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:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cultured human fibroblasts were exposed to various concentrations of actinomycin D, a DNA intercalating agent, and studied by various alkaline elution techniques for the presence of DNA lesions. DNA-protein crosslinks increased proportionately with increasing actinomycin D, with 1.53 crosslinks/10(6) nucleotides after 5 micrograms/ml exposure. However, in the single-strand DNA break assay, elution of DNA initially increased as expected with increasing actinomycin D but thereafter decreased, with only 0.09 breaks/10(6) nucleotides detected after 25 micrograms/ml exposure, suggesting the presence of DNA crosslinking. A standard alkaline elution assay for DNA-DNA crosslinking was performed, and lesions which mimicked such crosslinks were detected, with a relative crosslink frequency of 2.30 after 5 micrograms/ml exposure. These actinomycin D-associated lesions disappeared when the alkaline elution procedure was modified to include additional proteinase digestion and use of the detergent sodium dodecyl sulfate (SDS) in the elution buffer, suggesting that they represented undigested DNA-protein crosslinks or nonspecific protein on the filters inhibiting DNA elution. Greater than ten times as many DNA-protein crosslinks were detected in fibroblasts than the number of single-strand DNA breaks after cellular exposure to actinomycin, even after determining breaks using the modified methodology for decreasing cellular protein interference. The data suggest that the actinomycin-DNA complex is associated with the formation of DNA-protein crosslinks which represent lesions other than endonuclease-associated DNA strand scission.
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PMID:Actinomycin D-associated lesions mimicking DNA-DNA interstrand crosslinks detected by alkaline elution in cultured mammalian cells. 401 12

Mitochondrial DNA (mtDNA)-protein complexes were released from the organelles by sodium dodecyl sulfate-lysis and purified by Phenyl-Sepharose CL-4B chromatography. The mitochondrial DNA-binding protein P16 was the only detectable protein in the complex. Treatment of the complex with proteinase K, or subtilisin, revealed the presence of a protease-insensitive, submolecular domain (Mr approximately equal to 6,000) that retained the capacity to bind tenaciously to the DNA. Analysis of chemically fixed complexes by CsCl isopycnic gradient centrifugation showed that P16 was bound to a large subpopulation of mtDNA enriched in displacement loops (D-loops). Based upon the effective buoyant density of the complex in CsCl gradients and the molecular weights of P16 and mtDNA, it was estimated that a mean of 49 P16 molecules were bound per mtDNA. For this measurement, the variation in hydration of protein and DNA at different CsCl concentrations was ignored. Analysis of restriction endonuclease-digested complexes by glass fiber filters that bind only protein-associated DNA resulted in the retention of a single fragment regardless of the enzyme, or enzymes, used. In each case, the retained fragment was the D-loop-containing fragment. With direct electron microscopy, the protein was readily visualized on the displaced single strand portions of D-loops and expanding D-loops. The nucleoprotein fibers were approximately 12 nm in diameter without correcting for the thickness of tungsten coating and roughly 1/3 the length of the double strand segment of the corresponding D-loop structure. In addition, occasional molecules with the characteristics of gapped circles were seen exhibiting a nucleoprotein fibril, presumably containing the single strand gap segment, linking the ends of double strand DNA. P16 was not seen on the double strand portions in any of the complexes.
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PMID:The rat liver mitochondrial DNA-protein complex: displaced single strands of replicative intermediates are protein coated. 403 99

