The circular dichroism of calf thymus deoxyribonucleohistone was compared with that of samples partially depleted of histone in solutions of sodium chloride. Differences in the near ultraviolet circular dichroism of DNA and deoxyribonucleohistone were associated with the presence of the f2b, f2a2 and f 3 histone fractions in the complex. The near ultraviolet circular dichroic spectra of both DNA and deoxyribonucleohistone were very sensitive to the solvent environment. Thus differences in the circular dichroic spectra of DNA and deoxyribonucleohistone cannot necessarily be associated with long-range effects, such as supercoiling, but may be due to short-range effects, such as might be caused by histone-DNA interactions.The absorbance and the circular dichroic melting profiles of deoxyribonucleohistone were biphasic. A new dichroic species was formed during the melting of the first of these steps and evidence is presented that this is a new histone-DNA complex produced by the migration of some or all of the fractions f2b, f2a2 and f 3 from denatured t o native regions of the DNA during the melting process. Thus the biphasic melting profile of deoxyribonucleohistone does not necessarily imply that these fractions are arranged heterogeneously on the DNA.Studies on the hydrodynamic properties of nucleohistone have shown that certain histone fractions maintain nucleohistone in a more compact conformation than DNA [l]. It has been suggested that this conformation is a supercoil [2]. Marked differences are also found in the optical rotatory dispersion and circular dichroic spectra of DNA and nucleohistone [3-51. I n order to see whether the histone fractions reponsible for the characteristic difference in hydrodynamic properties were also responsible for the difference in optical properties, we have investigated the circular dichroic spectra of nucleohistone partially depleted of specific histone fractions by salt.The histone fractions which are implicated in the supercoiling of nucleohistone also produce biphasic melting profiles in the complex [1,6]. Therefore we have studied the circular dichroism of native and partially depleted nucleohistone as a function of temperature in order to gain further information about the contribution of specific histone fractions to the conformation of the native nucleoprotein. Apreliminary account of this work has been published [7]. EXPERIMENTAL PROCEDUREThe preparation, characterisation and experimental methods, other than circular dichroism, have been described previously [11. The solvent used in all cases was 0.7 mM sodium phosphate, p H 6.8, except where otherwise statcd. Deoxyribonucleohistone samples that have been partially depleted of histone will be identified by the molarity of the sodium chloride solution used for the depletion. Thus a sample depleted in 0.7 M NaCl is designated 0.7 deoxyribonucleohistone. The extinction coefficients for the heat-treated samples were obtained by phosphorus analysis [S]. Circular dichroic spectra were measured in a Jouan Dicrographe Model C...
Calf thymus deoxyribonucleohistone was partially depleted of histone in salt solutions and the viscosity, sedimentation and thermal denaturation properties of the depleted samples were measured. The histones removed were characterised by polyacrylamide gel electrophoresis.Both the viscosity and sedimentation studies show DNA to be considerably more asymmetric than nucleohistone. This increase in asymmetry has been shown to be associated with the removal of the f2b, f2a2 and f3 fractions from the nucleohistone. It is concluded that a t least one of these fractions maintains the DNA in a supercoil in the native nucleohistone.All the histone fractions stabilised the DNA to thermal denaturation. A biphasic melting profile was observed for native deoxyribonucleohistone, which became monophasic on the removal of the same histone fractions as those implicated in supercoiling.It has been well established that sodium chloride a t concentrations increasing from about 0.5 M cause deoxyribonucleohistone to dissociate progressively into DNA and histone [l-41. Attempts to separate the dissociated protein from the depleted nucleohistone have been made using Sephadex column chromatography [5,6] and sedimentation [7] or combinations of both methods [8]. We have obtained complete separation of the dissociated histone from the nucleoprotein, using agarose beads as a gel filtration medium. This separation, which is similar to one recently described by Loeb [9], is described in the following together with studies of the viscosity, sedimentation and thermal