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 have examined in some detail the chromatin structure of a 6.2 kilobase pair (kbp) chromosomal region containing the chicken beta-globin gene. The chromatin structure was probed with three nucleases, DNase I, micrococcal nuclease, and DNase II, and the rate of digestion of specific subfragments of the region was compared with the rate of bulk DNA digestion. We have characterized the rate of digestion of each fragment in terms of a sensitivity factor which measures the sensitivity of a fragment to a particular nuclease relative to bulk DNA. The sensitivity factors were determined by a least squares curve fitting method based on target analysis. In nuclei isolated from 14-day-old chicken embryo red blood cells, the entire 6.2-kbp region shows approximately a 10- to 20-fold increase in sensitivity to DNase I, a 3-fold increased sensitivity to micrococcal nuclease, and a 6-fold increased sensitivity to DNase II. In addition to the adult beta-globin gene, this region contains 5' and 3' flanking sequences, the 5' half of the inactive, embryonic globin gene, epsilon, and some repeated sequences. There is no obvious correlation between these genetic elements and the overall chromatin structure as measured by the nuclease sensitivity. This same region shows little or no special sensitivity in nuclei isolated from 14-day-old chicken embryo brain. Furthermore, fragments of the inactive ovalbumin gene show little or no sensitivity in either red blood cells or brain. These results support the conclusion that the entire 6.2-kbp region is largely packaged as active chromatin in 14-day-old chicken embryo red blood cells.
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PMID:Chromatin structure of the chicken beta-globin gene region. Sensitivity to DNase I, micrococcal nuclease, and DNase II. 628 52

1. It has been reported that DNase I can be highly purified from pancreas extract by affinity chromatography on a dDNA-Sepharose column under non-digestive conditions. In the present study, the adsorption-elution of other nucleases on the column under non-digestive conditions was studied. 2. All the seven kinds of nucleases tested were adsorbed when applied on a dDNA-Sepharose column under conditions which did not allow the enzymes to hydrolyze the DNA. The non-digestive conditions were as follows. i) For DNase II (pI=10.2), pH 3.0 in the presence of 50 mM sodium sulfate (inhibitor), ii) for micrococcal nuclease (pI=9.6), pH 4.0 in the absence of Ca2+ (activator), iii) for restriction endonucleases Eco RI (pI=5+1), Hind III (pI=5+1), and Bam HI (pI=5+1), pH 4.0 in the presence of 20% glycerol and 0.1% Neopeptone (stabilizers), and iv) for nucleases S1 (pI=5+1) and nuclease P1 (pI=4.5), pH 7.0. At the respective pH's, the enzymes other than nucleases S1 and P1 were cationic so as to exhibit electrostatic attraction to the anionic dDNA-Sepharose. Although S1 and P1 were anionic, they still adsorbed to the column. 3. All the adsorbed nucleases described above were eluted by a concentration gradient of KCl without changing pH. The ionic strengths required for elution were 0.19 for DNase II, 0.53 for micrococcal nuclease, 0.73 for Eco RI, 0.72 for Hind III, 0.37 for Bam HI, 0.17 for P1, and 0.13 for S1. The fact that the ionic strength required for the elution of DNase I (pI=5.0) was 0.39 at pH 4.0 indicates that the former five enzymes except DNase II can be chromatographed with almost the same or higher efficiency than DNase I, because the proteins adsorbed with no-specific affinity could be mostly eluted at lower ionic strength. On the other hand, the fact that nucleases P1 and S1 were adsorbed in spite of electrostatic repulsion suggests that these two enzymes can also be effectively chromatographed, especially when other cationic proteins are previously removed by an appropriate method such as adsorption to a typical cation exchanger.
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PMID:Affinities of various nucleases to DNA-Sepharose under non-digestive conditions: survey for productive affinity chromatography. 628 26