A site-specific endonuclease (Endo.Sce I) which caused double-strand scission of DNA was highly purified from a eukaryote, Saccharomyces cerevisiae IAM4274. The molecular weight of the active form of Endo.Sce I was estimated to be 120,000 and 110,000 by sedimentation analysis on a glycerol density gradient and gel filtration on Ultrogel AcA34, respectively. Analysis of the fractions from the last column chromatography by polyacrylamide gel-electrophoresis in the presence of sodium dodecyl sulfate and by an assay of the endonucleolytic activities suggested that Endo.Sce I consists of two non-identical subunits with molecular weights of 75,000 and 50,000. Unlike restriction endonucleases, Endo.Sce I was active on chromosomal DNA of the cells which produced Endo.Sce I. Single-stranded DNA was not cleaved by Endo.Sce I, but inhibited the endonucleolytic activity of the enzyme on double-stranded DNA. The endonucleolytic activity of Endo.Sce I required the magnesium ions (Mg2+) as a sole cofactor; Mg2+ could not be replaced by Ca2+ or Zn2+. When Mg2+ was replaced by manganese ions (Mn2+), extensively purified Endo.Sce I cleaved double-stranded DNA at many other sites in addition to the sites at which DNA was cleaved in the presence of Mg2+. Experiments indicated that this is not the activation of contaminating endonuclease in the preparation of Endo.Sce I, but the result of relaxation in the site-specificity of cleavage.
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PMID:Purification of a eukaryotic site-specific endonuclease, Endo.Sce I, from Saccharomyces cerevisiae and effectors on its specificity and activity. 608 75

A restriction endonuclease designated EcoVIII, an isoschizomer of HindIII, was isolated from Escherichia coli E1585-68 and purified by dextran-polyethylene glycol (DPG) phase partition, ammonium sulfate precipitation, phospho- and DEAE-cellulose chromatography, and hydroxylapatite chromatography. The purified EcoVIII was stable during the purification procedure and its high specific activity required 10 mM Mg2+. Unlike HindIII, EcoVIII exhibited a high specific activity even at low pH (pH 6.3) and showed the highest activity at 48 degrees C. Transformation of purified plasmid DNA from E. coli E1585-68 into K-12 indicated that the EcoVIII gene was carried on a multicopy 4.4-kb miniplasmid. EcoVIII seems to be preferable to HindIII for its production and use because of easier handling of producer cells and a wider range of activity.
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PMID:Isolation of restriction enzyme EcoVIII, an isoschizomer of HindIII, produced by Escherichia coli E1585-68. 609 26

We have purified to near homogeneity the single DNA-dependent ATPase activity that we have identified in extracts of KB cell nuclei. The protein structure of the enzyme was defined by sodium dodecyl sulfate gel electrophoresis, which revealed a single protein band of 75000 daltons that was coincident with the profile of ATPase activity resolved by the final step of agarose-ATP chromatography or by isoelectric focusing. The enzyme has a pI of 8.5, a Stokes' radius by gel filtration of 3.8 nm, and a sedimentation coefficient in high salt of 5.3 S. At low ionic strength the enzyme activity sediments at 7.0 S, suggesting that it may dimerize under these conditions. The purified enzyme has a specific activity of 5.9 X 10(5) nmol of ATP hydrolyzed per h per mg of protein and is devoid of endonuclease, exonuclease, RNA or DNA polymerase, nicking-closing, and gyrase activities at exclusion limits of 10(-6)-10(-8) of the ATPase activity. The enzyme can hydrolyze only ATP or dATP, to generate ADP or dADP plus Pi, but the other NTPs and dNTPs are competitive inhibitors of the enzyme with respect to ATP. A divalent cation (Mg2+ greater than Mn2+ greater than Ca2+) as well as a nucleic acid cofactor is required for activity. Single-stranded DNA or deoxyhomopolymers are most effective, but blunt-ended linear and nicked circular duplex DNA molecules are also used at Vmax values approximately 20% of that obtained with single-stranded DNA. Intact duplex DNA and polyribonucleotides are unable to support ATP hydrolysis. Velocity gradient sedimentation studies corroborate the interpretations of the kinetic analyses and demonstrate enzyme binding to single-stranded DNA and nicked duplex DNA but not to intact duplex DNA. Although we have not succeeded directly in demonstrating DNA unwinding by this protein, preliminary results suggest that in the presence of ATP, the ATPase can stimulate the reactivity of homogeneous human DNA polymerases alpha and beta on nicked duplex DNA substrates.
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PMID:Structural and enzymological characterization of a deoxyribonucleic acid dependent adenosine triphosphatase from KB cell nuclei. 610 81

