Nucleosome core particles of calf thymus, Tetrahymena, and the reconstituted hybrid. Their structure reflects the nature of the histone octamer

The circular dichroism spectra and the thermal denaturation profiles of the nucleosome core particles isolated by micrococcal nuclease digestion from nuclei of calf thymus and the protozoan Tetrahymena pyriformis were compared with those of the homogeneous and hybrid core particles reconstituted from calf core DNA and either calf or Tetrahymena histone octamer. The core DNA was obtained from the calf core particle, and both the histone octamers were reconstituted from the acid-extracted four core histones of calf thymus or Tetrahymena, whose amino acid sequences show the largest differences hitherto known. The reconstituted homogeneous core particle was identical in both the physical properties with the isolated calf core particle, showing that the correct reconstitution was achieved. The circular dichroism spectra of the calf and Tetrahymena core particles and the hybrid core particle showed no essential differences, indicating that the three core particles have the same overall structure. The derivative thermal-denaturation profiles, however, clearly differed; the calf core particle showed two melting transitions at 60 degrees C and 72 degrees C, while the Tetrahymena and hybrid core particles showed the same three transitions at 48-50 degrees C, 60-61 degrees C, and 72 degrees C. Thus, the thermal denaturation properties of nucleosome core particles do not reflect the nature of DNA, but rather that of the histone octamer bound to the DNA. We conclude that the Tetrahymena histones are more weakly bound to the DNA than the calf thymus histones in the same overall structure of nucleosomes.     

Two new photoaffinity polyamines appear to alter the helical twist of DNA in nucleosome core particles

Two new photoaffinity derivatives of polyamines have been synthesized by the reaction of spermine or spermidine with methyl 4-azidobenzimidate. The new compounds were purified chromatographically and characterized by several methods including proton magnetic resonance spectroscopy. The spermine derivative is N1-ABA-spermine [(azidobenzamidino)spermine], and the spermidine derivative is a mixture of N1- and N8-ABA-spermidine. ABA-spermine stabilizes nucleosome core particles in thermal denaturation experiments, with similar but not identical effects when compared with the parent polyamine, spermine. In circular dichroism experiments, ABA-spermine was capable of producing a B----Z transition in poly(dG-m5dC) at a concentration of 30 microM, compared with 5 microM required to produce the same effect with spermine. On the other hand, ANB-spermine [(azidonitrobenzoyl)spermine; Morgan, J. E., Calkins, C. C., & Matthews, H. R. (1989) Biochemistry 28, 5095-5106] stabilized the B form of poly(dG-br5dC). ABA-spermine is a potent inhibitor of ornithine decarboxylase from Escherichia coli, giving 50% inhibition at 0.12 mM, while ANB-spermine is a modest inhibitor, comparable to spermine or spermidine. Under conditions of nitrogen-limited growth, yeast take up ABA-spermine and ABA-spermidine at approximately one-third to half the rate of spermidine or spermine. In contrast, ANB-spermine was not significantly taken up. The photoaffinity polyamines were used to photoaffinity label the DNA in nucleosome core particles, and the sites of labeling were determined by exonuclease protection. All photoaffinity reagents showed both nonspecific labeling and specific sites of higher occupancy. However, the positions of the sites varied: the ANB-spermine sites confirmed those previously reported (Morgan et al., 1989); the ABA-spermine and ABA-spermidine sites were spaced at 9.8 bp intervals from the 3' end of each DNA strand. This observation, together with the effect of spermine on the circular dichroism of DNA in nucleosome core particles, implies that polyamines alter the helical twist of DNA in nucleosome core particles. The ABA-polyamines are offered as general-purpose photoaffinity polyamine reagents.     

