Urea perturbation and the reversibility of nucleohistone conformation

Urea effect on conformation and thermal stabilities in nucleohistone and NaCl-treated partially dehistonized nucleohistones has been studied by circular dichroism (CD) and thermal denaturation. Urea imposes a CD change at 278mm of DNA base pairs in native and NaCl-treated nucleohistones which can be decomposed into two parts: a decrease in Δε₂₇₈ for histone-free base pairs and an increase for histone-bound base pairs. The reduction by urea of Δε₂₂₀ of bound histones is approximately proportional to the increase of Δε₂₇₈ of histone-bound base pairs. Urea also lowers the melting temperatures of base pairs both free and bound by histones. The presence of urea indeed destroys the secondary structure of bound histones, causing changes in the conformation and thermal stabilities of histone-bound base pairs in nucleohistone. Such a urea perturbation on nucleohistone conformation is reversible.     

Circular dichroism of histone-bound regions in chromatin.

Native, NaCl-treated, trypsin-treated, and polylysine-bound nucleohistones were studied in 2.5 × 10^?4 M EDTA, pH 8.0, using circular dichroism (CD) and thermal denaturation. Removal of histone I by 0.6 M NaCl has a much smaller effect on both Δε₂₂₀ and Δε₂₇₈ than the removal of other histones. This indicates that histone I has less helical content and less conformational effect on the DNA in nucleohistone. By extrapolating to 100% binding by histones other than I, the positive CD band near 275 nm is close to zero. Comparison is also made between the effects of binding by the more basic and the less basic halves of histones by trypsin-digestion and polylysine-binding experiments. Trypsin digestion of nucleohistone reduces melting band IV at 82°C much more than melting band III at 72°C. However, the CD changes of Δε₂₇₈ and Δε₂₂₀ induced by trypsin digestion are small, unless melting band III is also reduced by the use of a higher trypsin level. This implies that the less basic halves of histones, which stabilize DNA to 72°C (melting band III), have more helical structure and are more responsible for conformational change in DNA than are the more basic halves, which stabilize DNA to 82°C (melting band IV). Polylysine binding to nucleohistone diminishes melting band III but has no effect on melting band IV. This binding affects only slightly the Δε₂₂₀ of nucleohistone, indicating that polylysine interferes very little with the structure of the less basic halves of bound histones. The implications of these studies with respect to chromatin structure are discussed.     

Helix–coil transition in nucleoprotein—theory and applications

A general theory of helix–coil transition of irreversibly complexed nucleoproteins is presented. The equations are tested by experimental results in basic polypeptide–DNA complexes, nucleohistone I and pea bud nucleohistones. They show good agreement between theory and experiments. The theory provides direct measurement of a fraction of DNA base pairs covered by proteins, yielding a value of about 75% histone-covered base pairs in pea bud nucleohistone. It also provides a measurement of an average number of amino acid residues per nucleotide in protein-bound regions. This number varies from 1.0 to 1.4 in DNA–polylysine or DNA–polyarginine and from 2.9 to 3.3 in nucleohistone Ia, Ib, f1, and pea bud nucleohistone.     

Studies on interaction between histone V (f2c) and deoxyribonucleic acids.

Histone V (2fc) from chick erythroctes was used in the study of its interaction with DNA from various sources. Complexes between this histone and DNA were formed using the procedure of continuous NaCl gradient dialysis in urea. Two physical methods, namely thermal denaturation and circular dichroism (CD), were used as analytical tools. Thermal denaturation of nucleohistone V with chick or calf thymus DNA shows three melting bands: band I at 45-50 degrees corresponds to free base pairs; band II at 75-79 degrees, and band III at 90-93 degrees correspond to histone-bound base pairs. In histone-bound regions, there are 1.5 amino acid residues/nucleotide in nucleohistone V. In contrast, a value between 2.9 and 3.3 was determined for nucleohistone I (fl) (H. J. Li (1973), Biopolymers 12, 287). Similar melting properties have been observed for histone V complexed with bacterial DNA from Micrococcus luteus. Histone V binding to DNA induces a slight transition from a B-type CD spectrum to a C-type spectrum. Trypsin treatment of nucleohistone V reduces melting band III much more effectively than band II. Such a treatment also restores DNA to B conformation in the free state. Reduction of the melting bands of nucleohistone V by polylysine binding follows the order of I greater than II greater than III, accompanied by the increase of a new band at 100 degrees. When two bacterial DNAs of varied A + T (adenine + thymine) content simultaneously compete for the binding of histone V, the more (A " T)-rich DNA is selectively favored. Under experimental conditions described here, Clostridium perfringens DNA with 69% A + T is bound by histone V in preference to chicken DNA with 56% A + T although the latter has natural sequences for histone V binding.     

