The Structure of Nucleohistone

Abstract

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...

Covalent cross‐linking of histones in chromatin

“…The formation of discrete high mol. wt polymers in 0.7 and 1.2 M NaCl suggests that long-range conformational changes (which result from the flexible, polyelectrolyte behaviour of 1.2 DNH [6]) bring the histone dimers into close association and enable extensive cross-linking to occur. The very high mol.…”

The Modification of Deoxyribonucleohistone by Trypsin and Chymotrypsin

“…Cleavage of f 3 is accompanied by a loss in the rigid rod-like properties of the molecule which have been associated with the native supercoiled structure [5]. A more flexible structure is formed which is nevertheless still more compact, hydrodynamically, than DNA.…”

“…The evidence is based largely on studies of fibres in the solid state using X-ray diffraction [l] and electron microscopy [2] and solution studies using low-angle X-ray diffraction [3,4]. The hydrodynamic properties of nucleohistone show that a compact, rigid conformation, identified with the supercoiled structure, also exists in dilute solution [5,6].The folding of the linear complex of DNA and histone into a coiled-coil conformation means that the nucleic acid may be partially embedded in the middle of a protein matrix [5] thus giving rise to a structure in which all or part of any one histone fraction may be out of contact with the aqueous environment and thus not accessible t o probes such as enzymes.I n order to gain information about the gross topology of the supercoil in terms of the location of the different histones in relation to the surface we have followed the action of trypsin and chymotrypsin on the histones when bound in the native complex and compared their behaviour to free histones. Cleavage of peptide bonds has been monitored using polyacrylamide gel electrophoresis and changes in secondary structure have been followed using circular dichroism.…”

“…The evidence is based largely on studies of fibres in the solid state using X-ray diffraction [l] and electron microscopy [2] and solution studies using low-angle X-ray diffraction [3,4]. The hydrodynamic properties of nucleohistone show that a compact, rigid conformation, identified with the supercoiled structure, also exists in dilute solution [5,6].…”

“…The folding of the linear complex of DNA and histone into a coiled-coil conformation means that the nucleic acid may be partially embedded in the middle of a protein matrix [5] thus giving rise to a structure in which all or part of any one histone fraction may be out of contact with the aqueous environment and thus not accessible t o probes such as enzymes.…”