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Query: EC:3.1.31.1 (micrococcal nuclease)
 2,818 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Chromatin structure has been studied in the sites of attachment to the nuclear matrix in interphase mouse liver and spleen nuclei. The patterns of fragmentation of the DNA belonging to these sites (0.3-2% of total DNA in spleen and liver, respectively) with staphylococcal nuclease and DNAase I were very close to those of usual nucleosomal chains. Moreover, the nuclear matrix preparations contained all five major histones, including H1, in almost stoichiometric amounts. The histone/DNA ratios for the matrix were also similar to those found in nuclei. These findings and the size of the matrix-protected DNA indicated that interphase chromatin was attached to the nuclear matrix via matrix-bound nucleosomes and, to a much lesser extent, oligonucleosomes up to 5-6 units long. Two-dimensional electrophoretic separation of the matrix-bound histones revealed that modifications of histone H1 and, probably, of other histones were distinguished from those in bulk chromatin. Study of binding of exogenously added labeled histone octamers or mononucleosomal size DNA to nuclear matrix excluded the possibility of their artifactual trapping during the isolation procedure.
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PMID:Evidence for attachment of interphase chromatin to the nuclear matrix via matrix-bound nucleosomes. 672 65

Chromatin in the nuclei fixed in tissue and in the nuclei isolated by low ionic strength solutions in the presence of Mg2+ is represented by globular (nucleomeric) fibrils, 20-25 nm in diameter. The staphylococcal or endogenous nuclear nuclease splits the chromatin fibrils resulting in fragments corresponding to nucleomers and their multimers. Upon removal of firmly bound Mg2+ the nucleomers unfold to form chains consisting of 4-6-8 nucleosomes. Mild hydrolysis of nuclear chromatin by staphylococcal nuclease results in a split-off of mono-, di- and trimers of nucleomers sedimenting in a sucrose density gradient in the presence of EDTA as particles with the sedimentation coefficients of 37, 47 and 55S, respectively. The sedimentation coefficient for the mononucleomer in a sucrose density gradient with MgCl2 is 45S. Determination of the length of DNA fragments of chromatin split-off by staphylococcal nuclease showed that the nucleomer consists of 8 nucleosomes, while the dimer and trimer of the nucleomer consists of 14-16 and 21-24 nucleosomes, respectively. The nucleomeric monomer undergoes structural transition from the compact (45S) to the "loose" state (37S) after removal of Mg2+. This transition is completely reversible, when the nucleomer contains histone H1. The removal of the latter or dialysis of the nucleomer against EDTA in low ionic strength solutions results in a complete unfolding of the nucleomer into a nucleosomal chain fragment. A model for the nucleomer fibril structure in which the helical organization of the nucleosomal chain in the nucleomer (2 turns with 4 nucleosomes in each) is alternated with the impaired helical bonds between the nucleomers is discussed. The functional significance of the nucleomeric organization of chromatin may be an additional restriction of the site-specific recognition of DNA in chromatin with the possibility of local (at the level of one nucleomer) changes in chromatin conformation excluding this restriction.
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PMID:[Nucleomeric organization of chromatin]. 679 81

Cell cycle variations in the phosphorylation of chromatin-associated nonhistones were determined. Cells were radiolabeled with [32P]orthophosphate and chromatin was obtained by mild digestion of nuclei with micrococcal nuclease. The experiments were performed in the presence of a substrate inhibitor of alkaline phosphatase, beta-glycerophosphate. The results show that, while similar molecular weight species of phosphorylated nonhistones are associated with interphase chromatin through the HeLa cell cycle, the incorporation (32P cpm/micrograms of protein) profiles of selected major phosphononhistones show substantial changes. The most prominent peaks of specific radioactivity occur in the DNA synthesis phase (S phase). The phosphorylation states of the proteins of isolated metaphase chromosomes were also determined. Nonhistone proteins of isolated metaphase chromosomes are strikingly dephosphorylated, especially in comparison to histone H1. The phosphorylation of the major phosphononhistone of chromatin, which has a molecular weight of 55,000, was further characterized by techniques that included one-dimensional peptide mapping in sodium dodecyl sulfate-polyacrylamide gels and nonequilibrium pH gradient slab gel electrophoresis. Phosphoproteins are also components of the nuclear scaffold, and cell cycle variations in these proteins were investigated. The primary phosphorylated species has a molecular weight of 119,000. As with chromatin-associated nonhistones, this nuclear scaffold protein shows substantial incorporation of 32P in S phase, and a high level of incorporation also occurs close to mitosis.
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PMID:Phosphorylation of nonhistone proteins during the HeLa cell cycle. Relationship to DNA synthesis and mitotic chromosome condensation. 682 62

