Heterogeneity of chromatin fragments produced by micrococcal nuclease action.

Digestion of calf thymus chromatin with micrococcal nuclease produces a mixture of apparently well defined nucleoprotein fragments which have been partially resolved by sedimentation on linear (5-20%) sucrose gradients. Sedimentation patterns reveal a predominant peak at the 11S position, three slower components, which have not previously been reported, at the 3.4S, 5.3S and 8.6S positions, and three faster components at the 17S, 22S and 26S positions. DNA isolated from the 3S to 12S region of gradients has been resolved on polyacrylamide gels into nine to ten discrete components ranging from 47 to 156 base pairs in length. A nearly identical pattern of small DNA products was obtained from chromatin digested in intact nuclei. These data suggest that chromatin contains either several types of subunits or predominently a single type of subunit which can be asymmetrically cleaved at any one of four or more sites.     

Distribution of H1 histone in chromatin digested by micrococcal nuclease.

The relative amount of H1 histone associated with isolated nucleosomes from calf thymus was determined as a function of the extent of DNA digestion by micrococcal nuclease. Generally the amount of H1 histone associated with mononucleosomes decreases with increasing digestion until 60% of the original H1 remains associated with DNA 150 base pirs or less in size. Coincidentally, H1 histone increases relative to the other histones in aggregated material that sediments through sucrose gradients to form a pellet. However, the level of H1 histone remains at control values for oligonucleosomes (dimer to hexamer) over the 30% digestion range studied. An increase in ionic strength to 0.3 M NaCl in the density gradient reveals a different pattern of H1 binding, whereby the amount of H1 reflects the average size of the DNA fragments with which it is associated. Although there is significant binding to nucleosomes per se, it appears that the major ionic involvement of H1 is with internucleosomal spacer DNA.     

Digestion of insect chromatin with micrococcal nuclease, DNase I and DNase I combined with single-strand specific nuclease S1.

The chromatin of the lepidopteran Ephestia kuehniella was digested by micrococcal nuclease, DNase I and S1-nuclease combined with DNase I pretreatment. The resulting DNA fragments were analyzed by gel electrophoresis and compared with the DNA fragments of rat liver nuclei obtained by the same process. Extensive homology was revealed between insect and mammalian chromatin structure. The combined DNase I- S1-nuclease digestion yields double-stranded DNA fragments of lengths from 30 to 110 base-pairs. These DNA fragments are not obtained from nuclei predigested extensively with micrococcal nuclease. The results are discussed with respect to the internal structure of the chromatin subunit.     

Estrogen receptor in hen oviduct chromatin, digested by micrococcal nuclease.

Nuclei from laying hen oviduct were prepared according to Hewish and Burgoyne i.e. in the presence of spermine and spermidine and in the absence of divalent cations and were then moderately digested by micrococcal nuclease. When the resulting chromatin was analysed by ultracentrifugation on a sucrose gradient, a peak of specific estradiol-binding sites was observed, sedimenting slightly faster (13-14 S) than the mononucleosomes (12 S). When the chromatin was centrifuged on a gradient containing heparin (5 microngram/ml) the sedimentation coefficient of the estradiol receptor peak shifted to 7-8 S; it returned to the 13-14 S position in the absence of heparin, when target organ chromatin was also present in the gradient. The preparation of the chromatin is described and the validity of the method to explore receptor localisation is discussed, as is the specificity of the receptor-DNA interaction.     

Artifacts accompanying digestion of chromatin with micrococcal nuclease

DNA in the micrococcal nuclease limit digest of chromatin is completely resistant to DNAse II. At least part of this resistance is not a property of the untreated chromatin, but is acquired in the course of digestion.     

Internucleosome interaction: detection of dinucleosome fragmentation of chromatin by micrococcal nuclease. Analysis of the products of cleavage of chromatin from rat liver nuclei and L cells by micrococcal nuclease

In murine L-cell nuclei micrococcal nuclease causes chromatin fragmentation with predominant liberation of dinucleosomes. Analysis of dynamics of rat liver nuclear chromatin cleavage by micrococcal nuclease revealed that the "dinucleosomal" mode of fragmentation is due to the pretreatment of nuclei with the non-ionic detergent Triton X-100 in the course of the isolation procedure. The set of particles detected in nuclease hydrolysates of nuclear chromatin pretreated with Triton X-100 and those isolated by the standard procedure was shown to be significantly different. In Triton X-100 treated nuclei the dichromatosome is the main hydrolysate component under various experimental conditions of nuclease hydrolysis and the sole component under "mild" conditions, whereas sucrose-treated nuclei contain three types of dinucleosomes. In Triton-treated nuclei prolongation of hydrolysis results in the liberation of the chromatosome which is absent in chromatin hydrolysates of sucrose-treated nuclei. Hydrolysis of Triton-treated nuclear chromatin by micrococcal nuclease is unaccompanied by the liberation (up to the stage of "deep" hydrolysis) of the core particle, the major component of the "sucrose" nuclear hydrolysate under the conditions used. The sharp differences in the accessibility of various types of dinucleosomes observed during pretreatment of nuclei with Triton X-100 are interpreted in terms of the localization of histone H1. The non-random type of the histone H1 molecule orientation along the nucleosome fibril is postulated.     

Cesium chloride gradients of chromatin after treatment with micrococcal nuclease

Cesium chloride equilibrium density centrifugation shows that treatment of rat liver nuclei with low concetrations of micrococcal nuclease for extremely short periods of time results in the appearance of chromatin fractions of low protein/DNA ratio and even free DNA. The DNA of these chromatin fractions is shorter than the DNA moiety of one chromatin subunit. The amount of high buoyant density material is decreased with increasing digestion time. We conclude that this material belongs to the minor chromatin fraction which is not organized according to the subunit model.     

