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)

With the availability of direct genomic footprinting techniques the study of native genomes has been greatly facilitated. This review provides an overview of the techniques involved and gives also a description of the mode of action of different DNA modifying agents which can be used for such methods. These include exonuclease III, deoxyribonuclease, DNase I, micrococcal nuclease, dimethyl sulfate, diethyl sulfate, ethyl methanesulphonate, ethylnitrosourea, diethylpyrocarbonate, bromoacetaldehyde, potassium permanganate, osmium tetroxide, methidiumpropyl-EDTA-iron(II), formaldehyde, psoralen, 1,10 phenanthroline-copper and UV light. We also describe the limitations of the currently existing techniques and give some potential developments.
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PMID:Approaches to characterize protein-DNA interactions in vivo. 843 8

We describe a procedure for the determination of nucleosome boundaries that utilizes single-stranded mononucleosomal DNA obtained from fixed cells as the template in a primer extension assay. The procedure entails treatment of cells with formaldehyde, a reversible protein-DNA crosslinking agent used with the object of fixing the histone octamers to DNA in vivo, followed by preparation of nucleosomal DNA with micrococcal nuclease, reversal of the crosslinks, and isolation of the mononucleosomal material. Full-length single-stranded mononucleosomal DNA is then prepared and used as a template in a linear amplification primer extension assay. The use of single-stranded DNA templates eliminates interference from nicked DNA present in double-stranded preparations. Because of its reversibility, the use of formaldehyde permits the preparation of DNA suitable as a template in DNA synthesis. We present evidence demonstrating the efficiency of histone-DNA crosslinking and the reversibility of the crosslinking reaction as applied to the regeneration of native DNA, active in DNA synthesis. Use of this methodology removes the impact that mobility of the histone octamer and the presence of nicks on nucleosomal DNA have on the determination of nucleosome boundaries.
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PMID:Analysis of in vivo nucleosome positions by determination of nucleosome-linker boundaries in crosslinked chromatin. 899 37

Archaea contain histones that have primary sequences in common with eukaryal nucleosome core histones and a three-dimensional structure that is essentially only the histone fold. Here we report the results of experiments that document that archaeal histones compact DNA in vivo into structures similar to the structure formed by the histone (H3+H4)2 tetramer at the center of the eukaryal nucleosome. After formaldehyde cross-linking in vivo, these archaeal nucleosomes have been isolated from Methanobacterium thermoautotrophicum and Methanothermus fervidus, visualized by electron microscopy on plasmid and genomic DNAs, and shown by immunogold labeling, SDS/PAGE, and immunoblotting to contain archaeal histones, cross-linked into tetramers. Archaeal nucleosomes protect approximately 60 bp of DNA and multiples of approximately 60 bp from micrococcal nuclease digestion, and immunoprecipitation has demonstrated that most, but not all, M. fervidus genomic DNA sequences are associated in vivo with archaeal histones.
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PMID:Archaeal nucleosomes. 935 1

Minichromosome maintenance (Mcm) proteins and the constituents of the origin recognition complex (Orc) are essential components of the eukaryotic replication initiation apparatus. Published evidence strongly suggests that the binding of Mcm proteins to chromatin is contingent upon the prior binding of Orc proteins. Here we use two different approaches to investigate the presence of the human ORC2 protein and of Mcm proteins on chromatin of HeLa cells in various cell cycle phases. First, we mobilized chromatin-bound proteins by micrococcal nuclease and analyzed the resulting digestion products by sucrose gradient centrifugations. Under digestion conditions when Mcm proteins were almost entirely released from chromatin, ORC2 protein was found to be associated with chromatin fragments containing several hundred base pairs of DNA. Second, we used an in vivo cross-linking procedure to covalently link Mcm proteins and ORC2 to DNA by short exposure of intact HeLa cells to formaldehyde. Specific immunoprecipitations revealed that cross-linked nucleoprotein fragments carried either Mcm proteins or ORC2 protein, but not both. Based on the lengths of the DNA fragments in immunoprecipitates, we estimate that the distance between chromatin-bound ORC2 protein and chromatin-bound Mcm proteins must be at least 500-1000 base pairs in HeLa cells.
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PMID:Human minichromosome maintenance proteins and human origin recognition complex 2 protein on chromatin. 973 49

Packaging of mouse mammary tumor virus (MMTV) promoter sequences in nucleosomes modulates access of DNA binding proteins and influences the interaction among DNA bound transcription factors. Here we analyze the binding of histone H1 to MMTV mononucleosomes assembled with recombinant histones and study its influence on nucleosome structure and stability as well as on progesterone receptor (PR) binding to the hormone responsive elements (HREs). The MMTV nucleosomes can be separated into three main populations, two of which exhibited precise translational positioning. Histone H1 bound preferentially to the 5' distal nucleosomal DNA protecting additional 27-28 nt from digestion by micrococcal nuclease. Binding of histone H1 was unaffected by prior crosslinking of protein and DNA in nucleosomes with formaldehyde. Neither the translational nor the rotational nucleosome positioning was altered by histone H1 binding, but the nucleosomes were stabilized as judged by the kinetics of nuclease cleavage. Unexpectedly, binding of recombinant PR to the exposed distal HRE-I in nucleosomes was enhanced in the presence of histone H1, as demonstrated by band shift and footprinting experiments. This enhanced PR affinity may contribute to the reported positive effect of histone H1 on the hormonal activation of MMTV reporter genes.
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PMID:Asymmetric binding of histone H1 stabilizes MMTV nucleosomes and the interaction of progesterone receptor with the exposed HRE. 1244 85