We have examined the interaction of the estradiol receptor molecule with chromatin in MCF-7 cells, a human breast tumor cell line responsive to estradiol. Receptor was found associated with the various nucleosomal products produced by digestion with micrococcal nuclease. In order to determine whether these receptor binding sites were distributed in a random or nonrandom manner within the chromatin, we have fractionated MCF-7 cell chromatin into transcriptionally active and inactive fractions by limited micrococcal nuclease digestion followed by Mg(2+) precipitation. A comparison of the Mg(2+)-soluble and insoluble chromatin fractions showed that the Mg(2+)-soluble fraction: (i) was composed predominantly of mononucleosomes; (ii) was enriched in nonhistone proteins; (iii) apparently lacked histone H1; (iv) was enriched approximately 5-fold in transcribed sequences as measured by a cDNA probe to cytoplasmic poly(A)-RNA sequences; and (v) was depleted at least 5-fold of globin sequences, which is presumably a nontranscribed gene in these cells. When these cells were stimulated with beta-[(3)H]estradiol, the Mg(2+)-soluble fraction showed a significant enrichment in chromatin-bound estradiol receptor: the Mg(2+)-soluble mononucleosomes showed a 3- to 4-fold enrichment and the di- and trinucleosomes, a 7- to 19-fold enrichment, when compared to the corresponding subunits in the Mg(2+)-insoluble chromatin fraction. This cofractionation of chromatin enriched in transcribed sequences and bound estradiol receptor indicated that receptor binding to MCF-7 cell chromatin was not random but, rather, occurred preferentially in specific regions of the chromatin.
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PMID:Enrichment of estradiol-receptor complexes in a transcriptionally active fraction of chromatin from MCF-7 cells. 692 87

Histone deposition in HeLa cells has been studied by monitoring the fractionation and electrophoresis mobility of pulse-labeled histones under conditions that separate newly replicated from bulk chromatin DNA. The separation efficiency of these two methods is approximately 70%. Following micrococcal nuclease digestion, chromatin was fractionated by salt elution. 50-65% of the newly synthesized histones eluted with bulk chromatin at NaCl concentrations between 0.1 and 0.3 M and were further down to co-electrophorese with bulk chromatin DNA, not with the more extensively digested newly replicated chromatin DNA contained in those fractions. The remaining chromatin fractions, solubilized with 0.4-0.6 M NaCl, were several-fold enriched in nascent DNA (Annunziato, A. T., Schindler, R. K., Thomas, C. A., Jr., and Seale, R. L. (1981) J. Biol. Chem. 256, 11880-11886) and were correspondingly enriched for the balance (35-50%) of newly synthesized core histones. This fraction of newly synthesized core histone may be preferentially deposited onto newly replicated DNA. In contrast, histone H1 showed little tendency toward deposition onto new DNA. Within 15 min all new core histones attained the same solubility and electrophoretic mobility as bulk chromatin. We conclude that newly synthesized histones are deposited onto both replicating and nonreplicating regions of chromatin.
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PMID:Association of newly synthesized histones with replicating and nonreplicating regions of chromatin. 708 80

In the nuclei fixed in situ, as well as in nuclei in low-ionic-strength solutions containing magnesium ions, chromatin is represented by globular nucleomeric fibrils 20-25 nm in diameter. Staphylococcal or endogenous nucleases cleave chromatin fibrils to nucleomers and multinucleomers. On removal of firmly bound magnesium, the nucleomers unfold into chains of four, six or eight nucleosomes. Mild staphylococcal nuclease digestion of nuclear chromatin releases mononucleomers, dinucleomers and trinucleomers that sediment in the sucrose density gradient in the presence of EDTA as 37-S, 47-S and 55-S particles, respectively. The mononucleomers in the sucrose density gradient with MgCl2 sediment as 45-S particles. The determination of the length of staphylococcal-nuclease-digested DNAs contained in the chromatin fragments showed that a nucleomer is composed of 8, and a dimer and trimer of 14-16 and 21-24 nucleosomes, respectively. When deprived of Mg2+ ions, the monomers lose their compactness (45 S) and become loose particles (37 S). This transition is completely reversible if nucleomers contain histone H1. Removal of this histone or dialysis of the nucleomer against EDTA at low ionic strength results in the complete unfolding of the nucleomer into a chain of nucleosomes. A structural model of a nucleomer fibril is suggested where the helicity of the nucleosome chain in a nucleomer (two turns of four nucleosomes each) is periodically discontinued. Such an organization of chromatin apparently provides additional hindrances for site-specific recognition of DNA in chromatin but permits local changes (within a single nucleomer) in chromatin when a hindrance is abolished.
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PMID:Nucleomeric organization of chromatin. 709 16

