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)

Coagulase-negative staphylococci isolated from a patient with a pacemaker electrode infection were extensively evaluated by phenotypic and genotypic characterization. Findings from this evaluation were striking because different colony morphologic subtypes were recovered from blood and resected pacemaker electrodes. Staphylococci from each colony subtype (LBL, LBV, LBP, LBS) were identified as slime-producing strains of Staphylococcus epidermidis sensu stricto. Direct plating of isolates from a restricted electrode revealed a mixture of colony phenotypes when examined on a high-salt, low-glucose medium, Memphis agar. Bacteriophage typing employing 17 different phages and plasmid profile analysis were largely unsuccessful in further characterizing bacterial cells of each of the four colony morphotypes. On the other hand, restriction endonuclease analysis by EcoRI digestion of the chromosomal DNA demonstrated the probable common clonal origin of the four colony phenotypes.
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PMID:Phenotypic variation of Staphylococcus epidermidis in infection of transvenous endocardial pacemaker electrodes. 233 65

We have investigated whether active RNA polymerase I, the enzyme responsible for transcribing ribosomal RNA, is immobilized by attachment to a large subnuclear structure in HeLa cells. As unphysiological salt concentrations induce artifacts, we have used isotonic conditions throughout the preparative and analytic procedures. Cells are encapsulated in agarose microbeads and lysed in Triton and a 'physiological' buffer; then soluble proteins and RNA diffuse out through the agarose pores to leave encapsulated chromatin. This can be manipulated without aggregation but is accessible to molecular probes; it retains the replicational and transcriptional activities of the living cell. After treatment with a restriction endonuclease, most chromatin can be removed from beads by electrophoresis: then active ribosomal genes and polymerase I remain behind. Active ribosomal genes are very accessible to nuclease digestion whilst the rest are even more inaccessible than inactive globin genes. Our observations confirm the complex organization of rDNA within nucleoli and are compatible with transcription occurring at fixed sites. A model for transcription involving an attached polymerase is presented.
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PMID:Active RNA polymerase I is fixed within the nucleus of HeLa cells. 235 67

Three endonucleases from murine plasmacytoma cells that specifically nick DNA which was heavily irradiated with ultraviolet (UV) light were resolved by Sephacryl S-200 column chromatography. Two of these, UV endonucleases I and II, were purified extensively. UV endonuclease I appears to be a monomeric protein with a molecular mass of 43 kDa; UV endonuclease II has an S value of 2.9 S, with a corresponding molecular mass estimated at 28 kDa. Both enzymes act as a class I AP endonuclease, cleaving phosphodiester bonds via a beta-elimination mechanism, so as to form an unsaturated deoxyribose at the 3' terminus. Both have thymine glycol DNA glycosylase activity and their substrate specificities generally appear to be overlapping but not identical. UV endonuclease I acts on both supercoiled and relaxed DNAs, whereas II acts only on supercoiled DNA. Both enzymes are active in EDTA, but have different optima for salt, pH, and Triton X-100. Each enzyme is also present in cultured diploid human fibroblasts.
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PMID:Purification and characterization of UV endonucleases I and II from murine plasmacytoma cells. 246 92

An endonuclease that cleaves ultraviolet light (UV)-damaged, supercoiled plasmid DNA was partially purified from spinach leaves (Spinacia oleracea) by a series of column chromatography steps. Dialysis of the enzyme against EDTA resulted in a greater than 90% loss of activity which could be fully restored following the addition of Zn2+, suggesting that divalent cations are associated with the active enzyme. The spinach endonuclease cleaved duplex, UV-damaged, end-labelled DNA of defined sequence at positions of adenine in the presence of salt (KH2PO4 or NaCl) concentrations of 50 mM or higher. Cleavage of UV-irradiated DNA was dose-dependent and increased steadily within a fluence range of 50-10,000 J/m2. The UV damage requirement and adenine cleavage specificity could be eliminated with lower salt concentrations (0-25 mM), suggesting that the endonuclease recognizes and incises single-stranded DNA. The properties of this enzyme, which we have termed nuclease SP, suggest that it may mediate a role in DNA repair and/or recombination processes in spinach.
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PMID:Partial purification and characterization of an endonuclease from spinach that cleaves ultraviolet light-damaged duplex DNA. 249 26

Two apurinic/apyrimidinic- (AP-) specific endonuclease activities have been isolated from the cells of Dictyostelium discoideum by fractionation on DEAE-cellulose, CM-cellulose and Sephadex G-75. These activities, designated A and B, have apparent molecular weights of 49000 and 40000, respectively. Although their precise reaction optima differ somewhat, both A and B quantitatively nick AP DNA best at pH 8.0-8.5 in low salt (less than 100 mM NaCl); both require Mg2+. These activities are apparently specific only for AP sites in DNA. The low activities observed on heavily ultraviolet-irradiated DNA, gamma-irradiated DNA and osmium tetroxide-treated DNA are consistent with the small numbers of secondary AP sites expected in these DNAs. Both A and B produce single-strand nicks in AP DNA that result in termini that serve as good primers for Escherichia coli polymerase I. Hence, A and B appear to be Class II AP endonucleases which yield 3'-OH termini at nicks on the 5' side of baseless sugars. It is unclear whether A and B are independently coded proteins, different post-translational modifications of the same gene product, or whether one is an artifact arising from the isolation. Many of the properties of these D. discoideum AP endonuclease activities are similar to those of the predominant AP endonucleases observed in bacterial, plant and animal cells. They will be of use in the characterization of excision repair in this organism.
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PMID:Apurinic/apyrimidinic-specific endonuclease activities from Dictyostelium discoideum. 258 May 57