To map the genomic interaction sites of chromatin proteins, two related methods were developed and experimentally explored in Saccharomyces cerevisiae. The ChIC method (chromatin immunocleavage) consists of tethering a fusion protein (pA-MN) consisting of micrococcal nuclease (MN) and staphylococcal protein A to specifically bound antibodies. The nuclease is kept inactive during the tethering process (no Ca2+). The ChEC method (chromatin endogenous cleavage) consists of expressing fusion proteins in vivo, where MN is C-terminally fused to the proteins of interest. The specifically tethered nucleases are activated with Ca2+ ions to locally introduce double-stranded DNA breaks. We demonstrate that ChIC and ChEC map proteins with a 100-200 bp resolution and excellent specificity. One version of the method is applicable to formaldehyde-fixed nuclei, another to native cells with comparable results. Among various model experiments, these methods were used to address the conformation of yeast telomeres.
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PMID:ChIC and ChEC; genomic mapping of chromatin proteins. 1546 30

Methylation and other covalent modifications of nucleosome core histones are key regulators of chromatin structure and function, including epigenetic control of gene expression. For the human brain, however, very little is known about the regulation of histone modifications at specific genomic loci. Furthermore, chromatin immunoprecipitation protocols applicable to postmortem tissue are lacking, and the impact of potential confounds such as autolysis time or tissue pH is unknown. We treated cerebral cortex from human postmortem brain and mice by micrococcal nuclease digestion or, alternatively, by formaldehyde-crosslinking and sonication. We show that the bulk of nucleosomal DNA remains attached to histones during the first 30 h after death. Immunoprecipitation with antibodies against methylated histones was at least 10-fold more effective in unfixed, micrococcal nuclease-digested samples, in comparison to extracts prepared by fixation and sonication. Histone methylation differences across various genomic sites were maintained within a wide range of autolysis times and tissue pH. Therefore, immunoprecipitation of micrococcal nuclease-digested tissue extracts is a feasible approach to profile histone methylation at defined genomic loci in postmortem brain.
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PMID:Chromatin immunoprecipitation in postmortem brain. 1657 39

Chromatin immunoprecipitation (ChIP) has been used to detect binding of DNA-binding proteins to sites in nuclear and mitochondrial genomes. Here, we describe a method for detecting protein-binding sites on chloroplast DNA, using modifications to the nuclear ChIP procedures. The method was developed using the lac operator (lacO)/lac repressor (LacI) system from Escherichia coli. The lacO sequences were integrated into a single site between the rbcL and accD genes in tobacco plastid DNA and homoplasmic transplastomic plants were crossed with transgenic tobacco plants expressing a nuclear-encoded plastid-targeted GFP-LacI fusion protein. In the progeny, the GFP-LacI fusion protein could be visualized in living tissues using confocal microscopy, and was found to co-localize with plastid nucleoids. Isolated chloroplasts from the lacO/GFP-LacI plants were lysed, treated with micrococcal nuclease to digest the DNA to fragments of approximately 600 bp and incubated with antibodies to GFP and protein A-Sepharose. PCR analysis on DNA extracted from the immunoprecipitate demonstrated IPTG (isopropylthiogalactoside)-sensitive binding of GFP-LacI to lacO. Binding of GFP-LacI to endogenous sites in plastid DNA showing sequence similarity to lacO was also detected, but required reversible cross-linking with formaldehyde. This may provide a general method for the detection of binding sites on plastid DNA for specific proteins.
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PMID:Binding of lac repressor-GFP fusion protein to lac operator sites inserted in the tobacco chloroplast genome examined by chromatin immunoprecipitation. 2048 80

Sequence-specific DNA-binding proteins including transcription factors (TFs) are key determinants of gene regulation and chromatin architecture. TF profiling is commonly carried out by formaldehyde cross-linking and sonication followed by chromatin immunoprecipitation (X-ChIP). We describe a method to profile TF binding at high resolution without cross-linking. We begin with micrococcal nuclease-digested non-cross-linked chromatin and then perform affinity purification of TFs and paired-end sequencing. The resulting occupied regions of genomes from affinity-purified naturally isolated chromatin (ORGANIC) profiles of Saccharomyces cerevisiae Abf1 and Reb1 provide high-resolution maps that are accurate, as defined by the presence of known TF consensus motifs in identified binding sites, that are not biased toward accessible chromatin and that do not require input normalization. We profiled Drosophila melanogaster GAGA factor and Pipsqueak to test ORGANIC performance on larger genomes. Our results suggest that ORGANIC profiling is a widely applicable high-resolution method for sensitive and specific profiling of direct protein-DNA interactions.
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PMID:High-resolution mapping of transcription factor binding sites on native chromatin. 2433 59

MNase-seq allows the genome-wide examination of the nucleosome landscape by determination of nucleosome positioning and occupancy. Typically, native or formaldehyde fixed chromatin is subjected to digestion by micrococcal nuclease (MNase), which degrades linker DNA and yields mainly mono-nucleosomes. The resulting material can be processed directly or can be subjected to an optional chromatin immunoprecipitation step (MNase-ChIP-seq). De-crosslinked and purified DNA is then subjected to next-generation sequencing. The protocol presented here has been tailored for the analysis of nucleosome landscape in the malaria parasite, Plasmodium falciparum, but most steps are directly applicable to other cell types. We also discuss general considerations for experimental design and computational analysis, which are crucial for accurate investigation of the nucleosome landscape.
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PMID:Characterization of the Nucleosome Landscape by Micrococcal Nuclease-Sequencing (MNase-seq). 2902 67


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