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)

EcoRI methylase is able to catalyze methy incorporation into DNA at sequences other than the canonical EcoRI site. At high enzyme concentrations and over a wide range of pH and ionic strengths, EcoRI methylase modifies polyoma DNA (which contains one EcoRI site) at a number of sites. This modification prevents EcoRI endonuclease activity, and thus is presumably at or near the EcoRI sequences (5') NAATTN.
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PMID:Overmethylation of DNAs by the EcoRI methylase. 2 33

The substrate specificity of the EcoRI restriction endonuclease can be varied in vitro by changing the pH and the ionic environment of the reaction. Phosphodiester bond cleavage occurs at a DNA hexanucleotide sequence d(N-G-A-A-T-T-C-N)/d(N-C-T-T-A-A-G-N) when the ionic strength is high, 100 mM Tris-HCl, 50 mM NaCl, 5 mM MgCl2, and the pH is approximately 7.3. Lowering the ionic strength to 25 mM Tris-HCl, 2 mM MgCl2, and adjusting the pH to 8.5 reduces the recognition specificity of the EcoRI endonuclease to the tetranucleotide sequence, d(N-A-A-T-T-N)/d(N-T-T-A-A-N). The enzymatic activity responsible for this substrate recognition is referred to as EcoRI. Cleavage of pVH51 plasmid DNA under EcoRI conditions results in a number of partial digest fragments, some of which disappear slowly over a prolonged digestion period. This suggests that different recognition sites are cleaved at different rates. Comparison of DNA fragment patterns of modified and unmodified pVH51 DNA indicates that the canonical EcoRI sequence is the most rapidly cleaved site under EcoRI conditions. DNA modified in vivo by the EcoRI methylase is not cleaved by the EcoRI endonuclease under standard conditions, but is cleaved under EcoRI conditions at sites other than the standard EcoRI substrate.
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PMID:Specificity of substrate recognition by the EcoRI restriction endonuclease. 24 1

Site-specific genetic recombinations promoted in vivo by the EcoRI endonuclease has been demonstrated by using constructed hybrid plasmids in which the chloramphenicol resistance gene was inactivated by insertion of DNA fragments at an EcoRI site within the gene. Such recombination can involve either the joining of intracellularly generated cohesive termini of the same DNA fragment or intermolecular ligation of different DNA fragments. DNA cleavage and ligation in vivo are precise: recombinant DNA molecules show functional continuity of the gene sequence cleaved by the enzyme and regeneration of nucleotide recognition sites for both the EcoRI endonuclease and the EcoRI DNA methylase. In other experiments, EcoRI-generated fragments of eukaryotic DNA that had not been modified by the Escherichia coli K methylase were shown to be taken up by bacterial cells and to undergo intracellular ligation to segments of bacterial plasmid DNA.
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PMID:In vivo site-specific genetic recombination promoted by the EcoRI restriction endonuclease. 33 2

An endonuclease having EcoRI specificity is produced by bacteria containing the ColE1 plasmid. Such bacterial cells fail to express restriction or modification functions in vivo, and phage or plasmid DNA obtained from ColE1-containing cells has unmodified EcoRI sites that are cleaved in vitro by purified EcoRI endonuclease or by enzyme extracted from bacteria that carry ColE1. No EcoRI DNA methylase activity associated with ColE1 has been detected. The finding of phenotypically cryptic ColE1-dependent EcoRI endonuclease activity and the absence of any detectable EcoRI modification system in ColE1-containing cells suggest a control mechanism that appears to prevent functional expression of the ColE1-determined enzyme in vivo.
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PMID:Phenotypically cryptic EcoRI endonuclease activity specified by the ColE1 plasmid. 34 63

The genes for a Class II restriction-modification system (HhaII) from Haemophilus haemolyticus have been cloned in Escherichia coli. The vector used for cloning was plasmid pBR322 which confers resistance to tetracycline and ampicillin and contains a single endonuclease R-PstI site, (formula: see text), in the ampicillin gene. The procedure developed by Bolivar et al. (1977) was used to form DNA recombinants. H. haemolyticus DNA was cleaved with PstI endonuclease and poly(dC) extensions were added to the 3'-OH termini using terminal deoxynucleotidyl transferase. Circular pBR322 DNA was cleaved to linear molecules with PstI endonuclease and poly(dG) extensions were added to the 3'-OH termini, thus regenerating the PstI cleavage site sequences. Recombinant molecules, formed by annealing the two DNAs, were used to transfect a restriction and modification-deficient strain of E. coli (HB101 r-m-recA). Tetracycline-resistant clones were tested for acquisition of restriction phenotype (as measured by growth on plates seeded with phage lambdacI-0). A single phage-resistant clone was found. The recombinant plasmid, pD110, isolated from this clone, had acquired 3 kilobases of additional DNA which could be excised with PstI endonuclease. In addition to the restriction function, cells carrying the plasmid expressed the HhaII modification function. Both activities have been partially purified by single-stranded DNA-agarose chromatography. The cloned HhaII restriction activity yields cleavage patterns identical to HinfI. A restriction map of the cloned DNA segment is presented.
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PMID:Cloning of restriction and modification genes in E. coli: the HbaII system from Haemophilus haemolyticus. 35 Jul 14

