of tyrosine).

[Structure of illexine I2 and its complexes with DNA]. / Issledovanie struktury illeksina I2 i ego kompleksov s DNK.

Conformational peculiarities of illexine I2 both in the solution and in the complexes with DNA were studied by circular dichroism, UV-spectroscopy and spectrophotometric melting. IIlexine I2 is shown to have an extended left-handed helical conformation of poly-L-proline II type, that are stable in a wide range of experimental conditions. Upon interaction of illexine I2 with DNA, the parameters of conformation are somewhat distorted but the main peculiarities remain. The DNA double helix changes from B- to the divection of C-form at its interaction with illexine I2. The interaction of illexine I2 with DNA at low ionic strength is non-cooperative and is characterized by some specificity to A--T sequences of DNA. Illexine I2 strongly affects the DNA stability by increasing the melting temperature of DNA.

[Theoretical study of structural transition in a nucleosome at low ionic strength]. / Teoreticheskoe issledovanie strukturnogo perekhoda v nukleosome pri nizkoi ionnoi sile.

The theoretical analysis of nucleosome stability at low ionic strength has been performed on the basis of consideration of different contributions to the free energy of compact state of the nucleosome DNA terminal regions. The proposed model explains: the fact of low-salt structural change; the transition point (approximately 1.7 mM NaCl) and width (approximately 1 mM); the shift of the transition to the higher salt concentrations in the case of histones tails removal by trypsin. According to the model the increase of electrostatic repulsion between neighbouring turns of DNA superhelix is the main cause of the unwinding of nucleosomal DNA terminal regions in the course of low-salt structural change. The interactions between histone (H2A-H2B) dimer and (H3-H4)2 tetramer provide the compact state of the nucleosomal DNA terminal regions. The existence of electrostatic interactions of nucleosomal DNA terminal regions with tetramer was suggested. These interactions can provide the compact state of nucleosomal DNA at physiological ionic strength even in the absence of (H2A-H2B) dimer.