Nonhistone nuclear high mobility group proteins 14 and 17 stabilize nucleosome core particles

Nucleosome core particles form well defined complexes with the nuclear nonhistone proteins HMG 14 or 17. The binding of HMG 14 or 17 to nucleosomes results in greater stability of the nucleosomal DNA as shown by circular dichroism and thermal denaturation. Under appropriate conditions the binding is cooperative, and cooperativity is ionic strength dependent. The specificity and cooperative transitions of high mobility group (HMG) binding are preserved in 1 M urea. Specificity is lost in 4 M urea. Thermal denaturation and circular dichroism show a dramatic reversal of the effects of urea on nucleosomes when HMG 14 or 17 is bound, indicating stabilization of the nucleosome by HMG proteins. Complexes formed between reconstructed nucleosomes containing purified inner histones plus poly(dA-dT) and HMG 14 or 17 demonstrate that the HMG binding site requires only DNA and histones. Electron microscopy reveals no major structural alterations in the nucleosome upon binding of HMG 14 or 17. Cross-linking the nucleosome extensively with formaldehyde under cooperative HMG binding conditions does not prevent the ionic strength-dependent shift to noncooperative binding. This suggests mechanisms other than internal nucleosome conformational changes may be involved in cooperative HMG binding.     

Interaction of polyamines with chromatin and DNA: formation of compact structures

We have used flow linear dichroism (LD) and light scattering at 90 degrees to study the condensation of both DNA and calf thymus chromatin induced by spermine, triamines NH3+(CH2)iNH+(CH2)jNH3+, designated as much less than i, j much greater than: much less than 3, 4 much greater than (spermidine), much less than 3, 3 much greater than, much less than 2, 3 much greater than, much less than 2, 2 much greater than; the diamines putrescine and cadaverine and MgCl2. It is found that the different polyamines affected DNA and chromatin in a similar way. The degree of compaction of the chromatin fibers induced by spermine, triamines except much less than 2, 2 much greater than and Mg2+ has been found to be identical. The triamine much less than 2, 2 much greater than and the diamines studied do not condense either chromatin of DNA. Such a big difference in the action of the triamines indicates that not only the charge, but also the structure of the polycations are important for their interactions with DNA and chromatin. The stoichiometry of polyamine binding to chromatin at which condensation occurred is found to be 2 polyamine molecules per DNA helical turn. Polyamines are supposed to bind to the exposed sites of core DNA every 10 b.p. The extent of DNA phosphate neutralization by the histones is estimated to be about 55%. It has been shown that a mixture of mono- and multivalent cations affected DNA and chromatin condensation competitively and not synergistically, as claimed in a recent report by Sen and Crothers.     

The divalent cations Ca2+ and Mg2+ play specific roles in stabilizing histone-DNA interactions within nucleosomes that are partially redundant with the core histone tail domains

We previously reported that reconstituted nucleosomes undergo sequence-dependent translational repositioning upon removal of the core histone tail domains under physiological conditions, indicating that the tails influence the choice of position. We report here that removal of the core histone tail domains increases the exposure of the DNA backbone in nucleosomes to hydroxyl radicals, a nonbiased chemical cleavage reagent, indicative of an increase in the motility of the DNA on the histone surface. Moreover, we demonstrate that the divalent cations Mg(2+) and Ca(2+) can replace the role of the tail domains with regard to stabilization of histone-DNA interactions within the nucleosome core and restrict repositioning of nucleosomes upon tail removal. However, when nucleosomes were incubated with Mg(2+) after tail removal, the original distribution of translational positions was not re-established, indicating that divalent cations increase the energy barrier between translational positions rather than altering the free energy differences between positions. Interestingly, other divalent cations such as Zn(2+), Fe(2+), Co(2+), and Mn(2+) had little or no effect on the stability of histone-DNA interactions within tailless nucleosomes. These results support the idea that specific binding sites for Mg(2+) and Ca(2+) ions exist within the nucleosome and play a critical role in nucleosome stability that is partially redundant with the core histone tail domains.     