Thermal denaturation of nucleohistones--effects of formaldehyde reaction

Thermal denaturation of native or partially dehistonized nucleohistones shows two melting bands at 66 and 81° in 2.5 × 10^?4 M EDTA, pH 8.0. These correspond to the melting of DNA segments bound by the less basic and the more basic half-molecules of histones, respectively. These two melting bands combine into a broad melting band from around 70 to 85° when these nucleohistones are pre-treated with formaldehyde. A formaldehyde reaction which fixes histones on DNA by covalent bonds account for the effect. Formaldehyde fixation also increases the melting temperature of some free DNA segments from around 42 to around 55°. This is interpreted as a result of closed or rigid boundaries between free DNA and formaldehyde-reacted histone-bound DNA segments. MgCl₂ dissociates histones from DNA more effectively and leaves longer free DNA segments than does NaCl. Thermal denaturation of a formaldehyde-reacted nucleoprotein thus provides an effective tool for comparing the relative size of free DNA regions on nucleoproteins. The effect of reversible binding of ligands on helix-coil transition of DNA is descussed and found not adequate for thermal denaturation of nucleohistones.     

Helix-coil transition in nucleoprotein. Effect of ionic strength on thermal denaturation of polylysine-DNA complexes

Thermal denaturation of direct-mixed and reconstituted polylysine–DNA complexes in 2.5 × 10^?4 M EDTA, pH 8.0 and various concentrations of NaCl has been studied. For both complexes, increasing ionic strength of the solution raises T_m, the melting temperature of free base pairs. The linear dependence of T_m on log Na⁺ indicates that the concept of electrostatic shielding on phosphate lattice of an infinitely long pure DNA by Na⁺ can be applied to short free DNA segments in a nucleoprotein. For a direct-mixed polylysine–DNA complex, the melting temperature of bound base pairs T_m′ remains constant at various ionic strengths. On the other hand, the T_m′ in a reconstituted polylysine–DNA complex is shifted to lower temperature at higher ionic strength. This phenomenon occurs for reconstituted complex with long polylysine of one thousand residues or short polylysine of one hundred residues. It is shown that such a decrease of T_m′ is not due to a reduction of coupling melting between free and bound regions in a complex when the ionic strength is raised. It is also not due to intermolecular or intramolecular change from a reconstituted to a direct-mixed complex. It is suggested that this phenomenon is due to structural change on polylysine-bound regions by ionic strength. It is suggested further that Na⁺ may replace water molecules and bind polylysine-bound regions in a reconstituted complex. Such a dehydration effect destabilizes these regions and lowers T_m′. This explanation is supported by circular dichroism (CD) results.     

Circular dichroism as a probe of DNA structure inside reconstituted nucleohistones.

Reconstituted nucleohistones were obtained by mixing in given conditions acid extracted histones and eukaryotic DNA. The histone/DNA ratio (w/w) was in the range 0.35 - 0.95. With the four histones (H2A2B) we have been able to obtain subunits (nucleosomes or upsilon-bodies). The variation of cirsular dichroism signal with temperature at 280 nm was measured to follow structural changes of the DNA inside the complex. The true change of ellipticity (see article) of histone-bound DNA regions, is similar for reconstituted nucleohistone and H1-depleted chromatin, and is therefore a physical probe of the presence of nucleosomes.     

