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)

An endonuclease has been purified more than 300-fold from Escherichia coli infected with bacteriophage T4. The enzyme degrades rapidly sedimenting (greater than 1000 S) DNA in vitro by introducing a limited number of breaks. The substrate is the replicative DNA isolated from cells infected with gene-49-defective phage [Kemper, B, and Janz, E. (1976) J. Virol. 18, 992-999]. Molecules of approximately a third the size of unit-length T4 DNA are exclusively found in a limit digest. The enzyme also reacts with single-stranded DNA from various sources. Heat-denatured T4 DNA is converted into acid-soluble oligonucleotides. Circular single-stranded M13 DNA is linearized by endonucleolytic cleavage causing a reduction of infectivity during transfection. The enzyme behaves like a typical late-gene product. Its activity is 100-fold reduced in cells infected with gene-55-defective phage (defect in expression of late functions). A 30-fold reduction in its specific activity was found in cells infected with gene-49-defective phage suggesting that gene 49 codes for the enzyme or controls its expression. The purified enzyme binds to native or denatured DNA from various sources. The protein has a molecular weight of 42000 as determined by gel filtration and sedimentation analysis. Optimal activity on rapidly sedimenting DNA is obtained at pH 8.6 in Tris/HCl buffer in the presence of 10 mM MgCl2. Some 75% of the activity can be obtained with 7 mM MnCl2. 5 mM CaCl2 has a stimulatory effect on the reaction with MgCl2 or MnCl2 each present at its individual optimal concentration. The enzyme does not require the addition of sulfhydryl reagent for full activity. The reaction can be inhibited by compounds like KCl, spermidine, p-hydroxymercuribenzoate or tRNA.
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PMID:Studies on T4-head maturation. 1. Purification and characterization of gene-49-controlled endonuclease. 626 77

Exonuclease VIII is an enzyme whose synthesis is induced as a result of sbcA mutations. The enzyme has been purified to near homogeneity from an Escherichia coli strain containing an sbcA mutation and mutations in the structural genes for exonuclease III, exonuclease V, and endonuclease I. The enzyme specifically degrades linear duplex DNA in a reaction which requires magnesium ions and is susceptible to inhibition by other divalent cations and by sulfhydryl-blocking reagents. Enzyme activity occurs over a broad pH range with peak activity at pH 8.5 in Tris buffer. The protein has a subunit Mr = 140,000, a sedimentation coefficient of 8.4 +/- 0.6, and a Stokes radius of 142 +/- 6 A, which is consistent with its active form being a multimer. Exonuclease VIII has a frictional coefficient of 2.6 which indicates that it has an asymmetric structure.
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PMID:Exonuclease VIII of Escherichia coli. I. Purification and physical properties. 635 Feb 89

A deoxyribonuclease has been purified 570-fold from the 14-day-old chick embryos. The purified enzyme requires Mg2+ or Mn2+ ions for maximum activity. The optimum pH is 9.0 in 20 mM Tris-HCl buffer. Its isoelectric point is 6.7. NaCl and N-ethylmaleimide strongly inhibit the reaction. An apparent molecular weight of 45,000 is determined by sedimentation in a glycerol density gradient. The enzyme hydrolyzes denatured DNA 50 to 100 times more rapidly than duplex DNA. RNA and synthetic polyribonucleotides are not substrate for the enzyme. DNase A catalyzes the endonucleolytic and exonucleolytic cleavages of single-stranded DNA. The enzyme produces DNA fragments having 70 to 100 nucleotides long at early time of reaction and then degrades these DNA fragments to acid-soluble materials, of which more than 70% is mononucleotides. In the exonucleolytic attack, the enzyme initiates hydrolysis of a single-stranded DNA from 5' to 3' direction. Chick embryo DNA-binding protein gives an intensive effect on the DNase A reaction by inhibiting the endonuclease activity rather than exonuclease activity under the standard assay conditions.
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PMID:Deoxyribonuclease A of chick embryo. Partial purification and characterization of the enzyme. 682 17