A 12-mer oligodeoxynucleotide containing 10 methylphosphonate bonds and 1 phosphodiester bond was shown to bind specifically to the restriction endonuclease fragment containing complementary DNA in a Southern blot. This 12-mer as well as 14-mer oligodeoxynucleotides containing 3 methylphosphonate and 10 phosphodiester bonds was used to examine the effect of reduced charge on the thermodynamics of binding to complementary DNA or complementary oligodeoxynucleotides with additional nucleotides overlapping both the 3' and 5' ends. The 14-mer oligodeoxynucleotides were synthesized with one methylphosphonamidite (A, C, G, or T). Melting profiles were examined by spectrophotometry for the 14-mers and by a gel-shift assay for the 12-mer. Nearest-neighbor free energy values were compiled for predicting concentration-dependent melting temperatures for all oligodeoxynucleotide hybridizations, including those involving adjacent dG residues. The free energy contribution to duplex formation from the dangling ends was about 1 kcal/mol. The free energy decrement due to introduction of each methylphosphonate linkage was -0.75 kcal/mol in high salt independent of the methylphosphonamidite used for synthesis of the oligodeoxynucleotide. However, the change in charge per nearest-neighbor base pair decreased from 0.26 to 0.0 when the nearest-neighbor base pair contained one methylphosphonate. Thus at very low salt, methylphosphonate-substituted oligodeoxynucleotides form more stable hybrids than analogous phosphodiester sequences. The 12-mer with 10 methylphosphonate bonds outcompetes the analogous phosphodiester 12-mer below 0.01 M NaCl. The temperature of 50% dissociation of bound oligodeoxynucleotide after being washed for 30 min was measured with a dot-blot assay. These results, together with the thermodynamic results, indicate that the substitution of methylphosphonate linkages at high salt only affects the reverse rate constant.
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PMID:Effect of ionic strength on the hybridization of oligodeoxynucleotides with reduced charge due to methylphosphonate linkages to unmodified oligodeoxynucleotides containing the complementary sequence. 271 56

The gene encoding TaqI restriction endonuclease has been subcloned downstream from an inducible phoA promoter. Certain strains of Escherichia coli remain viable when endonuclease is expressed, even in the absence of (protective) methylation. Infecting lambda phage DNA is not restricted in vivo. One E. coli strain, MM294, exhibited a temperature-sensitive phenotype when TaqI endonuclease was induced. This allowed for design of an in vivo plate assay for identification of specially constructed two-codon insertion mutants in the endonuclease gene. These mutants exhibited a wide range of in vitro activities, including wild-type activity, greater activity in low-salt buffer, and sequence-specific nicking activity.
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PMID:A genetic system for isolation and characterization of TaqI restriction endonuclease mutants. 282 88

The restriction endonuclease Alu I induces chromosome-type aberrations in Chinese hamster ovary cells whose frequencies are considerably elevated in the presence of high concentrations of MgCl2, (NH4)2SO4, CaCl2 or NaCl. The most plausible explanation for these findings is that salt leads to partial dehistonization of the chromatin which makes more recognition sites available for Alu I.
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PMID:Potentiation of Alu I-induced chromosome aberrations by high salt concentrations in Chinese hamster ovary cells. 282 43

The vaccinia virus nicking-joining (NJ) enzyme has been purified to homogeneity from a preparation of virus cores. The virus-specific DNA-dependent enzyme, which does not require ATP, is a single polypeptide of Mr 50,000 and possesses both endonuclease and ligase activities. The principal end product of the enzyme activity, following incubation with closed circular DNA of sufficient linking deficiency, is a linear DNA in which one of the termini has become cross-linked by the in vitro formation of a hairpin. The ability of the NJ enzyme to cross-link DNA is significantly enhanced by in vitro proteolysis. The enzymatic properties of the proteolytic digestion product, a 44-kDa polypeptide, differ in several other ways from the intact NJ enzyme. In particular, the specific activity is enhanced and the ionic strength optimum is shifted toward higher salt concentrations. It is suggested that the purified 50-kDa species is a pronuclease that is activated by proteolytic processing.
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PMID:Activation of the vaccinia virus nicking-joining enzyme by trypsinization. 290 31

The structural gene for the single-stranded endonuclease coded for by gene 3 of bacteriophage T7 has been cloned in pGW7, a derivative of the plasmid pBR322, which contains the lambda PL promoter and the gene for the temperature-sensitive lambda repressor, cI857. The complete gene 3 DNA sequence has been placed downstream of the PL promoter, and the endonuclease is overproduced by temperature induction at mid-log phase of Escherichia coli carrying the recombinant plasmid pTP2. Despite the fact that cell growth rapidly declines due to toxic effects of the excess endonuclease, significant amounts of the enzyme can be isolated in nearly homogeneous form from the induced cells. An assay of nuclease activity has been devised using gel electrophoresis of the product DNA fragments from DNA substrates. These assays show the enzyme to have an absolute requirement for Mg(II) (10 mM), a broad pH optimum near pH 7, but significant activity from pH 3 to pH 9, and a 10-100-fold preference for single-stranded DNA (ssDNA). The enzyme is readily inactivated by ethylenediaminetetraacetic acid or high salt. The differential activity in favor of ssDNA can be exploited to map small single-stranded regions in double-stranded DNAs as shown by cleavage of the melted region of an open complex of T7 RNA polymerase and its promoter.
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PMID:Cloning, expression, and purification of gene 3 endonuclease from bacteriophage T7. 293 91


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