Poly(L–lysine)–DNA interactions in NaCl solutions: B → C and B → ψ Transitions

Thermal denaturation and circular dichroism (CD) properties of poly(L -lysine)–DNA complexes vary greatly when these complexes are prepared differently, that is, whether by NaCl-gradient dialysis starting from 2.0 M NaCl or by direct mixing at low salt. These differing properties were investigated in more detail by examining complexes, made by direct mixing in the presence of various concentrations of NaCl, both before and after the NaCl was dialyzed out of the complex solution. The precipitation curves of DNA due to polylysine binding indicate that such binding is noncooperative at zero salt; from 0.1 up to 1.0 M NaCl they exhibit varying degrees of cooperatively. Starting from zero salt, as the NaCl concentration used for complex formation is increased, both the CD and the melting properties of the complexes are shifted from those of directly mixed at zero salt to those of reconstitution: in the CD spectra there is a gradual shift from a B → C transition to a B → ψ transition; thermal denaturation results show a gradual increase in the melting temperatures of both free DNA (t_m) and polylysine-bound DNA (t′_m). The progressive shift from B → C to B → ψ suggests a close relationship between these two transitions. Large aggregates of the complexes do not warrant the appearance of ψ-type CD spectra: ψ-spectra have been obtained in the supernatants of polylysine–DNA complexes made and measured at 1.0 M NaCl while slightly perturbed CD spectra in B → C transition have been observed in turbid solutions of fully covered complexes made at very low salt. If the complexes are made at intermediate salts and dialyzed to a very low salt, although up to 60% of the DNA is still bound by polylysine, the CD spectra of the complexes are shifted back to the B-type CD characteristic of pure DNA.     

Investigation of huge negative circular dichroism spectra of some nucleoproteins

Under certain conditions of preparation, DNA, whether free or complexed with polylysine or histone KAP (I, fl), produce huge negative circular dichroism (CD) spectra with maxima at about 270nm. In order to investigate the cause of these spectra, reconstituted polylysine-DNA complex was used as a model system. It was found that the CD change of DNA in the complex is not a linear function of the fraction of base pairs bound. Such a CD spectrum is not changed despite dilution up to 128 folds for as long as 12 hours. Difference CD spectra taken between free DNA and any of the complexes are qualitatively the same, and are similar to those of free DNA and nucleohistone KAP (Fasman et al., Biochemistry 9, 2814-2822, 1970), free DNA and direct mixed polylysine-DNA complexes, or free DNA in high salt (Chang et al., Biochemistry12, 3028-3032, 1973). The suggestion is made that this CD spectrum might be caused by specific conformational changes in DNA, perhaps belonging to the family of B to C transitions followed by a further structural distortion of DNA due to aggregation of the nucleoprotein molecules.     

Protein-DNA crosslinking in reconstituted nucleohistone, nuclei and whole cells by picosecond UV laser irradiation.

A picosecond UV laser was used to cross-link proteins to DNA in nuclei, whole cells and reconstituted nucleohistone. Irradiation of the nucleohistone resulted in crosslinking 15-20% of bound histones to DNA in a very short time (one or several picosecond pulses), the efficiency of crosslinking to single stranded DNA being higher than to double stranded DNA. All histones as well as high mobility group 1 proteins were identified in the covalently linked protein-DNA complexes upon irradiation of isolated nuclei and whole cells. A method is suggested for isolation of crosslinked material from cells and nuclei in amounts sufficient for further analysis. Experiments with reconstituted nucleohistones showed that upon irradiation at a constant dose the efficiency of crosslinking depended on the intensity of the light, thus suggesting a two-quantum process is involved in the reaction.     

Localization of chromatin proteins within DNA grooves by methylation of chromatin with dimethyl sulphate

The use of the comparative modification with ³H-dimethyl sulphate (DMS) of free DNA and DNA in different complexes is proposed to evaluate the shielding of the minor and major grooves of the DNA double helix and to determine the presence of single-stranded DNA in the complexes.Glucosyl groups in DNA of T6 phage protect, as expected, the major groove, and actinomycin d in its complex with DNA shields the minor groove against methylation with DMS.The data obtained suggest that histones and protamine in reconstituted nucleohistone and nucleoprotamine are allocated within partly the major groove leaving the minor groove open, while polylysine does not seem to be buried within either of the grooves, and cations of cetyltrimethylammonium lie within the minor groove of DNA.     