A DNA ligase has been purified from a subnuclear soluble replication complex isolated from adenovirus type 2-infected human KB cells. DNA ligase activity could not be demonstrated using an exogenous template until the complex was dissociated, suggesting that the ligase activity may be a component of the complex. The purified enzyme was free of endonuclease, exonuclease, 5'-nucleotidase, and phosphatase activities, and had a molecular weight of 105 000, as estimated by sedimentation in a glycerol gradient. The ligase requires ATP and a divalent cation for activity. The optimum of the reaction is at pH 7.8 in 50--100 mM Tris-HCl buffer and 10--20 mM MgCl2. Monovalent salts greatly stimulate ligase activity and the optimum was found at 150 mM. The reaction is very sensitive to high temperature; maximum activity was observed at 25--30 degrees C. ATP is the sole required cofactor and NAD, dATP and GTP could not replace the requirement for ATP. The Km for ATP is 60 microM. The Km for DNA is 250 microgram/ml or 1.6 nmol of terminal phosphate/ml and thus the enzyme shows relatively weak affinity for exogenous DNA. The maximum conversion of 32P into a phosphatase-resistant form is approximately 1.3% of the total, whereas T4 ligase, under the same conditions, can convert more than 25% of phosphate into a resistant form.
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PMID:Purification and properties of a DNA ligase from a soluble DNA replication complex. 735 2

Apoptosis is a distinct form of cell death characterized by internucleosomal cleavage of DNA, cell membrane blebbing, condensation of nuclear chromatin in the nuclear periphery, and the formation of apoptotic, condensed nuclear bodies. The finding of internucleosomal cleavage of chromatin, perhaps caused by endonuclease activation, has become accepted as a hallmark of this form of cell death. We describe the incidental and artifactual finding of internucleosomal cleavage of chromatin from kidney tissue from normal animals. Nephrectomy was performed in living animals, and renal tissue was digested with proteinase K in 10 mmol/L Tris, 1 mmol/L ethylenediaminetetraacetic acid (EDTA), 10 mmol/L NaCl, and 0.5% sodium dodecyl sulfate. Agarose gel electrophoresis of extracted DNA showed internucleosomal cleavage. Internucleosomal cleavage of DNA was not tissue specific but was evident also in liver DNA from a number of animals. Histologic examination of kidney tissue where DNA exhibited internucleosomal cleavage showed normal morphology, with no evidence of either apoptotic or necrotic cell death. Cleavage was not completely prevented by immediate freezing of kidney tissue in liquid nitrogen before DNA extraction, nor was it prevented by the addition of spermidine, of ethyleneglycol-bis-(beta-aminoethylether)-N,N,N',N'-tetraace tic acid, of phenylmethylsulfonylfluride, or by an increased concentration of NaCl to 100 mmol/L in the digestion buffer. Internucleosomal cleavage of DNA was mostly, although not invariably, inhibited by the use of a digestion buffer containing 10 mmol/L Tris, 25 mmol/L EDTA, and 100 mmol/L NaCl. "Apoptotic" chromatin changes (internucleosomal fragmentation) are not always associated with histologic evidence of apoptosis and may occur artifactually.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Internucleosomal cleavage of DNA as the sole criterion for apoptosis may be artifactual. 803 5

The DNA-binding properties of the FokI restriction endonuclease were studied using the gel-mobility-shift assay. Specific recognition of the cognate sequence and cleavage of DNA are distinguishable functions and can be separated. FokI binds to its recognition site predominantly as a monomer. At high concentrations, FokI exhibits a cooperative recognition sequence-dependent aggregation. In 20 mM KCl/10 mM Tris.HCl buffer, the binding constant of FokI to its cognate site is equal 6.0-7.9 x 10(8)/mol and is lower than the values for most gene-regulatory proteins. FokI binding is 600-1500 times weaker to non-cognate double-stranded DNA than to the GGATG site, and 30,000 times weaker to single-stranded DNA or tRNA. The method of Bading [Nucleic Acids Res. 16 (1988) 5241-5248], used for determining the stoichiometry of protein bound to DNA by gel-mobility-shift assay, is extended.
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PMID:Atypical DNA-binding properties of class-IIS restriction endonucleases: evidence for recognition of the cognate sequence by a FokI monomer. 816 73