Structural organization of the meiotic prophase chromatin in the rat testis

Pachytene nuclei were isolated from rat testes by the unit gravity sedimentation technique and contained histone variants H1a, H1t, TH2A, TH2B, and X2 in addition to the somatic histones H1bde, H1c, H2A, H2B, H3, and H4. The basic organization of the pachytene chromatin namely the nucleosome repeat length and the accessibility to micrococcal nuclease, was similar to that of rat liver interphase chromatin. However, when digested by DNase I, the susceptibility of pachytene chromatin was 25% more than liver chromatin under identical conditions. Nucleosome core particles were isolated from both liver and pachytene nuclei and were characterized for their DNA length and integrity of the nucleoprotein on low ionic strength nucleoprotein gels. While liver core particles contained all the somatic histones H2A, H2B, H3, and H4, in the pachytene core particles, histone variants TH2A, X2, and TH2B had replaced nearly 60% of the respective somatic histones. A comparison of the circular dichroism spectra obtained for pachytene and liver core particles indicated that the pachytene core particles were less compact than the liver core particles. Studies on the thermal denaturation properties of the two types of core particles revealed that the fraction of the pachytene core DNA melting at the premelting temperature region of 55-60 degrees C was significantly higher than that of the liver core DNA.     

Use of selectively trypsinized nucleosome core particles to analyze the role of the histone "tails" in the stabilization of the nucleosome

Using immobilized trypsin and an appropriate fractionation procedure, we have been able to prepare, for the first time, nucleosome core particles containing selectively trypsinized histone domains. The particles thus obtained: [(H3T-H4T)2-2(H2AT-H2BT)].DNA; [(H3-H4)2-2(H2AT-H2BT)].DNA; [H3T-H4T)2-2(H2A-H2B)].DNA (where T means trypsinized), together with the non-trypsinized controls have been characterized using the following techniques: analytical ultracentrifugation, circular dichroism, thermal denaturation and DNAse I digestion. The major aim of this study was to analyze the role of the amino-terminal regions (the histone "tails") on the stability of the nucleosome in solution. The data obtained from this analysis clearly show that stability of the nucleosome core particle to dissociation (below a salt concentration of 0.7 M-NaCl) is not affected by the presence or the absence of any of the N-terminal regions of the histones. Furthermore, these histone regions make very little contribution, if any, to the conformational transition that nucleosomes undergo in this range of salt concentrations. They play, however, a very important role in determining the thermal stability of the particle, as reflected in the dramatic alterations exhibited by the melting profiles upon selective removal of these tails by trypsinization. The melting data can be explained by a simple hypothesis that ascribes interaction of H2A/H2B and H3/H4 tails to particular regions of the nucleosomal DNA.     

Thermal transition of core particle is not a two-state process

Thermal transition of core particle which occurs before melting of DNA and can be followed by circular dichroism is not a two-state process; it is the result of two processes which cannot be dissociated in static experiments: unfolding of core particles is immediately followed by their aggregation. It is thus impossible to get thermodynamic parameters of core particle unfolding from its thermal transition monitored by circular dichroism. Thermal denaturation kinetics of core particles gives some information about their stability. Finally core particle structure is more stable in chromatin than in its isolated state.     

Analysis of chromatin reconstitutiion.

The ability of high molecular weight chicken erythrocyte chromatin to spontaneously self-assemble into native-like material, after dissociation by high ionic strength and reassociation by salt gradient dialysis, was critically examined. The native conformational state of the reassembled nucleoprotein complex was regenerated to the extent reflected by circular dichroism spectra and thermally induced helix--coil transition of the nucleoprotein DNA. However, internucleosomal packing of approximately 205 base pairs of DNA per repeating unit, as probed by digestion with micrococcal nuclease, was not regenerated upon reassembly and was replaced by a packing of approximately 160 base pairs per repeating unit. Thus, high molecular weight chromatin containing only lysine-rich histones (H1 and H5) and core histones (H2A, H2B, H3, and H4) is not a true self-assembling system in vitro using the salt gradient dialysis system used herein. Circular dichroism and thermal denaturation studies on core chromatin (lysine-rich histones removed) showed that core histones alone are not capable of reassembling high molecular weight DNA into native-like core particles at low temperature (4 degree C). Reassembly at 21 degree C restored the circular dichroism but not the thermal denaturation properties to those characteristic of undissociated core chromatin. Nonetheless, micrococcal nuclease digestions of both reassembled core chromatin products were identical with undissociated native core chromatin. Ressembly in the presence of the complete complement of histones, followed by removal of the lysine-rich histones, did regenerate the thermal denaturation properties of undissociated native core particles. These results indicated multiple functions of the lysine-rich histones in the in vitro assembly of high molecular weight chromatin.     