Studies on poly(L-lysine50, L-tyrosine50)-DNA interaction

Interaction between poly(Lys⁵⁰, Tyr⁵⁰) and DNA has been studied by absorption, circular dichroism (CD), and fluorescence spectroscopy and thermal denaturation in 0.001M Tris, pH 6.8. The binding of this copolypeptide to DNA results in an absorbance enhancement and fluorescence quenching on tyrosine. There is also an increase in the tyrosine CD at 230 nm. The CD of DNA above 250 nm is slightly shifted to the longer wavelength which is qualitatively similar to, but quantitatively much smaller than, that induced by polylysine binding. At physiological pH the poly(Lys⁵⁰, Tyr⁵⁰)–DNA complex is soluble until there is one lysine and one tyrosine per nucleotide in the complex. The same ratio of amino acid residues to nucleotide has also been observed in copolypeptide-bound regions of the complex. The addition of more poly(Lys⁵⁰, Tyr⁵⁰) to DNA yields a constant melting temperature, T_m′, for bound base pairs at 90°C which is close to that of polylysine-bound DNA under the same condition. The melting temperature, T_m, of free base pairs at about 60°C on the other hand, is increased by 10°C as more copolypeptide is bound to DNA. As the temperature is raised, both absorption and CD spectra of the complexes with high coverage are changed, suggesting structural alteration, perhaps deprotonation, on bound tyrosine. The results in this report also suggest that intercalation of tyrosine in DNA is unlikely to be the mode of binding.     

Circular dichroism of native and reconstituted nucleohistones

Circular dichroism (CD) spectra of histone, DNA and native, partial and reconstituted nucleohistone complexes in 0.01 M Tris buffer in which nucleohistone complexes appear to be intact and in 2 M NaCl, in which complexes are mostly dissociated, have been compared. The CD spectra in the longwave region, where the protein contribution is negigible, reveal differences in the spectra for DNA in nucleohistone complexes as compared with that of free DNA, the difference being roughly proportional to the protein DNA ratio. An analysis of the shape of the first positive CD maximum suggests that we are probably dealing here with slight conformational changes on DNA. Minor differences between the behavior of native and reconstituted complexes may be due to the effect of different conformational states of the protein component.     

One hundred-fold acceleration of DNA renaturation rates in solution

Solvents which accelerate DNA renaturation rates have been investigated. Addition of NaCl or LiCl to DNA in 2.4M Et₄NCl initially increases renaturation rates at 45°C and then leads to a loss of second-order behavior. The greatest accelerations are seen with LiCl and dilute DNA. Volume exclusion by dextran sulfate is the most effective method of accelerating DNA renaturation with concentrated DNA. Addition of dextran sulfate beyond 10–12% in 2.4M Et₄NCl fails to increase the acceleration beyond approximately 10-fold. Accelerations of 100-fold may be achieved with 35–40% dextran sulfate in 1M NaCl at 70°C. No other mixed solvent system was found to be more effective, although acceleration may be achieved in solvents containing formamide or other denaturants. The acceleration in 2M NaCl occurs without loss of the normal concentration and temperature dependence of DNA renaturation and is also independent of dextran sulfate concentration if sufficient dextran sulfate is used. Dextran sulfate may be selectively precipitated by use of 1M CsCl.     

Conformation and properties of DNA–(Lys33, Leu67)100–(Orn)20 complex: A nucleohistone model

The synthesis and characterization of the block copolypeptide (Leu⁶⁷, Lys³³)₁₀₀Orn₂₀, a synthetic model of histone, are reported. In neutral aqueous solutions, 80% of the etheropolypeptide block assumes an α-helical conformation, whereas the polyornithine block is in a random-coil conformation. In the association complexes with DNA, melting and titration experiments, as well as CD results, indicate that the polyornithine block interacts with DNA, whereas at least 2/3 of the lysine residues of the (Leu, Lys) moiety are excluded from the direct binding with DNA. CD spectra of the association complexes reveal significant differences from those obtained with DNA–polyornithine and DNA–polylysine complexes but substantial similarities with CD spectra of native and reconstituted nucleohistones. In contrast to DNA–polyornithine complexes, the CD spectra of the ternary complexes, copolypeptide–DNA–ethidium bromide, indicate a strong reduction of the dye intercalation. The low-angle x-ray diffraction pattern, reminiscent of that of chromatin, reveals the presence of a superstructure in these complexes. The results obtained are discussed in connection with the expected structural features of the model.     