Additional evidence is presented to support our recently reported conclusion that the mitotic factors of mammalian cells, which induce germinal vesicle breakdown and chromosome condensation when injected into fully grown Xenopus laevis oocytes, are localized on metaphase chromosomes. Chromosomes isolated from mitotic HeLa cells were further purified on sucrose gradients and digested for varying periods with either the micrococcal nuclease or DNase II. At each time point of digestion the amount of mitotic factors released was determined by injecting a supernatant of these fractions, obtained by high-speed centrifugation, into oocytes. The amount of DNA rendered acid soluble under the conditions of digestion used was 3% ot 5% of the total chromosomal DNA. The extent of release of mitotic factors with both nucleases was estimated to be about 30% to 40% as evidenced by the reextraction of the undigested chromosomal pellet with 0.2 M NaC1. Similar results were obtained when nuclei from G2 cells were digested under identical conditions. The release of these chromosome-bound mitotic factors by mild digestion with these nucleases though only partial, clearly demonstrates that a significant proportion of these factors are localized on metaphase chromosomes.
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PMID:Chromosome-bound mitotic factors: release by endonucleases. 628 33

Relative to nonreplicating DNA in mature simian virus 40 (SV40) chromosomes, newly synthesized DNA in replicating SV40 chromosomes was found to be hypersensitive to the nonspecific endonucleases, micrococcal nuclease (MNase), DNase I, and DNase II. Nascent DNA, pulse labeled in either intact cells or nuclear extracts supplemented with cytosol, was digested about 5-fold faster and about 25% more extensively than uniformly labeled DNA in mature viral chromosomes. Pulse-chase experiments in vitro revealed a time-dependent chromatin maturation process that involved two distinct steps: (i) conversion of prenucleosomal DNA (PN-DNA) into immature nucleosomal oligomers and (ii) maturation of newly assembled chromatin into a structure with increased nuclease resistance. PN-DNA was hypersensitive to MNase, releasing short DNA fragments which were subsequently solubilized by the nuclease. However, when the nascent PN-DNA was specifically removed by digestion of replicating viral chromosomes with Escherichia coli exonuclease III (3'-5') and phage T7 exonuclease (5'-3'), subsequent digestion of the remaining chromatin with MNase revealed the same degree of hypersensitivity observed prior to exonuclease treatment. Furthermore, newly assembled nucleosomal oligomers, isolated after a brief MNase digestion of replicating viral chromosomes, were also hypersensitive to MNase relative to oligomers isolated from mature chromosomes. Hybridization analysis of the DNA in these immature oligomers revealed that it originated from both sides of replication forks. Inhibition of DNA polymerase alpha by aphidicolin inhibited conversion of PN-DNA into nucleosomes but did not inhibit loss of nucleosomal hypersensitivity to MNase. In contrast, components in the soluble fraction of the subcellular system ("cytosol") were required for both DNA replication and chromatin maturation. Analysis of the nucleoprotein products from a MNase digestion of replicating and mature SV40 chromosomes failed to detect a change in nucleosome structure that corresponded to the loss of nuclease hypersensitivity. However, the results presented demonstrate that both PN-DNA and newly assembled immature chromatin, present on both arms of SV40 replication forks, contribute to the commonly observed hypersensitivity of newly replicated chromatin to endonucleases.
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PMID:Structure of chromatin at deoxyribonucleic acid replication forks: nuclease hypersensitivity results from both prenucleosomal deoxyribonucleic acid and an immature chromatin structure. 631 Dec 55

