Conformational studies of two non-histone chromosomal proteins and their interactions with DNA.

The conformational properties of two non-histone chromosomal proteins (high-mobility-group proteins 1 and 2) have been studied by spectroscopic methods. The interaction of high-mobility-group protein 1 with DNA has also been studied. 1. Circular dichroism results indicate that in the presence of salt both proteins are 40-50% helical between pH 1 and 9. Above pH 9 denaturation takes place. In the absence of salt the proteins denature below pH 4. 2. Nuclear magnetic resonance spectra show the presence of ring-current shifted peaks and perturbed aromatic resonances, demonstrating that the helix formation is accompanied by specific tertiary folding. 3. Nuclear magnetic resonance spectra of compelxes between high mobility group protein 1 and DNA demonstrate that a low ionic strength a portion of the molecule rich in lysine and containing all the aromatic residues is bound to DNA, whilst a more acidic region of the chain remains free from the DNA.     

The mechanism of nucleosome assembly onto oligomers of the sea urchin 5 S DNA positioning sequence

We have used a model system composed of tandem repeats of Lytechinus variegatus 5 S rDNA (Simpson, R. T., Thoma, F., and Brubaker, J. M. (1985) Cell 42, 799-808) reconstituted into chromatin with chicken erythrocyte core histones to investigate the mechanism of chromatin assembly. Nucleosomes are assembled onto the DNA template by mixing histone octamers and DNA in 2 M NaCl followed by stepwise dialysis into very low ionic strength buffer over a 24-h period. By 1.0 M NaCl, a defined intermediate composed of arrays of H3.H4 tetramers has formed, as shown by analytical and preparative ultracentrifugation. Digestion with methidium propyl EDTA.Fe(II) indicates that these tetramers are spaced at 207 base pair intervals, i.e. one/repeat length of the DNA positioning sequence. In 0.8 M NaCl, some H2A.H2B has become associated with the H3.H4 tetramers and DNA. Surprisingly, under these conditions DNA is protected from methidium propyl EDTA.Fe(II) digestion almost as well as in the complete nucleosome, even though these structures are quite deficient in H2A.H2B. By 0.6 M NaCl, nucleosome assembly is complete, and the MPE digestion pattern is indistinguishable from that observed for oligonucleosomes at very low ionic strength. Below 0.6 M NaCl, the oligonucleosomes are involved in various salt-dependent conformational equilibria: at approximately 0.6 M, a 15% reduction in S20,w that mimics a conformational change observed previously with nucleosome core particles; at and above 0.1 M, folding into a more compact structure(s); at and above 0.1 M NaCl, a reaction involving varying amounts of dissociation of histone octamers from a small fraction of the DNA templates. In low ionic strength buffer (less than 1 mM NaCl), oligonucleosomes are present as fully loaded templates in the extended beads-on-a-string structure.     

Analysis of the binding of high mobility group protein 17 to the nucleosome core particle by 1H NMR spectroscopy

The binding of high mobility group (HMG) protein 17 to the nucleosome core particle has been studied in D2O solution using 1H NMR at 500 MHz. Spectra were obtained for purified HMG 17, purified nucleosome core particles, and the reconstituted HMG 17-nucleosome core particle complex at 0.1, 0.2, 0.3, and 0.4 M NaCl. Subtraction of the core particle spectra from spectra of the core particle reconstituted with HMG 17 demonstrated those regions of HMG 17 which interact with the nucleosome at different ionic strengths; the resonance peaks of interacting groups are broadened due to their restricted mobility. At 0.1 M NaCl, the mobility of all the amino acid side chains of HMG 17 was restricted, indicating complete binding of HMG 17 to the much larger nucleosome core particle. At 0.2 M NaCl most of the amino acids were free with the exception of arginine and proline which are confined to or predominant in the basic central region of HMG 17. These amino acids were completely free only at 0.4 M NaCl. We conclude that the entire HMG 17 molecule interacts with the nucleosome core particle at physiological ionic strength. The acidic COOH-terminal region of HMG 17 is released from interaction with the core histones at an NaCl concentration between 0.1 and 0.2 M and so binds weakly at physiological ionic strength. The basic central region binds more strongly to the core particle DNA, being completely released only at much higher ionic strength, between 0.3 and 0.4 M NaCl.     

Spermidine-Deoxyribonucleic acid interaction in vitro and in Escherichia coli.

