Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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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)

We investigated the mechanism by which polyanions gelatinized nuclei of some mouse lymphocytes and ruptured these cells. The gel produced by the addition of dextran sulfate (DS) to mouse lymphocyte nuclei was composed of histones (H1, H2A, H2B, H3, and H4), DS, and DNA. Adding DS to the chromatin obtained from nuclei by micrococcal nuclease (MNase) digestion also produced a gel containing a complex of DS-histones-DNA. When this mixture was further digested by MNase, DNA debris of random sizes was observed instead of the 150-bp repeating units of DNA usually observed when normal nucleosomes are digested with MNase. Removal of DS from the chromatin-DS gel resulted in the regeneration of nucleosomes. These results suggest the following: After entering the cells with damaged cellular membrane, DS extracts histones from nucleosomes to form DS-histone complexes, which then aggregate with the liberated DNA to form a macromolecular gel. Finally, the swelling pressure of the gel destroys the cells.
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PMID:Interaction between polyanions and cell nuclei: mechanism of gelatination of nuclei. 894 64

The spermatozoa of a dasyurid marsupial, Sminthopsis crassicaudata, have two distinct nuclear regions: uniformly electron-dense chromatin (C1) in the interior and fissured chromatin (C2) at the periphery. To investigate whether the differences in morphology are due to incorporation of different packaging proteins, spermatozoa nuclear proteins were characterised by acetic acid-urea polyacrylamide gel electrophoresis (PAGE) and fractionated by reverse-phase high-pressure liquid chromatography (HPLC). The main protein component was protamine I, but a complete histone complement (H1, H2A, H2B, H3, and H4) was also detected. Immunocytochemistry showed localisation of H4, H2B, and H2A histones to the periphery of the nuclei, a region that corresponded to the C2 chromatin. The fissures in the chromatin of this region disappeared following incubation with fish protamines, indicating that the nucleohistone C2 region may be incompletely condensed relative to nucleoprotamines. This observation is consistent with the view that 60% of phosphodiester charges remain negative in nucleohistone DNA, whereas all DNA charges are neutralised in highly compact nucleoprotamines. Treatment of spermatozoa with micrococcal nuclease showed that the C1 chromatin was resistant to digestion, whereas the C2 region was cleaved into 30- to 38-nm agglomerates and 11-nm nucleosomal-size structures. Thus, this study demonstrates that spermatozoa nuclei of this marsupial species contain peripherally localised histones, and the nucleohistone chromatin accounts for the different morphology of the C2 region compared with the rest of the nucleus.
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PMID:Isolation of histones and related chromatin structures from spermatozoa nuclei of a dasyurid marsupial, Sminthopsis crassicaudata. 921 75

Upstream binding factor (UBF) is a vertebrate RNA polymerase I transcription factor that can bend and wrap DNA. To investigate UBF's likely role as an architectural protein of rRNA genes organized in chromatin, we tested UBF's ability to bind rRNA gene enhancers assembled into nucleosome cores (DNA plus core histones) and nucleosomes (DNA plus core histones plus histone H1). UBF bound with low affinity to nucleosome cores formed with enhancer DNA probes of 162 bp. However, on nucleosome cores which contained approximately 60 bp of additional linker DNA, UBF bound with high affinity similar to its binding to naked DNA, forming a ternary DNA-core histone-UBF complex. UBF could be stripped from ternary complexes with competitor DNA to liberate nucleosome cores, rather than free DNA, suggesting that UBF binding to nucleosome cores does not displace the core histones H2A, H2B, H3, and H4. DNase I, micrococcal nuclease, and exonuclease III footprinting suggests that UBF and histone H1 interact with DNA on both sides flanking the histone octamer. Footprinting shows that UBF outcompetes histone H1 for binding to a nucleosome core and will displace, if not dissociate, H1 from its binding site on a preassembled nucleosome. These data suggest that UBF may act to prevent or reverse the assembly of transcriptionally inactive chromatin structures catalyzed by linker histone binding.
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PMID:Nucleosome binding by the polymerase I transactivator upstream binding factor displaces linker histone H1. 931 41

