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Title: Studies on the structure of deoxyribonucleohistone
Author: Skidmore, Christopher J.
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 1973
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The chromatin of eukaryotic nuclei can be separated into two classes on morphological grounds. The heterochromatin is genetically inert and remains condensed while the euchromatin contains the genetically active DNA. Changes in the ionic strength of the medium can affect the balance between these two classes. The structure of chromatin in the electron microscope is also affected by ionic changes. The ionic composition of the medium is critical for the structure of extracted nucleohistone. Increasing ionic strength will first precipitate the DNH and then dissociate the histone from the complex. Nucleohistone was extracted from calf thymus glands by a standard procedure. The final product had a protein:DNA weight ratio of 1.30. It contained a full complement of histones. Fluorescence analysis of the DNH showed that there was little or no tryptophan present - denoting the absence of non-histone proteins. A protease activity was noted in the preparation which was responsible for its eventual degradation. f1 histone was completely digested from the complex in 4-6 weeks. During the study of the effects of neutral salts on the DNH, several physicochemical methods were used to detect changes in the various structural levels of the complex. Polyacrylamide gel electrophoresis analysed the complement of histones present, circular dichroism examined the secondary structure of the DNA and hydrodynamic methods observed changes in the shape of the molecule. Controlled proteolysis was used to examine the arrangement of the histones on the surface of the complex. The dissociation of histone from DNH by salt was investigated by separating the products of the dissociation on a column of agarose gel. No equilibrium method was available for studying the extent of dissociation without separating the components. It was assumed that the equilibrium was not disturbed to any great extent by passage down the column. The dissociation curve - the plot of amount of protein dissociated against salt concentration - varied from preparation to preparation. However, the main features remained constant. The first histone fraction to be dissociated was f1 at 0.4-0.6M sodium chloride - this dissociation correlates with the return to solution of the DNH. No further protein was removed until at greater than 0.7M salt, f2a2, f3b and f3 were dissociated together in a very narrow salt range. The position of this dissociation on the salt axis was variable. Finally f2a1 was removed in a discrete step at around 1.2M sodium chloride. Removal of all the protein from DNH was accomplished in 2M salt. If a 2M salt sample was dialysed back to a lower salt concentration then a nucleohistone was obtained which was identical with a-nucleohistone obtained by depleting the DNH at that salt concentration. This reversibility of the dissociation is held to argue for the view that DNH is the stable form of the DNA-histone system. Some workers have argued that DNH is metastable. Some characteristics of the f1-depleted nucleohistone were investigated. It has a lower andDelta;andepsilon;275 than DNH and a higher s20. Fibres drawn from this DNH give the low-angle X-ray diffraction pattern described as the 'supercoil rings'. This degree of structure is also visible in a depleted nucleohistone containing only f2a1 and DNA. If only one histone is responsible for this 'supercoiled' structure then it is f2a1. This is in contrast with other reports that the compact hydrodynamic structure of DNH is dependent on f2a2 binding. The two structures are not identical. After removal of f1, the CD of DNH increases at both 275 and 225nm, linearly with protein removal, until a protein-free DNA is obtained. The structure represented andDelta;andepsilon;275 is dependent non-specifically on binding of protein to DNA. Analysis of the CD spectra of depleted nucleohistones below 250nm using reference spectra derived from proteins of known structures shows that the extent of structure in the histone moiety of DNH does not vary from histone to histone. There is approximately 40% α-helix, 10% β-structure and 50% unordered residues. This estimate agrees well with other studies using different methods. The dissociation of protein from DNH by salt was further investigated by treating it as a reversible equilibrium. The initial DNH concentration and the temperature were varied independently and dissociation curves measured under the different conditions. A variation of DNH between 0.16 and 1.23mM (with respect to DNA phosphate) gave a shift in the position of the dissociation curves on the salt axis to higher salt concentrations. This was just outside the noise limits of the experiment and in a good experiment could be as much as 50-100mM. This shift was not sufficient to explain the heterogeneity of preparations with respect to dissociation. This must be due to a true structural heterogeneity. Application of the model to the central portion of the dissociation concerning f2a2, f2b and f3, gave good straight lines but with a .wide degree of variance. The value for x+1 obtained was 1.53±0.56, leading to a value for the histone exponent of 0.53. There is little or no cooperativity in the binding of these histones to DNA. A massive cooperativity was found, however, with respect to salt. A value for n was obtained as 14.7±3.5, leading to a value for the number of sodium chloride molecules required to dissociate a molecule of histone of 28. This closely agrees with the number of salt linkages between histone and DNA, thus strongly suggesting that the dissociation is mediated by the displacement of histone from DNA phosphate by sodium ions. The variations in the extent of dissociation with temperature differ greatly from preparation to preparation. This lack of agreement together with the possibility of proteolytic artefacts when working at high temperature made the application of the model to these data dubious. Other workers' claims to have observed cooperative binding of histones to DNA are not borne out in this study. There are some indications that the binding of f2al may be cooperative. We propose that the binding of f2a1 to DNA occurs cooperatively to form a template to which the following fractions can bind specifically. Variation of the salt used to dissociate the histone from DNH can produce large differences in the extent of dissociation. A range of salts were used and the extent of dissociation at a single salt concentration was measured. The differences, which could not be accounted for by the differences in activity coefficients of the salts, were normalised with respect to sodium chloride and an order of effectiveness in dissociation was obtained. This emphasises that displacement of histones by metal cations is an important part of the mechanism of dissociation. However, variation of the anion has quantitatively the greater effect on the dissociation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available