The binding of spermidine to deoxyribonucleic acid (DNA) was studied by equilibrium dialysis in a wide range of salt concentrations. The association constants ranged from 6 x 10(5) M-1 in 1 mM sodium cacodylate, pH 7.5, to 3 x 10(2) M-1 in 0.3 M NaCl. MgCl2 reduced spermidine-DNA interaction even more than NaCl so that in moderate-ionic-strength solutions (0.3 M NaCl, 0.002 M MgCl2) there was little detectable binding. Low-ionic-strength media were used to isolate DNA from Escherichia coli by a method shown to minimize loss of spermidine from the DNA. Considerable spermidine was associated with E. coli DNA, but control experiments indicated that complex formation had taken place during or after lysis of the cells. Exogenous DNA or ribonucleic acid added to spheroplasts at the time of their lysis caused most of the cellular spermidine to be scavenged by the extra nucleic acid. The data suggest that spermidine is relatively free in the cell and thereby capable of strong (high-affinity) associations with nucleic acids only after the ionic strength of the cell environment is lowered.     

Thermodynamic and spectral study of DNA-prospidin complex

By the methods of heat denaturation and luminescence the interaction between an antitumor drug prospidine and DNA in aqueous solutions at two ionic strengths (0.1 and 0.001 M NaCl) and at various prospidine concentrations was studied. For the first time it has been demonstrated that the interaction occurs at 0.1 M NaCl and therapeutic prospidine concentrations. In the framework of Frank-Kamenetsky's theory of melting of a polymer with stabilizing ligands the size of the binding site and binding constants (K) with the decrease of ionic strength, the lack of alterations in the DNA UV absorption spectrum on complex formation and the data on the competitive binding of ethydium bromide suggest that at the first stage of the reaction an external complex is formed due to electrostatic interactions between quaternary nitrogen atoms of prospidine and DNA phosphate groups. Incubation of the complex at 37 0 C leads to a decrease of the DNA melting temperature and hyperchromic effect. Presumably this is due to the relatively slow formation of chemical bonds between alkylating groups of prospidine and nucleophilic groups of DNA bases, which results in the destabilization and denaturation of DNA. It is concluded that the interaction between prospidine and DNA must be taken into consideration when studying the molecular mechanism of prospidine antitumour activity.     

Nucleosome dissociation at physiological ionic strengths.

Monomer nucleosomes purified on isokinetic sucrose gradients are shown to dissociate into component DNA and histones at physiological ionic strength upon dilution to a DNA concentration below 20 microgram/ml. The starting material is 11S, contains 145-190 BP DNA, and equimolar amounts of the four core histones with slightly less H1. Dilution of monomers in the presence of 0.14 M NaCl results in the rapid conversion of 10-40% of the 3H thymidine labeled material from 11S to 5S (5S is coincident with the S value of monomer length DNA). The proportion of nucleosomes which dissociate increases with increasing NaCl concentration between 0.15 M and 0.35 M and decreases with increasing DNA concentration above 1 microgram/ml. Recycling 11S monomers, which remain after dissociation, through a second dilution in salt generates an equivalent proportion of 5S material as seen after the initial dilution. Thus, the dissociation does not result from special properties of a subset of nucleosomes. An equilibrium between intact monomer and free DNA and histones appears to be rapidly established under the conditions described and the dissociated DNA will reassociate with histones to form 11S monomers if conditions of high DNA concentration and low ionic strength are established.     

Cytochemical characterization of deoxyribonucleoprotein by UV-microspectrophotometry on heat denatured cell nuclei

Denaturation of double-stranded DNA into a single-stranded state can be studied by heating fixed cells attached to quartz slides and then determining the increase in nuclear UV-absorption at 265 nm by microspectrophotometry at room temperature. In order to prevent renaturation as the slides are cooled, formaldehyde is added to the solution in which heat denaturation is performed. The influence of formaldehyde concentration, duration of heating and ionic strength on the stability of DNA to heat denaturation has been examined. The standard method involves heating of ethanol/acetone fixed cells to temperatures between 22°C and 100°C in 0.15 M NaCl, 0.015 M sodium citrate containing 4% formaldehyde for 20 min followed by cooling to room temperature and mounting in glycerol.     

The conformation of the chromatin core particle is ionic strength-dependent.

We have examined the susceptibilities of the histones within the HeLa chromatin core particle to covalent modification by a diol-epoxide derivative of the carcinogenic polycyclic aromatic hydrocarbon benzo(a)-pyrene. Core-particle histones exhibit substantial variation in their relative susceptibilities to modification, depending upon the ionic strength of the environment. In contrast, the relative susceptibilities of either purified histones or histones in urea-denatured core particles are insensitive to changes in ionic strength. The variations in the pattern of modification of core particle histones occur primarily at ionic strengths at which the histones remain associated with core-particle DNA (0 to 0.6 M NaCl). Non-histone proteins influence the ionic strength-dependent variations in histone modification. The results imply that the ionic strength of the environment affects the conformation of the core particle and that the nucleoprotein has a flexible structure.     