The positions and relative frequencies of the primary cleavages made by micrococcal nuclease on the DNA of nucleosome core particles have been found by fractionating the double-stranded products of digestion and examining their single-stranded compositions. This approach overcomes the problems caused by secondary events such as the exonucleolytic and pseudo-double-stranded actions of the nuclease and, combined with the use of high resolution gel electrophoresis, enables the cutting site positions to be determined with a higher precision than has been achieved hitherto. The micrococcal nuclease primary cleavage sites lie close (on average, within 0.5 nucleotide) to those previously determined by Lutter (1981) for the nucleases DNase I and DNase II. These similarities show that the accessible regions are the same for all three nucleases, the cleavage sites being dictated by the structure of the nucleosome core. The differences in the final products of the digestion are explained in terms of secondary cleavage events of micrococcal nuclease. While the strongly protected regions of the nucleosome core DNA are common to all three nucleases, there are differences in the relative degrees of cutting at the more exposed sites characteristic of the particular enzyme. In particular, micrococcal nuclease shows a marked polarity in the 3'-5' direction in the cutting rates as plotted along a single strand of the nucleosomal DNA. This is explained in terms of the three-dimensional structure of the nucleosome where, in any accessible region of the double helix, the innermost strand is shielded by the outermost strand on the one side and the histone core on the other. The final part of the paper is concerned with the preference of micrococcal nuclease to cleave at (A,T) sequences in chromatin.
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PMID:Location of the primary sites of micrococcal nuclease cleavage on the nucleosome core. 663 65

The DNA sequences targeted by a complete homologous series of aromatic diamidines have been determined at single-nucleotide resolution via protection from cutting by the endonucleases DNase I, DNase II and micrococcal nuclease. Propamidine, pentamidine and to a lesser extent hexamidine bind selectively to nucleotide sequences composed of at least four consecutive A-T base pairs. In contrast, the binding to DNA of butamidine, heptamidine, octamidine and nonamidine is poorly sequence-selective. Sequences composed of only three consecutive A-T base pairs do not afford a potential binding site for propamidine or the longer homologues, and none of the drugs tolerate the presence of a G-C base pair within the binding site. Experiments with DNA molecules containing inosine in place of guanosine and 2,6-diaminopurine in place of adenine reveal that the lack of binding of propamidine to GC-containing sites is attributable to an obstructive effect of the exocyclic 2-amino group of guanosine. The present data support the view that the local conformation of the double helix (in particular the width of the minor groove) plays a dominant role in the binding reaction and that the capacity of diamidines to recognize AT-rich sequences selectively varies considerably depending on the length of the alkyl chain. The evidence indicates that binding to AT-tracts in DNA must play a role in the biological activity of these diamidines, but there is no simple correlation between binding and pharmacological efficacy.
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PMID:Sequence-selective binding to DNA of bis(amidinophenoxy)alkanes related to propamidine and pentamidine. 917 86

A polyclonal antibody against purified bull seminal plasma Ca2+, Mg2+-dependent endonuclease was raised in a rabbit. The antibody specifically cross-reacted with chromatin-bound Ca2+, Mg2+-dependent endonucleases from bovine thymus, human placenta, and bovine, rat and mouse liver in addition to the bovine seminal enzyme. The antibody did not cross-react with other endonucleases examined, including the acid-endonucleases from bovine thymus and liver, porcine spleen DNase II, micrococcal nuclease, and bovine pancreas DNase I, a known Ca2+ and Mg2+ requiring endonuclease. The present results indicate that this antibody specifically recognizes a class of so-called Ca2+, Mg2+-dependent endonuclease, which is localized in cell nuclei of various tissues and is probably involved in chromatin degradation during apoptosis. The antibody will be used to study the functional role of this class of endonuclease.
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PMID:Anti-Ca2+, Mg2+-dependent endonuclease antibody detects specifically a class of chromatin-bound endonuclease. 924 Apr 53