The assembly of an H2A2,H2B2,H3,H4 hexamer onto DNA under conditions of physiological ionic strength

A novel nucleohistone particle is generated in high yield when a complex of DNA with the four core histones formed under conditions that are close to physiological (0.15 M NaCl, pH 8) is treated with micrococcal nuclease. The particle was found to contain 102 base pairs of DNA in association with six molecules of histones in the ratio 2H2A:2H2B:1H3:1H4 after relatively brief nuclease treatment. Prolonged nuclease digestion resulted in a reduction in the DNA length to a sharply defined 92-base pair fragment that was resistant to further degradation. Apparently normal nucleosome core particles containing two molecules each of the four core histones in association with 145 base pairs of DNA and a particle containing one molecule each of histones H2A and H2B in association with approximately 40 base pairs of DNA were also generated during nuclease treatment of the histone-DNA complexes formed under physiological ionic strength conditions. Kinetic studies have shown that the hexamer particle is not a subnucleosomal fragment produced by the degradation of nucleosome core particles. Furthermore, the hexamer particle was not found among the products of nuclease digestion when histones and DNA were previously assembled in 0.6 M NaCl. The high sedimentation coefficient of the hexameric complex (8 S) suggests that the DNA component of the particle has a folded conformation.     

A comparison of the interactions of proflavine with DNA and with deoxyribonucleohistone using circular dichroism spectroscopy

Complexes of proflavine with DNA and deoxyribonucleohistone from calf thymus show different optical activity in the visible and the ultraviolet. Although the visible CD spectra of both complexes arise from interactions of dye molecules, the variation in the optical activity with the amount of dye bound suggests that a lesser conformational mobility exists in DNH. This is confirmed by the ultraviolet CD spectra of the complexes, and it is suggested that the conformation of DNA within nucleohistone is altered by separation of the base pairs by a greater extent than occurs in DNA in free solution. Even if the protein is unequally distributed along the DNA, the conformation of all of the DNA is altered by its incorporation into the nucleoprotein complex, since no evidence could be detected to show that DNA in a “free” conformation existed.     

Studies on interaction between poly(L-lysine) and DNA of varied G + C contents

Interaction between polylysine and DNA's of varied G + C contents was studied using thermal denaturation and circular dichroism (CD). For each complex there is one melting band at a lower temperature t_m, corresponding to the helix–coil transition of free base pairs, and another band at a higher temperature t′_m, corresponding to the transition of polylysine-bound base pairs. For free base pairs, with natural DNA's and poly(dA-dT) a linear relation is observed between the t_m and the G + C content of the particular DNA used. This is not true with poly(dG)·poly(dC), which has a t_m about 20°C lower than the extrapolated value for DNA of 100% G + C. For polylysine-bound base pairs, a linear relation is also observed between the t′_m and the G + C content of natural DNA's but neither poly(dA-dT) nor poly(dG)·poly(dC) complexes follow this relationship. The dependence of melting temperature on composition, expressed as dt_m/dX_G·C, where X_G·C is the fraction of G·C pairs, is 60°C for free base pairs and only 21°C for polylysine-bound base pairs. This reduction in compositional dependence of T_m is similar to that observed for pure DNA in high ionic strength. Although the t′_m of polylysine-poly(dA-dT) is 9°C lower than the extrapolated value for 0% G + C in EDTA buffer, it is independent of ionic strength in the medium and is equal to the t_m⁰ extrapolated from the linear plot of t_m against log Na⁺. There is also a noticeable similarity in the CD spectra of polylysine· and polyarginine·DNA complexes, except for complexes with poly(dA-dT). The calculated CD spectrum of polylysine-bound poly(dA-dT) is substantially different from that of polyarginine-bound poly(dA-dT).     

Thermal stability and renaturation of DNA in dimethyl sulfoxide solutions: Acceleration of the renaturation rate

The thermal stability and renaturation kinetics of DNA have been studied as a function of dimethyl sulfoxide (DMSO) concentration. Increasing the concentration of DMSO lowers the melting temperature of DNA but results in an increased second-order renaturation rate. For example, in a DNA solution containing 0.20M NaCl, 0.01M Tris (pH 8.0), and 0.001M EDTA, the addition of 40% DMSO lowers the melting temperature of the DNA by 27°C and approximately doubles the optimal renaturation rate. The effect of DMSO on the renaturation rate is shown to be at least partially due to its effect on the solution dielectric constant and to be consistent with the polyelectrolyte counterion condensation theory of Manning [(1976) Biopolymers 15 , 1333–1343].