Protein dissociation from DNA in model systems and chromatin.

Salt induced dissociation of protamine, poly(L-lysine) and poly(L-arginine) from DNA was measured by relative light scattering at theta = 90 degrees and/or centrifugation. Dissociation of histones from DNA was studied using relative light scattering and intrinsic tyrosine fluorescence. Protamine was dissociated from DNA at 0.15 M MgCl2 (ionic strength mu = 0.45) or 0.53 M NaCl (mu = 0.53) based on light scattering data and at approximately 0.2 M MgCl2 (mu = 0.6) or 0.6 M NaCl based on centrifugation data. NaCl induced dissociation of poly(Lys) or poly(Arg) from natural DNAs measured by light scattering did not depend on the guanine plus cytosine content. To dissociate poly(Arg) from DNA higher ionic strength using NaCl, MgCl2, or CaCl2, similar ionic strength using NaClo4, and lower ionic strength using Na2SO4 was needed then to dissociated poly(Lys). Both the decrease in light scattering and the enhancement of tyrosine fluorescence of chromatin occurred between 0.5 and 1.5 M NaCl when histones were dissociated.     

Some aspects of the copper(II)-DNA interaction

The Cu(II) ion interaction with calf-thymus DNA was studied by means of differential pulse polarography and sweep voltammetry as well as chromatography and viscosimetry. Most of the complexes formed at high ionic strength (0.2 M) and lower Cu(II) concentrations are of a nondenaturing nature. Their formation has but a minor effect on unwinding process of the DNA double helix. The excess of Cu(II) (P = 5) leads, however, to distinct denaturation of the DNA structure. Metal ions have little effect on the denaturation induced by the polarographic reduction of DNA on the mercury electrode. This conclusion is consistent with the character of the polarographic process and with the fact that Cu(II) ions are not very effective in the interaction with AT pairs. Cupric ions have no renaturing ability towards thermally denatured DNA at 0.2 M ionic strength but distinct renaturation was observed at low ionic strength (0.05 M).     

Salt-induced structural changes in nucleosomes

Ehrlich ascites tumor (EAT) nucleosomes treated with increasing NaCl concentrations were analyzed by sucrose density gradient centrifugation. Two events were found to take place in the course of the salt treatment: a) increasing amounts of nucleosomes dissociated into free DNA and protein in the interval 0.6M–1.5M NaCl, and b) the sedimentation coefficient of the nucleosomes decreased from 11S to 8S in the interval 0.6M-1M NaCl. This decrease was not caused by loss of protein and was fully reversible upon slow and gradual lowering of the ionic strength. This shows that before dissociation of the protein core from DNA, nucleosomes undergo a structural transition. The electron microscopic observations revealed that it consisted in detachment of the ends of nucleosomal DNA from the protein core. It is suggested that an arginine-rich domain in the protein core exists, which holds more tightly the central part of the nucleosomal DNA, while its ends are relatively loosely bound to lysine-rich domains.     

Detection of 5-bromodeoxyuridine (BrdUrd) incorporation by monoclonal antibodies: role of the DNA denaturation step

Immunochemical detection of cells that incorporate 5-bromodeoxyuridine (BrdUrd) requires prior denaturation of DNA in situ to make BrdUrd binding sites accessible to the antibodies. A technique is described in which the DNA denaturation step is facilitated by a) prior dissociation of histones from DNA and b) the use of low ionic strength buffer in which the cells are suspended during heating. Dissociation of histones is achieved by cell treatment with 0.08N HCl at 0 degree C, which a) increases accessibility of DNA to propidium iodide (and following the denaturation to the antibodies); b) lowers stability of DNA to thermal denaturation; c) decreases differences between various cell types due to variability in chromatin structure; and d) ensures more complete DNA denaturation. Cell heating (80-95 degrees C) at low ionic strength (1 mM Na+) eliminates the need for formamide and results in extensive and rapid DNA denaturation. The method was applied in Friend leukemia, L1210 and HL-60 cell lines, and to bone marrow, experimental animal tumor and primary human tumor cells.     