Interaction of the alternating double stranded copolymer poly(dA-dT) x poly(dA-dT) with NiCl(2) and CdCl(2): solution behavior

The thermal denaturation of the synthetic high molecular weight double stranded polynucleotide poly(dA-dT) x poly(dA-dT) has been studied in aqueous buffered solution (Tris 1.0 mM; pH 7.8+/-0.2) in the presence of increasing concentrations of either Ni(2+) (borderline cation) or Cd(2+) (soft cation) at four different constant ionic strength values (NaCl), making use of UV and circular dichroism (CD) spectroscopies. The experimental results show that the B-type double helix of the polymer is stabilized against thermal denaturation in the presence of both cations at low concentrations, relative to the systems where only NaCl is present, in the same conditions of ionic strength and pH. The effect is more pronounced for Ni(2+) than for Cd(2+). At higher concentrations, both cations start to destabilize the double helix, with Cd cations inducing larger variations of T(m). In many cases, when denaturation starts, interstrand cross-linking occurs with formation of aggregates that precipitate.     

Effect of DNA length and H4 acetylation on the thermal stability of reconstituted nucleosome particles

To examine the factors involved with nucleosome stability, we reconstituted nonacetylated particles containing various lengths (192, 162, and 152 base pairs) of DNA onto the Lytechinus variegatus nucleosome positioning sequence in the absence of linker histone. We characterized the particles and examined their thermal stability. DNA of less than chromatosome length (168 base pairs) produces particles with altered denaturation profiles, possibly caused by histone rearrangement in those core-like particles. We also examined the effects of tetra-acetylation of histone H4 on the thermal stability of reconstituted nucleosome particles. Tetra-acetylation of H4 reduces the nucleosome thermal stability by 0.8 degrees C as compared with nonacetylated particles. This difference is close to values published comparing bulk nonacetylated nucleosomes and core particles to ones enriched for core histone acetylation, suggesting that H4 acetylation has a dominant effect on nucleosome particle energetics.     

Polyamines permit the preparation of stable Physarum core particles which have a structure similar to those from higher eukaryotes

The inherent instability of Physarum nucleosome core particles prepared by micrococcal nuclease digestion in Na⁺/Ca²⁺ buffers can be overcome by the addition of 0.15 mM spermine and 0.5 mM spermidine. Neutron scattering, circular dichroism, nuclease digestion and thermal denaturation studies carried out on these stable monosomes show them to be very similar to those obtained from higher eukaryotes.     

Structural transition from the random coil to quadruplex of AG(3)(T(2)AG(3))(3) induced by Zn(2+)

Structures of G-quadruplex DNAs can be typically stabilized by monovalent cations such as K(+), Na(+). Some divalent and trivalent cations, such as Sr(2+), Pb(2+), Tb(3+) and Eu(3+), can also induce the formation of G-quadruplex DNA. Here we show that Zn(2+) can induce the human telomeric sequence AG(3)(T(2)AG(3))(3) to fold the G-quadruplex structure by UV absorbance difference spectra and circular dichroism (CD) spectroscopy. At micromolar concentrations, the Zn(2+)-induced changes in the UV absorbance difference spectra and CD spectra are the characteristics of antiparallel G-quadruplexes although the long wavelength CD maximum is around 285 nm rather than the typical value of 295 nm. The binding stoichometry of Zn(2+) per one AG(3)(T(2)AG(3))(3) molecule is four. To our knowledge, the structural transition of human telomeric sequence induced by Zn(2+) was observed for the first time.     