The transiently altered DNA-histone interaction of parental chromatin during replication was studied by micrococcal nuclease digestion. A large amount of nuclease-resistant pulse-labeled DNA and a small fraction of nonreplicating DNA are released from chromatin fragments by treatment with 0.5 M NaCl and appear as protein-free DNA. As shown by reconstitution experiments, the salt sensitivity of digested nascent chromatin is most probably a consequence of the shorter DNA fragment size (55 +/- 15 base pairs) in these complexes. This new DNA is associated with parental chromatin fragments which are structurally changed in such a way that parts of nucleosomal DNA were more susceptible to nuclease attack. The core histones of these particles are probably not distinct from those of salt-stable nucleosomes. However, histone H1 and probably high-mobility group proteins appear to be more weakly bound during replication as shown by electrophoresis under nondenaturing conditions. The results agree with the assumption that the transient alteration of nucleosomal conformation describes a state in which DNA could be replicated without leaving the associated core histone complexes. A possible attachment of pulse-labeled chromatin with nuclear matrix is discussed.
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PMID:Replicative conformation of parental nucleosomes: salt sensitivity of deoxyribonucleic acid-histone interaction and alteration of histone H1 binding. 710 18

Some of the properties of chromatographically purified satellite chromatin are compared with those of unfractionated, control chromatin. Nucleosomes were present in the purified satellite chromatin as verified by digestion with micrococcal nuclease and DNase I and by electron microscopy. Average nucleosome DNA repeat lengths of 186 +/- 7 and 193 +/- 5 base pairs were obtained through micrococcal nuclease digestion of the purified satellite chromatin and control chromatin, respectively; nucleosome spacer lengths were equally heterogeneous for the two chromatin samples. The distribution of Eco RI-produced chromatin fragments of different size in the satellite chromatin was the same as that calculated assuming random cleavage at each Eco RI site, consistent with the notion that nucleosomes do not have specific locations on the 1.715 g ml-1 satellite DNA. The purified satellite chromatin contained little non-histone protein, but did contain all five histones and all detectable histone sequence variants. Amounts of the core histones were identical in the satellite chromatin and the control chromatin, but the amount of histone H1 was 30% less in the satellite chromatin than in the control. Although the molar ratios for the major sequence variants of both histones H3 and H2A differed between kidney and thymus, the ratios were the same for satellite and control chromatin isolated from a single tissue.
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PMID:Chromatin fragments containing bovine 1.715 g ml-1 satellite DNA. Nucleosome structure and protein composition. 711 10

A new procedure is described for the reassociation of histone H1 to rat liver polynucleosomes selectively depleted of H1 and nonhistone proteins. The fidelity of reconstitution was scrutinized by nuclease digestion and sedimentation studies monitoring the regeneration of structural features which disappear following removal of H1. The results demonstrate that the 166-base pair barrier of digestion with micrococcal nuclease was restored in polynucleosomes reconstituted at the same ratio of H1 per octamer as that found in native nuclei, while association of twice that amount of H1 produced a barrier of digestion at about 178-180 base pairs. Polynucleosomes associated with polylysine exhibited a similar barrier as H1-depleted polynucleosomes. Digestion of H1-reconstituted polynucleosomes with DNase I produced preferentially dinucleosomal DNA fragments in the same manner as that of untreated polynucleosomes. The rate of digestion of H1-reconstituted polynucleosomes by micrococcal nuclease and DNase I was also comparable to that of untreated polynucleosomes. Sedimentation of H1-reconstituted polynucleosomes in a quasi-physiological ionic milieu revealed the regeneration of disassembly-refractory particles, a structural feature of untreated polynucleosomes. We conclude that the nucleosomal and supra-nucleosomal structure of polynucleosomes was reconstituted with fidelity.
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PMID:Reassociation of histone H1 to H1-depleted polynucleosomes. 713 Jan 95

A previous study on the distribution of histone H1 subfractions in chromatin suggested that these proteins differ in the protection they confer to DNA. To elucidate further this suggestion, reconstitution experiments were carried out with purified H1 subfractions (H1-1, H1-2, H1o) and H1-depleted chromatin. We have studied the structural properties of H1o as compared to those of other H1 fractions by electrophoretic analysis of DNA and mononucleosomes obtained after micrococcal nuclease digestion, thermal denaturation, and electron microscopy. The three fractions studied reassociate to H1-depleted chromatin. However, differences in the extent of DNA protection are observed between H1o and the other fractions: H1o induces a more rapid degradation of long oligomers into mononucleosomes; these mononucleosomes bearing H1o only, have a greater electrophoretic mobility; furthermore, thermal denaturation shows that a small fraction of DNA is less efficiently protected by H1o than by the other fractions. Electron microscopy, on the other hand, shows that these differences are not due to areas of chromatin devoid of H1o in the reconstitute and that the reconstituted samples are able, under proper ionic conditions, to refold in a higher-order structure.
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PMID:The structural role of histone H1: properties of reconstituted chromatin with various H1 subfractions (H1-1, H1-2, and H1o). 718 52


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