denaturation of the partially depleted nucleohistone complex. The hydrodynamic behaviour of whole nucleohistone implies that the molecule is more compact than DNA [10] and it has recently been suggested that this greater compactness is due to supercoiling of the nucleohistone molecule [ 1 I]. We have examined the hydrodynamic properties of partially depleted nucleohistone in order to see whether any particular fraction can be implicated in the supercoiling ofthe native complex. Histone has been shown to stabilise DNA towards thermal denaturation [12,13], an effect which has been interpreted in terms of the decrease in net electrostatic charge of the DNA when complexed to histone [14]. I n the following, the thermal denaturation behaviour of the partially deproteinised nucleohistones is studied as a possible source of information concerning the arrangement of the various histone fractions along the DNA backbone. A preliminary account of this work has been published [15]. EXPERIMENTAL PROCEDURE Preparation of Deoxyribonucleohistone and of D N ADeoxyribonucleohistone was prepared from calf thymus by the method of Zubay and Doty [lo], and then dialysed against 0.7 mM sodium phosphate, p H 6.8. DNA was prepared as described previously P61.Partial Dissociation and Removal of Protein from Deoxyribonucleohistone by Agarose gel Chromatography Deoxyribonucleohistone (15 ml) was dialysed a t room temperature for 3 hours against large volumes of sodium chloride (0.5-1.0 M). Aliquots (10 ml) o...
The viscosity of histone-depleted nucleohistone has been measured a t high and low ionic strength. Native nucleohistone a t low ionic strength and f l -depleted nucleohistone a t both low and high ionic strength all have the same intrinsic viscosity of 10.0 dl/g. This implies that these molecules are not flexible but behave in solution as rigid particles. On the assumption that they are rods they have a weight-average length of 1160 f 120 nm and diameter 5.6 & 0.3 nm.The DNA is compressed in the rod to give an increase in mass per unit length for the DNA of 5.0 0.5.By contrast the intrinsic viscosity of nucleohistone depleted of f l , f2a2 and 75O/, of F2b and F3, varies with ionic strength in the manner typical of a flexible polyelectrolyte. The shapes of the sedimentation boundaries measured at high ionic strength of depleted nucleohistone show that the dissociation of histone from DNA takes place so that all molecules are depleted to the same extent. It thus has a conformation intermediate between that of native nucleohistone and DNA.Polyacrylamide gel electrophoresis of histones removed by 1 .O M NaCl using a technique capable of resolving all five main histone fractions, shows that if one fraction only is responsible for supercoiling nucleohistone it must be f2a2.There is some evidence that in fibres of nucleohistone the DNA is held in the form of a compact supercoil by the histones [l]. The hydrodynamic properties of nucleohistone suggest that the compact conformation is also present in solutions of the complex as both the viscosity and the frictional coefficient of nucleohistone increased sharply as histone was dissociated in salt [3]. These changes were interpreted as an increase in asymmetry of the molecule resulting from the loss of the supercoiled structure. However, the hydrodynamic studies were carried out in solutions of low ionic strength and it can be argued that the increased electrostatic charge on the complex consequent on the removal of histones could give rise to polyelectrolyte effects which might account €or all the changes observed. I n this study we present viscosity data measured a t high ionic strength where polyelectrolyte effects are negligible. New data on the selectivity of the salt dissociation of histones, together with the viscosity observations a t high ionic strength have enabled us to make a more complete interpretation of the properties of the nucleohistone molecule in solution. EXPERIMENTAL PROCEDUREThe preparation and characterisation of deoxyribonucleohistone and deoxyribonucleohistone partially depleted of histone have been described else-1 Eur. 3. Biochem.. Vol.22 where [3]. Histone-depleted samples are referred to by the concentration of NaCl used to dissociate the protein. Thus 0.6 deoxyribonucleohistone has been depleted in 0.6 M NaCl. Dissociated histone was separated from the nucleohistone complex using gel exclusion chromatography. Relative viscosities and sedimentation coefficients were measured as described previously [3]. The procedure for polyacrylamide gel...
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