Interaction of rat testis protein, TP, with nucleosome core particle

Circular dichroism studies have revealed that addition of testis specific protein, TP in vitro, to rat testes nucleosome core particle resulted in a decrease in the compaction of the core particle DNA. This was also corroborated by thermal denaturation analysis. Addition of TP to nucleosome core particle resulted in the conversion of a biphasic transition towards a single phase. However, at the same time there was a 20% reduction in the overall hyperchromicity of core particle DNA at core particle to TP molar ratios of 1:2 and 1:3. These observations along with our earlier report, showing the DNA melting properties of TP, suggest that TP may play an important role in the disassembly process of nucleosome core particle during spermiogenesis.     

Effects of zinc and other metal ions on the stability and activity of Escherichia coli alkaline phosphatase

Measurement of the ultraviolet circular dichroism of apo-(alkaline phosphatase) in urea solutions showed substantial denaturation in 3m-urea. A zinc-deficient mutant alkaline phosphatase behaved similarly. The stability of the enzyme in 6m-urea was followed as a function of its zinc content and was found to be dependent on the first two of the four zinc atoms bound by apoenzyme. Phosphatase activity was mostly dependent on a second pair of zinc atoms. Mn(2+), Co(2+), Cu(2+) or Cd(2+) also restored structural stability. Sedimentation-velocity and -equilibrium experiments revealed that dissociation of the dimer accompanied apoenzyme denaturation in urea concentrations of 1m or higher, without treatment with disulphide-reducing agent.     

Stabilization of RNA tertiary structure by monovalent cations

The effects of monovalent cations (Li(+), Na(+), K(+), Rb(+), Cs(+), and NH4(+)) on the thermal stability of RNA tertiary structure were investigated by UV melting. We show that with the RNA used here (nucleotides 1051-1108 of Escherichia coli 23 S rRNA with four base substitutions), monovalent cations and Mg(2+) compete in stabilizing the RNA tertiary structure, and that the competition takes place between two boundaries: one where Mg(2+) concentration is zero and the other where it is maximally stabilizing ("saturating"). The pattern of competition is the same for all monovalent cations and depends on the cation's ability to displace Mg(2+) from the RNA, its ability to stabilize tertiary structure in the absence of Mg(2+), and its ability to stabilize tertiary structure at saturating Mg(2+) concentrations. The stabilizing ability of a monovalent cation depends on its unhydrated ionic radius, and at a low monovalent cation concentration and saturating Mg(2+), there is a (calculated) net release of a single monovalent cation/RNA molecule when tertiary structure is denatured. The implications are that under these conditions there is at least one binding site for monovalent cations on the RNA, the site is specifically associated with formation of stable tertiary structure, K(+) is the most effective of the tested cations, and Mg(2+) appears ineffective at this site. At high ionic strength, and in the absence of Mg(2+), stabilization of tertiary structure is still monovalent-cation specific and ionic-radius dependent, but a larger number of cations ( approximately eight) are released upon RNA tertiary structure denaturation, and NH(4)(+) appears to be the most effective cation in stabilizing tertiary structure under these conditions. In the majority of the experiments, methanol was added as a cosolvent to the buffer. Its use allowed the examination of the behavior of monovalent ions under conditions where their effects would otherwise have been too weak to be observed. Methanol stabilizes tertiary but not secondary structure of the RNA. There was no evidence that it either causes qualitative changes in cation-binding properties of the RNA or a change in the pattern of monovalent cation/Mg(2+) competition.     

Histone hyperacetylation: its effects on nucleosome conformation and stability

We have prepared nucleosome particles from HeLa cells that have been subjected to butyrate treatment. Fractions containing different levels of acetylation have been obtained within the range 7-17 acetyl groups per nucleosome. We have put special emphasis in the characterization of the particles with the highest level of histone acetylation. At low to physiological ionic strengths, these nucleosomes exhibit only small differences in hydrodynamic behavior and circular dichroism from control particles with minimal acetylation. There are, however, significant differences in thermal denaturation and nuclease sensitivity. In terms of stability toward high salt, the hyperacetylated and control particles behave identically. A model that reconciles these results is proposed. The major conclusion from our results, however, is that, at physiological ionic strength and in the absence of factors other than acetylation, the highly hyperacetylated nucleosomes remain essentially folded.     