It was shown that E. coli C, E. coli MRE 600 DNA, and also plasmid DNA of Col E1, RSF 2124 from E. coli K-12, and plasmid DNA from E. coli MRE 600 were completely resistant against restriction endonuclease R. Eco RII. Plasmid DNAs of Col E1, RSF 2124 amplificated for 4 hours in the presence of chloramphenicol are sensitive to R. Eco RII but after 16-hour amplification in the presence of chloramphenicol these DNAs acquire complete resistance against R. Eco RII. These data point to the slower rate of modification of DNA in vivo by DC-methylases of Eco RII type in comparison with DNA methylase Eco RII.
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PMID:[Sensitivity of chromosomal and plasmid E. coli DNA to restriction endonuclease Eco RII]. 36 77

A type II restriction endonuclease (endo R . Bsp) has been purified from Bacillus sphaericus to electrophoretic homogeneity. The enzyme appears to be a single polypeptide chain with a molecular weight of 35000. Its pH optimum is around 8.2, it requires 20 mM Mg2+ for optimal activity and it is inhibited by Zn2+. The yield of the enzyme is higher than that of any type II restriction endonuclease so far reported. The enzyme also cleaves single-stranded DNA, albeit at a slower rate. It seems likely that single-stranded DNA is cleaved at the same sequences as double-stranded DNA. Bacillus sphaericus also contains a modification methylase (meth M . Bsp) which completely protects the cell's own DNA against cleavage by its restriction endonuclease. The methylase activity has been partially purified, it copurifies with the nuclease until the next to the last step. The enzyme does not require ATP or Mg2+, it transfers the methyl group of S-adenosyl-methionine to cytosine residues of DNA. As the action of this methylase completely protects any DNA from endo R . Bsp cleavage, it seems likely that the methylase recognizes and methylates the same sequence (dG-dG-dC-dC) as the nuclease.
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PMID:Biochemical characterization of the restriction-modification system of Bacillus sphaericus. 71 Apr 8

Genetic analyses of DNA restriction and modification mechanisms have been encumbered by the inability to rigorously select for mutant phenotypes associated with these systems. The application of restriction endonucleases has now proved to be a successful approach to the genetic analyses of small genomes that are recalcitrant to the more standard genetic techniques. Restriction endonucleases EcoRI and HindIII were used to analyze the structure of the plasmid genome responsible for the EcoRI restriction endonuclease and modification methylase. This plasmid in the original clinical isolate of Escherichia coli appears to be identical to the ColE 1 plasmid except for a 1.95 kilobase pair segment which contains these genes. A preliminary restriction map of this plasmid is presented.
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PMID:A restriction endonuclease analysis of the bacterial plasmid controlling the ecoRI restriction and modification of DNA. 77 5

The nonrestricting/nonmodifying strain Bacillus subtilis 222 (r-m-) can be induced to synthesize a DNA-modifying activity upon treatment with either mitomycin C (MC) or UV light. This is shown by the following facts. (i) Infection of MC-pretreated 222 cells with unmodified SPP1 phage yields about 3% modified phage that are resistant to restriction in B. subtilis R (r+m+). The induced modifying activity causes the production of a small fraction of fully modified phage in a minority class of MC-treated host cells. (ii) The MC-pretreated host cells contain a DNA cytosine methylating activity: both bacterial and phage DNAs have elevated levels of 5-methylcytosine. (iii) The MC-induced methylation of SPP1 DNA takes place at the recognition nucleotide sequences of restriction endonuclease R from B. subtilis R. (iv) Crude extracts of MC-pretreated 222 cells have enhanced DNA methyltransferase activities, with a substrate specificity similar to that found in modification enzymes present in (constitutively) modifying strains.
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PMID:Restriction and modification in Bacillus subtilis: inducibility of a DNA methylating activity in nonmodifying cells. 82 59

The effects of substituents at position 5 in the pyrimidine ring of a variety of phage DNAs upon EcoRI endonuclease and methylase activities have been examined. The replacement of cytidine in DNA with glucosylated hydroxymethylcytidine confers resistance to cleavage by the EcoRI endonuclease. Substitution of thymidine in DNA by hydroxy-methyluridine(a change in the methyl at position 5 of thymidine for a hydroxymethyl) lowers the maximal velocity of endonucleolytic cleavage 20-fold, but has no detectable effect upon the Km. Substitution of thymidine in DNA by uridine (a change in the methyl at position 5 of thymidine for a hydrogen atom) has no effect upon either the maximal velocity or the Km. The effect of these modifications upon EcoRI methylase activity was markedly different. DNA containing glucosylated hydroxymethylcytidine is methylated as well as normal DNA. DNA containing uridine or hydroxy-methyluridine, in place of thymidine, is much more poorly methylated than normal DNA. These different sensitivities of the EcoRI endonuclease and methylase to modifications in the pyrimidine rings of DNA suggest there are significant differences in the manner by which these enzymes recognize and bind to the canonical EcoRI sequence.
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PMID:EcoRI cleavage and methylation of DNAs containing modified pyrimidines in the recogintion sequence. 86 78


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