Thermal denaturation and fluorescence study of nucleosomes containing non-histone chromosomal protein HMG2

Interaction of calf thymus non-histone chromosomal protein HMG2 with H1,H5-depleted nucleosomes from chicken erythrocytes was studied by means of thermal denaturation and an N-(3-pyrene)maleimide fluorescence probe. Under low ionic conditions (2 mM Tris buffer plus EDTA) addition of 1-2 molecules of HMG2 per nucleosome markedly stabilized the segment of the linker DNA against thermal denaturation. Under approximately physiological ionic conditions (0.1 M NaCl) addition of two HMG2 molecules per nucleosome, labeled by N-(3-pyrene)maleimide at the sulfhydryl groups of Cys-110 of histones H3, resulted in a decrease of the pyrene excimer fluorescence corresponding to the slight movement of the sulfhydryl groups of the two histone H3 molecules apart.     

Hydroxyapatite-mediated transfer of DNA from diluted solutions to nitrocellulose or nylon membranes

Transfer of DNA (from 0.1 to 10 micrograms) from diluted solutions of variable volumes (1-10 ml) and various composition (2 M NaCl; 4 M LiCl, 8 M urea; 4 M CsCl; 20% sucrose) to nitrocellulose or nylon membranes was achieved with the use of hydroxyapatite. This absorbent that binds nucleic acids effectively and independently of ionic strength and composition of solution (except for chelators and phosphate ions) easily dissolves in small volumes of acids (for example, in 10% TCA). This phenomenon provides the opportunity to deliver the acid-insoluble precipitates to membrane filters. After alkaline denaturation on the filter followed by a fixation step (baking or UV irradiation for nitrocellulose or nylon filters, respectively), DNA hybridizes effectively with nick-translated DNA probes. The method is simple, reproducible, sensitive, and useful for working with diluted DNA solutions containing interfering substances.     

Novikoff hepatoma deoxyribonucleic acid polymerase. Identification of a stimulatory protein bound to the beta-polymerase.

The Novikoff hepatoma DNA polymerase-beta sediments as a 7.3S form in crude extracts but during purification sediments as a 4.1S form (after diethylaminoethyl-Sephadex chromatography) or as a 3.3S form (after DNA-cellulose chromatography). If 0.25 M ammonium sulfate or 0.5 M NaCl is included in the sucrose gradients, the 7.3S form sediments at 3.3 S; after removal of the salt, it sediments again at 7.3 S, indicating the reversibility of the aggregation phenomenon. By careful adjustment of ionic strength in the gradient, four distinct and reproducible forms of the enzyme sedimenting at 7.3, 5.8, 4.1, and 3.3 S can be generated. The isoelectric point of the DNA polymerase also changes during purification; the 7.3S form has a pI of 7.5, while the 4.1S form isoelectrically focuses at a pH of 8.5. During DNA-cellulose chromatography, the Novikoff beta-polymerase is separated from a stimulatory factor designated as Novikoff factor IV. Factor IV is a protein as shown by its sensitivity to protease and resistance to nucleases. It is responsible for converting the 3.3S enzyme to the 4.1S form since the 3.3S homogeneous DNA polymerase-beta sediments at 4.1 S in the presence of factor IV. Factor IV confers stability to the polymerase in low ionic strength buffers as well as stability to heat denaturation. Factor IV has the ability to increase the activity of the 3.3S homogeneous polymerase by about fourfold.     

Analysis of the effectiveness of sodium chloride in dissociating non-histone chromatin proteins of cultured hepatoma cells

We examined the ability of NaCl (at 0.15 to 3 M) to release non-histone proteins from chromatin of cultured rat hepatoma cells. The percentage of the non-histones released increased with increasing NaCl concentrations up to 0.75 M; 1 and 3 M NaCl were not significantly more effective. A maximum of 50% of the non-histone protein was recovered free of DNA. The release of non-histones from sheared and unsheared chromatin was similar. The electrophoretic patterns of the non-histone proteins released by NaCl resembled that of the non-histones released by sodium dodecyl sulfate, which indicates that many of the detectable components were at least partially released by NaCl. Some non-histones (especially low molecular weight polypeptides) were fully released by NaCl and other proteins were relatively resistant to NaCl release. Higher recoveries of NaCl-dissociated non-histones were obtained with sucrose gradient centrifugation than with centrifugation in the absence of sucrose.     

Binding of NR1 plasmid DNA to membrane during replication in Escherichia coli mini-cells

Studying the replication of NR1 plasmid in E. coli mini-cells it was shown that the character of bond between plasmid DNA and membrane depends on the stage of replication cycle of the plasmid. On initiation the DNA-membrane complex is sensitive to the action of ionic force. In the process of elongation the bond of DNA molecules with the membrane is unstable if exists at all, and can be broken even by the nonionic detergent. At the final stage of replication the newly synthesized molecules form a complex with the membrane structures which is unstable in the presence of 0,5 M NaCl. The destruction of the complex followed by the open cycle of plasmid DNA coming out of it takes place under the action of ionic detergent.     