Sin mutations in Saccharomyces cerevisiae alleviate transcriptional defects that result from the inactivation of the yeast SWVI/SNF complex. We have investigated the structural and functional consequences for the nucleosome of Sin mutations in histone H3. We directly test the hypothesis that mutations in histone H3 leading to a SWI/SNF-independent (Sin) phenotype in yeast lead to nucleosomal destabilization. In certain instances this is shown to be true; however, nucleosomal destabilization does not always occur. Topoisomerase I-mediated relaxation of minichromosomes assembled with either mutant histone H3 or wild-type H3 together with histones H2A, H2B, and H4 indicates that DNA is constrained into nucleosomal structures containing either mutant or wild-type proteins. However, nucleosomes containing particular mutant H3 molecules (R116-H and T118-I) are more accessible to digestion by micrococcal nuclease and do not constrain DNA in a precise rotational position, as revealed by digestion with DNase I. This result establishes that Sin mutations in histone H3 located close to the dyad axis can destabilize histone-DNA contacts at the periphery of the nucleosome core. Other nucleosomes containing a distinct mutant H3 molecule (E105-K) associated with a Sin phenotype show very little change in nucleosome structure and stability compared to wild-type nucleosomes. Both mutant and wild-type nucleosomes continue to restrict the binding of either TATA-binding protein/transcription factor IIA (TFIIA) or the RNA polymerase III transcription machinery. Thus, different Sin mutations in histone H3 alter the stability of histone-DNA interactions to various extents in the nucleosome while maintaining the fundamental architecture of the nucleosome and contributing to a common Sin phenotype.
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PMID:Sin mutations of histone H3: influence on nucleosome core structure and function. 937 28

The Holliday junction is a key intermediate in genetic recombination. Here, we examine the effect of a nucleosome core on movement of the Holliday junction in vitro by spontaneous branch migration. Histone octamers consisting of H2A, H2B, H3, and H4 are reconstituted onto DNA duplexes containing an artificial nucleosome-positioning sequence consisting of a tandem array of an alternating AT-GC sequence motif. Characterization of the reconstituted branch migration substrates by micrococcal nuclease mapping and exonuclease III and hydroxyl radical footprinting reveal that 70% of the reconstituted octamers are positioned near the center of the substrate and the remaining 30% are located at the distal end, although in both cases some translational degeneracy is observed. Branch migration assays with the octamer-containing substrates reveal that the Holliday junction cannot migrate spontaneously through DNA organized into a nucleosomal core unless DNA-histone interactions are completely disrupted. Similar results are obtained with branch migration substrates containing an octamer positioned on a naturally occurring sequence derived from the yeast GLN3 locus. Digestion of Holliday junctions with T7 endonuclease I establishes that the junction is not trapped by the octamer but can branch migrate in regions free of histone octamers. Our findings suggest that migration of Holliday junctions during recombination and the recombinational repair of DNA damage requires proteins not only to accelerate the intrinsic rate of branch migration but also to facilitate the passage of the Holliday junction through a nucleosome.
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PMID:A histone octamer blocks branch migration of a Holliday junction. 937 46

Histone-DNA templates for bacteriophage T7 RNA polymerase were assembled from a plasmid containing a promoter and a terminator for this polymerase, (H3 x H4)2 tetramers deprived of their tail domains, and H2A x H2B dimers. Histone (H3 x H4)2 tetramers lacking their terminal domains were obtained from trypsin-digested nucleosomal cores. The oligonucleosomal templates containing (H3 x H4)2 tetramers lacking their tail domains, like the control templates with intact core histone octamers, protect approximately 146 base pairs of DNA against micrococcal nuclease digestion. The transcriptional inhibition caused by the association of DNA with core histone octamers is significantly reduced upon elimination of the tail domains of the (H3 x H4)2 tetramers. Apparently, the terminal domains of (H3 x H4)2 must be present to block transcription efficiently. These results show the important inhibitory role played by the tail domains of the histone (H3 x H4)2 tetramers, suggesting the involvement of these regions in transcriptional regulation.
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PMID:Transcriptional inhibitory role of the tail domains of histone (H3 x H4)2 tetramers. 975 Jan 70

Transcription of the sea urchin early histone genes occurs transiently during early cleavage, reaching the maximum at the morula stage and declining to an undetectable level at the gastrula stage. To identify the regulatory elements responsible for the timing and the levels of transcription of the H2A gene, we used promoter binding studies in nuclear extracts and microinjection of a CAT transgene driven by the early H2A promoter. We found that morula and gastrula nuclear proteins produced indistinguishable DNase I footprint patterns on the H2A promoter. Two sites of interactions, centred on the modulator/enhancer and on the CCAAT box respectively, were detected. Deletion of the modulator or coinjection of an excess of modulator sequences severely affected the expression of two transgenes driven by the enhancer-less and modulator-containing H2A promoter. Finally, a DNA fragment containing 3' coding and post-H2A spacer sequences, where upon silencing three micrococcal nuclease hypersensitive sites were previously mapped, specifically repressed at the gastrula stage the expression of the transgene driven by the H2A promoter. These results indicate that the modulator is essential for the expression of early H2A gene and that sequences for downregulation are localized near the 3' end of the H2A gene.
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PMID:Regulation of the sea urchin early H2A histone gene expression depends on the modulator element and on sequences located near the 3' end. 1019 23