We prepared synthetic 50-mer DNA duplexes, each containing four mismatched base-pairs in similar positions. We examined their cleavage by DNases I and II, micrococcal nuclease (MNase), methidiumpropyl-EDTA-Fe(II) [MPE-Fe(II)] and hydroxyl radicals. We find that single mismatches only produce subtle changes in the DNase I-cleavage pattern, the most common of which is attenuated cleavage at locations 2-3 bases on the 3'-side of the mismatch. Subtle changes are also observed in most of the DNase II-cleavage patterns, although GT and GG inhibit the cleavage over longer regions and generate patterns that resemble footprints. MNase cleaves the heteroduplexes at the mismatches themselves (except for CC), and in some cases cleaves CpG and CpC steps. None of the mismatches causes any change in the cleavage patterns produced by hydroxyl radicals or MPE-Fe(II). We also examined the cleavage patterns of fragments containing tandem GA mismatches in the sequences RGAY/RGAY and YGAR/YGAR (R, purine; Y, pyrimidine). RGAY causes only subtle changes in the cleavage patterns, which are similar to those seen with single mismatches, except that there are no changes in MNase cleavage. However, YGAR inhibits DNases I and II cleavage over 4-6 bases, and attenuates MPE-Fe(II) and hydroxyl radical cleavage at 2 bases. These changes suggest that this mismatch has a more pronounced effect on the local DNA structure. These changes are discussed in terms of the structural and dynamic effects of each mismatch.
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PMID:Cleavage of fragments containing DNA mismatches by enzymic and chemical probes. 1255 99

Nuclease footprinting techniques were initially developed to investigate protein-deoxyribonucleic acid (DNA) interactions but these tools of molecular biology have also become instrumental for probing sequence-selective binding of small molecules to DNA. Here, the method is described and technical details are given for performing deoxyribonuclease (DNase) I footprinting with DNA-binding drugs. An example is presented where DNase I is used (as well as DNase II and micrococcal nuclease) to probe the patterns of sequence-selective recognition of DNA by the anticancer antibiotic actinomycin D. DNase I is a convenient endonuclease for detecting and locating the position of actinomycin-binding sites within GC-rich sequences.
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PMID:DNase I footprinting of small molecule binding sites on DNA. 1533 13

Apoptosis is commonly associated with the catabolism of the genome in the dying cell. The chromatin degradation occurs in essentially two forms: (1) internucleosomal DNA cleavage to generate oligonucleosomal-length fragments (180-200 bp and multiples thereof), and (2) cleavage of higher order chromatin structures to generate approximately 30-50 Kb fragments. To investigate this component of apoptosis and identify the nuclease(s) responsible, we have developed and utilized an in vitro assay that recapitulates the genomic destruction seen during apoptosis in vivo and allows the simultaneous analysis of both forms of DNA degradation from the same sample. Using this assay we evaluated the digestion patterns of several candidate apoptotic nucleases: DNase I, DNase II, and cyclophilin (NUC18) as well as the bacterial enzyme micrococcal nuclease (not thought to be involved in apoptosis). Chromatin degraded by DNase I formed a smear of DNA on conventional static-field agarose gels and approximately amp;30 - 50 Kb DNA fragments on pulsed field gels. In contrast, DNase II, at a physiologically relevant pH, had no effect on the integrity of HeLa chromatin in either analysis. Similar to DNase I, cyclophilin C produced only approximately 30-50 Kb DNA fragments but did not generate internucleosomal fragments. In contrast, micrococcal nuclease generated both oligonucleosomal and approximately 30-50 Kb DNA fragments. Nuclear extracts from glucocorticoid-treated apoptotic thymocytes generated oligonucleosomal DNA fragments and the larger approximately 30-50 Kb DNA fragments, fully recapitulating both types of apoptotic DNA degradation. Previously, differential sensitivity of nucleases to inhibition by Zn2+ was used to argue that two distinct enzymes mediate approximately 30-50 Kb DNA cleavage and internucleosomal DNA degradation. While, the nuclease activity present in thymocyte nuclear extracts was differentially sensitive to inhibition by Zn2+ during short term incubations it was not during prolonged digestions, suggesting that differences in DNA detection are likely to account for previous results. Together our studies show that none of the nucleases commonly associated with apoptosis could fully recapitulate the DNA degradation seen in vivo.
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PMID:Utilization of an in vitro assay to evaluate chromatin degradation by candidate apoptotic nucleases. 1646 29


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