Structural changes of nucleosomal particles and isolated core-histone octamers induced by chemical modification of lysine residues

Treatment of nucleosomal particles and isolated core-histone octamers with dimethylmaleic anhydride, but not with acetic anhydride, is accompanied by a biphasic release of the two H2A.H2B dimers, the first dimer being more easily released than the second. With both kinds of particles, 50% of histones H2A and H2B are released for modification of approximately 35% of the histone amino groups. The similar behavior of nucleosomal particles and isolated core-histone octamers is consistent with the same structure of the histone octamer in the nucleosomal particle and in the free octamer in 2 M NaCl. The described release of H2A.H2B dimers allows the preparation of nucleosomal particles deficient in one H2A.H2B dimer and of the histone hexamers H2A.H2B.(H3.H4)2. For more extensive modifications, both reagents, acetic and dimethylmaleic anhydrides, cause the dissociation of nucleosomal particles with liberation of double-stranded DNA, which suggests that lysine amino groups are involved in the binding of histones to DNA. The modified nucleosomal particles are more sensitive to ionic strength than those untreated, and the presence of salt (NaCl) increases the extent of DNA release. The histones corresponding to the liberated DNA, except H2A and H2B released with dimethylmaleic anhydride, are apparently bound to the DNA-containing particles as extra histones.     

Association of DNA with the nuclear lamina in Ehrlich ascites tumor cells

We have studied in vitro binding of DNA to nuclear lamina structures isolated from Ehrlich ascites tumor cells. At low ionic strength in the presence of Mg++, they bind considerable amounts of mouse and bacterial DNA, forming complexes stable in 2 M NaCl. Single-stranded DNA and pulse-labeled DNA show higher binding efficiencies than native uniformly labeled DNA. When mixing occurs in 2 M NaCl, complex formation is inhibited. When nuclei are digested with DNAse I under conditions that favor chromatin condensation, DNA associated with matrices subsequently prepared from such nuclei is markedly enriched in satellite DNA. If digestion is carried out with DNAse II while nuclei are decondensed in EDTA, no enrichment in satellite DNA is observed. Preparations of purified, high-molecular weight, double-stranded DNA contain variable amounts of fast-sedimenting aggregates, which are insoluble in 2 M NaCl but are dispersed by DNA fragmentation or denaturation. These results point at some artifacts inherent in studies of DNA bound to residual nuclear structures in vivo and suggest conditions expected to avoid these artifacts. Further, using controlled digestion with DNAse II, we have studied the in vivo association of DNA with nuclear lamina isolated from Ehrlich ascites tumor cells. In the course of DNA fragmentation from above 50 kbp to about 20 kbp average size, the following events were observed. The DNA of high molecular weight (much longer than 50 kbp) behaved as if tightly bound to the nuclear lamina, as judged by sedimentation in sucrose and metrizamide density gradients, electron microscopy, and retention on glass fiber filters. As the size of DNA decreased, it was progressively detached from the nuclear lamina, and at about 20 kbp average length practically all DNA was released. The last 1-4% of DNA, although cosedimenting with the nuclear lamina in sucrose gradients, behaved as free DNA, banding at 1.14 g/cm3 in metrizamide density gradients and showing less than 4% retention on filters. At no stage of digestion did the DNA cosedimenting with nuclear lamina show changes in satellite DNA content relative to that of total DNA or enrichment in newly replicated DNA. It was shown, however, that digestion of nuclear lamina-DNA complex with EcoRI or Hae III led to the formation of DNA-protein aggregates, which banded at 1.35 g/cm3 in high salt containing metrizamide density gradients and which were strongly enriched in satellite DNA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Salt induced conformational transition between A and Z forms of r(CGCGCG) as revealed by a Raman spectroscopic study

Solution conformation in different conditions of r(CGCGCG) has been studied by a Raman spectroscopic method. In NaCl solution, r (CGCGCG) takes only an A-form duplex in which guanosine and cytidine have C3'endo-anti conformation even at 5M salt concentration. In much higher ionic strength condition (5M NaCl plus 1M MgCl2 or 6M NaClO4), it undergoes a transition to a left-handed Z-form. The Raman spectrum of the Z-form RNA was found to be very similar to that of Z-form DNA, suggesting that Z-RNA involves a C3'endo-syn guanosine and an in between form of C2'endo-Cl'exo-anti cytidine.