A rapid screening assay for Campylobacter fetus in bull semen was developed using the polymerase chain reaction (PCR) and restriction endonuclease analysis (REA) to complement isolation by culture. An oligonucleotide primer pair (C1/C2) from the hypervariable region of 16S rRNA of C. fetus was used to amplify a 362 base pair fragment by PCR. The PCR/REA assay, which is completed in 10 hours, detected as few as three C. fetus subsp. venerealis cells in experimentally infected raw bull semen and in semen diluted with milk or egg yolk Tris (EYT). All the strains tested, of both subspecies of C. fetus, were amplified, as were some other Campylobacter species. Restricting the amplified products by AluI differentiated C. fetus from the other organisms. There was no visible product generated by PCR from C. sputorum subsp. bubulus, a saprophytic organism found in the prepuce of bulls, or from seven other species of bacteria found in semen. A modification of the PCR assay, using another primer pair (C3/C2) and two temperature PCR cycling conditions, increased the probability of detecting C. fetus subsp. venerealis. PCR amplification followed by REA could be used to screen bovine semen rapidly for C. fetus. In most cases, sequencing of C1/C2 PCR generated products would be preferable for distinguishing between the two subspecies of C. fetus.
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PMID:A detection assay for Campylobacter fetus in bovine semen by restriction analysis of PCR amplified DNA. 854 Feb 37

We purified an apurinic/apyrimidinic (AP) endonuclease from mouse ascites sarcoma (SR-C3H/He) cells. The enzyme showed nicking activity on acid-depurinated DNA but not on untreated, intact DNA. It also showed priming activity for DNA polymerase on both acid-depurinated and bleomycin-damaged DNA. The priming activity on bleomycin-damaged DNA was two times higher than that on an acid-depurinated DNA. The enzymatic properties indicate that the enzyme is a class II AP endonuclease having DNA 3' repair diesterase activity. The purified enzyme has a molecular weight of 39,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimal pH for AP endonuclease activity was 8.0 in 50 mM Tris-HCl buffer. The AP endonuclease activity depended on divalent cation such as Mg2+ and Co2+ ions, and was inhibited by 2 mM EDTA with no addition of the divalent cation. An appropriate concentration of sodium or potassium salt stimulated the activity. Partial digestion of the AP endonuclease with Staphylococcus aureus V8 protease produced 4 major peptide fragments which may be used for protein sequencing.
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PMID:Purification and characterization of a 39kDa apurinic/apyrimidinic endonuclease from mouse ascites sarcoma cells. 880 52

A new restriction endonuclease was isolated from the Bacillus cereus BKM B-814 by means of the cell disruption with ultrasonication, ammonium sulfate fractionation of the cell-free extract, and chromatography on DEAE-Sepharose to give about 1400 U of the enzyme per gram of cells. The enzyme revealed the maximum activity at 30-37 degrees C, pH 7.6-8.2, and 5-10 mM MgCl2 under a high ionic strength (50 mM Tris-HCl, 100 mM NaCl). The site-specific endonuclease BcuAI was found to recognize the 5' G decreases G(A/T)CC sequence in double-stranded DNA and cleave it as shown with the arrow, thus being a true isoschisomer of the AvaII restriction endonuclease.
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PMID:[Site-specific BcuAI endonuclease from Bacillus cereus A]. 899 58

The chromatographic purification of (His)6EcoRV, a fusion protein consisting of a hexahistidine affinity domain and restriction endonuclease EcoRV produced from recombinant Escherichia coli, led to high product concentrations (> or = 1 mg/ml) in the preparative mode. Increasing the amount of applied crude cell homogenate caused competition with host-specific proteins was achieved by pre-adsorption on to a DEAE anion-exchange sorbent. This, in combination with 0.5-1 M NaCl in the adsorption buffer, assured a purity > 95% and a total protein recovery of approximately 34% in the preparative mode. Contamination of the product with about 2 mol of Ni(11)/mol of (His)6EcoRV was found due to metal-ion transfer to the N-terminal high-affinity binding site at (His)6. Tris(carboxymethyl)ethylenediamine (TED)-Sepharose was employed as an Ni(11) adsorber. One passage of Ni(11)-contaminated protein solutions through the TED-Sepharose column resulted in a decrease in the Ni(11) content in the (His)6EcoRV fractions below the detection limit (approximately 0.02 mg/l) of the atomic-adsorption spectrophotometer.
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PMID:Purification of (His)6EcoRV [recombinant restriction endonuclease EcoRV fused to a (His)6 affinity domain] by metal-chelate affinity chromatography. 912 87


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