NF-Y is a CCAAT-binding trimer with two histonic subunits, NF-YB and NF-YC, resembling H2A-H2B. We previously showed that the short conserved domains of NF-Y efficiently bind to the major histocompatibility complex class II Ea Y box in DNA nucleosomized with purified chicken histones. Using wild-type NF-Y and recombinant histones, we find that NF-Y associates with H3-H4 early during nucleosome assembly, under conditions in which binding to naked DNA is not observed. In such assays, the NF-YB-NF-YC dimer forms complexes with H3-H4, for whose formation the CCAAT box is not required. We investigated whether they represent octamer-like structures, using DNase I, micrococcal nuclease, and exonuclease III, and found a highly positioned nucleosome on Ea, whose boundaries were mapped; addition of NF-YB-NF-YC does not lead to the formation of octameric structures, but changes in the digestion patterns are observed. NF-YA can bind to such preformed DNA complexes in a CCAAT-dependent way. In the absence of DNA, NF-YB-NF-YC subunits bind to H3-H4, but not to H2A-H2B, through the NF-YB histone fold. These results indicate that (i) the NF-Y histone fold dimer can efficiently associate DNA during nucleosome formation; (ii) it has an intrinsic affinity for H3-H4 but does not form octamers; and (iii) the interactions between NF-YA, NF-YB-NF-YC, and H3-H4 or nucleosomes are not mutually exclusive. Thus, NF-Y can intervene at different steps during nucleosome formation, and this scenario might be paradigmatic for other histone fold proteins involved in gene regulation.
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PMID:NF-Y associates with H3-H4 tetramers and octamers by multiple mechanisms. 1056 83

We have examined salt-soluble chromatin released by micrococcal nuclease from a 15-day-old chicken embryo erythrocyte nuclei for histone acetyltransferase (HAT) activities. This chromatin is enriched in transcriptionally active sequences from within the active beta-globin locus and contains elevated levels of acetylated core histones. HAT activities present in this fraction target histones H4, H3, and H2A when the chromatin itself is used as the substrate. In gel HAT activity assay demonstrates that the salt-soluble chromatin fraction contains four acetyltransferase molecules distinguished by their different molecular masses (47, 33, 32, and 28 kDa). Further separation of the chromatin by centrifugation through sucrose gradients shows that the acetyltransferases segregate into chromatin-bound and chromatin-free populations. The 32- and 28-kDa HATs are associated with chromatin, whereas the 47- and 33-kDa HAT molecules are not. The chromatin-bound HAT activities predominantly target H4 to give the diacetyl and triacetyl species; some acetylation of H2A can also be seen. Our results suggest that the chromatin-associated acetyltransferases have a role in gene regulation.
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PMID:Multiple histone acetyltransferases are associated with a chicken erythrocyte chromatin fraction enriched in active genes. 1089 66

MacroH2A histones have an unusual hybrid structure, consisting of an N-terminal domain that is approximately 65% identical to a full-length histone H2A and a large C-terminal nonhistone domain. To develop an in vitro approach for investigating the effects of macroH2A proteins on chromatin structure and function, we reconstituted nucleosomes with recombinant macroH2A1.2, substituting for conventional H2A. Recombinant macroH2A1.2 was able to efficiently replace both of the conventional H2As in reconstituted nucleosomes. The substitution of macroH2A1.2 for H2A did not appear to grossly perturb the basic structure of the nucleosome core, as assessed by sedimentation and by digestion with micrococcal nuclease or DNase I. However, two differences were observed. First, the region around the midpoint of the nucleosomal core DNA was more resistant to digestion by DNase I in nucleosome core particles reconstituted with macroH2A1.2. Second, preparations of core particles reconstituted with macroH2A1.2 had a greater amount of material that sedimented more rapidly than mononucleosomes, suggesting that macroH2A1.2 may promote interactions between nucleosomes. Recombinant macroH2A proteins should be valuable tools for examining the effects of macroH2A on nucleosome and chromatin structure.
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PMID:Reconstitution of nucleosomes with histone macroH2A1.2. 1177 15


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