Copper-rich nucleoprotein generated by micrococcal nuclease

Nuclei from calf thymus tissue digested with micrococcal nuclease under nonchelating conditions yielded soluble nucleoprotein enriched in copper. Following limited digestion, the ratio of μg Cu:mg DNA was inversely related either to percent solubility of chromatin or to levels of enzyme maintaining an enzyme:A ₂₆₀ ratio of 0.059. The enzyme appeared to cleave preferentially regions of chromatin where copper is localized, releasing no additional metal upon further digestion. Moreover, the highest copper: DNA ratio was always associated with the least-digested sample. The distribution between copper and angiotensin II (AII) in chromatin fragments following slight nuclease digestion suggests a possible link between copper and nuclear AII binding. When nuclei are incubated with AII prior to digestion and dialysis, solubilized chromatin contained about three times more copper than buffer control. Metal profiles generated from gel (A-5 M) chromatography for these samples were distinctive: copper peaks appeared near or adjacent to linker DNA regions, and in the case of AII, coincided with fragments containing specific AII receptors; thus, there appears to be an enrichment of copper in these active nucleoprotein fragments.     

Sequence specific cleavage of DNA by micrococcal nuclease.

Micrococcal nuclease is shown to cleave DNA under conditions of partial digestion in a specific manner. Sequences of the type 5'CATA and 5'CTA are attacked preferentially, followed by exonucleolytic degradation at the newly generated DNA termini. GC-rich flanking sequences further increase the probability of initial attack. Unexpectedly, long stretches containing only A and T are spared by the nuclease. These results, which were obtained with spared by the nuclease. These results, which were obtained with mouse satellite DNA and two fragments from the plasmid pBR22, do not support the previous contention that it is the regions of high At-content which are initially cleaved by micrococcal nuclease. This specificity of micrococcal nuclease complicates its use in experiments intended to monitor the nucleoprotein structure of a DNA sequence in chromatin.     

Fractionation of chromatin of liver cell nuclei after mild micrococcal nuclease digestion

Mild micrococcal nuclease treatment of rat and mouse nuclei and fractionation were based on the method of Tata and Baker. Three chromatin fractions, S, P1, P2, were separated, and for each of these fractions the sensitivity to the DNase 1 action was determined. The relative content in these fractions of non-transcribed DNA sequences was established by hydridization with a mouse satellite DNA, and the relative content of transcribed DNA sequences--by hydridization with DNA synthesised on the total poly (A) mRNA. None of the fractions displayed the properties characteristic of active chromatin.     

Another potential artifact in the study of nucleosome phasing by chromatin digestion with micrococcal nuclease

We show that, contrary to expectations, restriction enzyme cleavage of chicken erythrocyte nucleosome core particle DNA generates a series of distinct subnucleosome fragments. These fragments do not result from bulk nucleosome phasing in vivo, but arise from micrococcal nuclease cleavages internal to the core particle, at roughly 10-base pair intervals and at AT-rich sequences. Those 145-base pair DNA fragments remaining intact are a biased population in which the guanine content can fluctuate by as much as 10%, with a 10-base pair period. We suggest that these same considerations, when applied to a unique DNA sequence, are the true explanation for several previous claims for nucleosome phasing.     

High sequence specificity of micrococcal nuclease.

The substrate specificity of micrococcal nuclease (EC 3.1.4.7.) has been studied. The enzyme recognises features of nucleotide composition, nucleotide sequence and tertiary structure of DNA. Kinetic analysis indicates that the rate of cleavage is 30 times greater at the 5' side of A or T than at G or C. Digestion of end-labelled linear DNA molecules of known sequence revealed that only a limited number of sites are cut, generating a highly specific pattern of fragments. The frequency of cleavage at each site has been determined and it may reflect the poor base overlap in the 5' T-A 3' stack as well as the length of contiguous A and T residues. The same sequence preferences are found when DNA is assembled into nucleosomes. Deoxyribonuclease 1 (EC 3.1.4.5.) recognises many of the same sequence features. Micrococcal nuclease also mimics nuclease S1 selectively cleaving an inverted repeat in supercoiled pBR322. The value of micrococcal nuclease as a "non-specific" enzymatic probe for studying nucleosome phasing is questioned.     

Superstructural differences between chromatin in nuclei and in solution are revealed by kinetics of micrococcal nuclease digestion.

Digestion of chromatin in nuclei by micrococcal nuclease, measured as the change in the concentration of monomer-length DNA with time, displays Michaelis-Menten kinetics. Redigestion of soluble chromatin prepared from nuclei by micrococcal nuclease treatment, however, is apparently first order in enzyme and independent of chromatin concentration. This qualitative difference results from an increase in the apparent second order rate constant, kcat/Km, for liberation of monomer DNA: the apparent Km for soluble chromatin is lower by close to 3 orders of magnitude than that for chromatin in nuclei, whereas kcat decreases by less than 1 order of magnitude. Neither the integrity of the nuclear membrane nor the presence of histone H1 contributes to the high Michaelis constant characteristic of chromatin in nuclei. Moreover, differences due to the buffers used for digestion and redigestion are minimal. Low catalytic efficiency is, however, correlated with the presence of higher order chromatin superstructure. Micrococcal nuclease added to soluble chromatin under nondigesting conditions at low ionic strength (I = 0.002) co-sediments with chromatin in sucrose gradients. In 0.15 M NaCl, added nuclease no longer sediments with chromatin and redigestion kinetics become first order in both enzyme and substrate. Kinetic analysis of this type may afford an assay for native, higher order structures in chromatin. Our results suggest that micrococcal nuclease binds to soluble chromatin through additional interactions not present in nuclei, which may be partly ionic in nature.