DNA binding and cleavage properties of certain tetrammine ruthenium(II) complexes of modified 1,10-phenanthrolines--effect of hydrogen-bonding on DNA-binding affinity

A series of ruthenium(II) mixed ligand complexes of the type [Ru(NH(3))(4)(L)](2+), where L=imidazo[4,5-f][1,10]phenanthroline (ip), 2-phenylimidazo[4,5-f][1,10]phenanthroline (pip), 2-(2-hydroxyphenyl)imidazo[4,5-f][1,10]phenanthroline (hpip), 4,7-diphenyl-1,10-phenanthroline (dip), naphtha[2,3-a]dipyrido[3,2-h:2',3'-f]phenazine-5,18-dione (qdppz), 5,18-dihydroxynaphtho[2,3-a]dipyrido[3,2-H:2',3'-f]phenazine (hqdppz), have been isolated and characterized. The interaction of these complexes with calf thymus DNA (CT DNA) has been explored by using absorption, emission, and circular dichroic spectral methods, thermal denaturation studies and viscometry. All these studies suggest the involvement of the modified phenanthroline 'face' rather than the ammonia 'face' of the complexes in DNA binding. An intercalative mode of DNA binding, which involves the insertion of the modified phenanthroline ligands in between the base pairs, is suggested. The results from absorption spectral titration and circular dichroism (CD), thermal denaturation and viscosity experiments indicate that the qdppz and hqdppz complexes (K(b) approximately 10(6) and Delta T(m)=11-13 degrees C) bind more avidly than the ip, pip and hpip complexes (K(b) approximately 10(5), Delta T(m)=6-8 degrees C). Intramolecular hydrogen bonding in the hpip and hqdppz complexes increases the surface area of the intercalating diimines and enhances the DNA binding affinity substantially. The ammonia co-ligands of the complexes are possibly involved in hydrogen bonding with the intrastrand nucleobases to favour intercalation of the extended aromatic ligands. Circular dichroism spectral studies reveal that all the complexes effect certain structural changes on DNA duplex; [Ru(NH(3))(4)(ip)](2+) induces a B to A transition while [Ru(NH(3))(4)(qdppz)](2+) a B to Psi conformational change on CT DNA. Cleavage efficiency of the complexes were determined using pBR322 supercoiled plasmid DNA. All the complexes, except hqdppz complex, promote the cleavage of supercoiled plasmid (form I) to relaxed circular form (form II).     

The action of chymotrypsin on nucleosome cores. Histone products and conformational effects of limited digestion

alpha-Chymotrypsin was used to probe accessible hydrophobic amino acid residues in nucleosome cores. Small amounts of chymotrypsin rapidly and selectively cleaved at leucine 20 of histone H3. Cleavage at this site caused partial unfolding of the nucleosome core at low ionic strengths indicated by a small decrease in sedimentation coefficient and increase in circular dichroism in the 265-285-nm range. Unfolding did not occur at moderate ionic strengths, probably because of more effective electrolyte screening of residual negative charge on the nucleosome core. More extensive treatment with chymotrypsin partially degraded other core histones in nucleosome cores at similar rates. The primary sites of cleavage were assigned to Leu115 of H2a, Val18 or Gln22 of H2b, and Leu10 plus Leu22 of H4. We conclude that these primary sites of chymotrypsin cleavage of the four core histones lie on or near the nucleosome core surface, while the large number of other hydrophobic histone residues located in more central sequences must be inaccessible. Extensive chymotrypsin treatment yielded a set of "limit" products approximately 80-100 residues long that were similar to the limit products of trypsin digestion. Sedimentation coefficients and circular dichroism spectra of nucleosome cores treated to near limits with chymotrypsin or chymotrypsin followed by trypsin were not consistent with significant unfolding of the proteolyzed cores at moderate ionic strength. These results indicate that the amino-terminal 20-30 residues of H2b, H3, and H4 and the amino- and carboxyl-terminal approximately 12 residues of H2a, in toto, interact weakly if at all with DNA of isolated nucleosome cores. These histone termini stabilize less than two turns and perhaps only one turn on each DNA terminus.