A differential screening procedure was employed to isolate a cDNA clone corresponding to a major phenobarbital (PB)-inducible form of rat hepatic cytochrome P-450. The G-C homopolymer-tailing technique was utilized to construct a cDNA library in the PstI site of plasmid pBR322. The library represented PB-induced poly(A+)RNA sequences from hepatic polysomes of 150-g male Sprague-Dawley rats. Hybrid-selection experiments against total PB-inducible RNA were performed with plasmid DNA derived from clones enriched in PB-inducible information. The mRNA molecules that specifically hybridized were subjected to in vitro translation, were immunoprecipitated with antibody raised in rabbits against purified cytochrome P-450b (P. E. Thomas, D. Korzeniowski, D. Ryan, and W. Levin (1979) Arch. Biochem. Biophys. 192, 524-532), and were electrophoresed under sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoretic conditions. One cDNA clone, designated PB-8, contained a 600-bp insert partially coding for a PB-inducible cytochrome P-450 species that comigrated on SDS-gel electrophoresis with highly purified P-450b. A single injection of PB, 15-18 h before sacrifice, increased the level of polysomal poly(A+)RNA complementary to the isolated cDNA clone by approximately 16-fold. Northern blot hybridizations of polysome-derived poly (A+)RNA, electrophoresed in denaturing agarose gels, demonstrated that the size of the mRNA corresponding to the isolated clone was 4 kb. Isolated heteronuclear RNA species demonstrated a time-dependent increase in the synthesis of a similar 4-kb RNA molecule. By genomic blot hybridization to EcoRI-restricted DNA, at least three complementary DNA fragments migrating at 5.1, 3.2, and 2.9 kb were observed with 32P-labeled PB-8 as a probe. These data, together with restriction endonuclease mapping and partial cDNA sequence information of the PB-8 cDNA, suggest that the PB-8 clone represents a previously unreported cDNA clone for a form of cytochrome P-450 inducible by PB.
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PMID:Molecular induction by phenobarbital of a rat hepatic form of cytochrome P-450: expression of a 4-kilobase messenger RNA. 614 64

Neisseria meningitidis was isolated from the throat, blood, and cerebrospinal fluid of six patients and from the throat and blood of two patients. All 22 isolates were of serogroup B, serotype 15, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis type IV. Isolates from a single patient always possessed identical DNA restriction endonuclease patterns; this observation strongly suggested genomic identity. In spite of this apparent genomic identity, however, significant differences in piliation and adherence were observed among isolates from different sites in the same patient. Isolates from the throat were significantly more piliated (P less than .001) and adhered to human buccal epithelial cells in vitro in significantly higher numbers (P less than .001) than did isolates from the blood and the cerebrospinal fluid of the same patient. These results indicate that phase shift occurs in N. meningitidis in vivo.
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PMID:Isolates of Neisseria meningitidis from different sites in the same patient: phenotypic and genomic studies, with special reference to adherence, piliation, and DNA restriction endonuclease pattern. 614 70

pppA(2'p5'A)n-1 ((2'-5')(A)n) synthetase is one of the mediators of interferon action. On activation by double-stranded RNA, it converts ATP into (2'-5')(A)n; in turn, (2'-5')(A)n activates an endonuclease (RNase L) which cleaves single-stranded RNA. We report a simple procedure for the isolation of pure (2'-5')(A)n synthetase from interferon-treated Ehrlich ascites tumor cells. The procedure involves differential precipitation of the ribosomal salt wash fraction with ammonium sulfate and chromatography on DEAE-cellulose and CM-cellulose. The apparent molecular weight of the enzyme is 105,000 as determined by gel electrophoresis in sodium dodecyl sulfate and about 85,000 when determined by centrifugation through a glycerol gradient. The size range of the (2'-5')(A)n produced by the enzyme extends from the dimer to at least the pentadecamer.
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PMID:Interferon, double-stranded RNA, and RNA degradation. Isolation of homogeneous pppA(2'p5'A)n-1 synthetase from Ehrlich ascites tumor cells. 615 3

SV80 cells, a simian virus 40 (SV40)-transformed derivative of a strain of human fibroblasts, synthesize an 8-kilodalton anti-T reactive polypeptide in addition to large T and small t antigens. Although not observed during lytic infection carried out under a variety of conditions, an anti-T reactive molecule which comigrated with the SV80 8-kilodalton protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis was synthesized by one of five other SV40-transformed cell lines studied. The SV40 8-kilodalton protein was present in lysates of cells exposed to a brief pulse of radioactive methionine and did not accumulate during an extended chase period. This polypeptide could not by generated by mixing an unlabeled extract of SV80 cells with a labeled extract of infected monkey cells. The 8-kilodalton molecule reacts with antibody raised against homogeneous large T antigen, is present only in the cytoplasm, is not complexed with T, lacks DNA-binding properties, and is not phosphorylated. This protein could be translated in a cell-free system programmed by SV40-specific mRNA. At least two messenger species (approximately 19S and approximately 22S) directed its synthesis. Tryptic peptide analysis of [35S]methionine-labeled proteins demonstrated that the 8-kilodalton protein contains all eight of the common T/t peptides and one additional peptide not present in the maps of t or T. It lacks both of the t-unique peptides. The organization of the integrated viral sequences which encode this molecule was determined by restriction endonuclease analysis. In particular, SV80 cells contain at least two integrated SV40 genomes which are oriented in tandem, with an intervening cellular sequence..
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PMID:Identification and initial characterization of a new low-molecular-weight virus-encoded T antigen in a line of simian virus 40-transformed cells. 615 82

One of the mediators of interferon action is an endonuclease system. This consists of (2'-5')(A)n synthetase, which, if activated by double-stranded RNA, converts ATP into (2'-5')(A)n and RNase L, a latent endoribonuclease, which binds (2'-5')(A)n and is thereby activated. We report here that a derivative of (2'-5')(A)n (i.e. (2'-5')pppApApA[32P]pCp) can be covalently cross-linked by UV irradiation to a protein in cytoplasmic extracts from mouse Ehrlich ascites tumor cells. This protein has an apparent molecular weight of 77,000 as determined by gel electrophoresis in sodium dodecyl sulfate. It appears to be identical with RNase L according to the following criteria: co-chromatography on DEAE-cellulose and Sephacryl S300. The gel filtration in Sephacryl S300 reveals that the apparent molecular weight of the protein is between 70,000 and 90,000, indicating that it is a monomer. The cross-linking is oligonucleotide specific. It is inhibited by 10 nM (2'-5')pppApApA or 1 microM (2'-5')ApApA, i.e, compounds known to block, even at low concentration, the binding of (2'-5')pppApApApCp to RNase L. (3'-5')ApApA inhibits only at a 0.1-1 mM concentration, and 1 mM ATP, 2'-AMP, or 5', 3'-pCp have no effect. (2'-5')pppApApApCp was also cross-linked to a protein with a molecular weight of about 78,000 (as determined by gel electrophoresis in sodium dodecyl sulfate) in cytoplasmic extracts from human (HeLa) cells and to protein(s) with molecular weight(s) of 75,000-77,000 (as determined by gel electrophoresis in sodium dodecyl sulfate) in nuclear extracts from Ehrlich ascites tumor cells.
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PMID:Interferon action. Covalent linkage of (2'-5')pppApApA(32P)pCp to (2'-5')(A)n-dependent ribonucleases in cell extracts by ultraviolet irradiation. 617 33


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