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)

Electron microscopy of UV-irradiated circular DNA molecules which had been treated with T4 endonuclease V revealed the formation of multimeric DNA structures in addition to the expected conversion of the superhelical DNA molecules into nicked circular and linear forms. The multimeric DNA molecules could be distinguished in electron micrographs from catenated molecules which were present in the original DNA preparation by a combination of rotary and single angle heavy metal shadowing. The complexity and frequency of these structures increased with time of reaction with endonuclease V. Their formation, as well as the endonuclease activity of enzyme, was dependent on UV irradiation of the DNA, and the complexes could be disrupted by prior phenol extraction and ethanol precipitation. Preparations of endonuclease V estimated to be 98% pure by mass promoted the same complex formation between DNA molecules as did preparations estimated to be only 5-10% pure. In addition to these intermolecular structures, the formation of complexes between regions on the same DNA molecules was manifest as discrete double-stranded 'loops' 200-300 base pairs in length. DNA 'bubble structures' were also observed and may represent folding of the 'loops' onto adjacent segments of DNA. These results suggest that at least one active form of T4 endonuclease V may be a multimeric complex of enzyme molecules in association with DNA.
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PMID:T4 endonuclease V promotes the formation of multimeric DNA structures. 354 3

With the use of the strain-overproducer restriction endonuclease R.EcoRV was isolated and purified to homogeneity. The molecular mass of the enzyme was determined by gel filtration and polyacrylamide gel electrophoresis to be 25 000 daltons. According to the data of immunological tests R.EcoRV differs in its antigenic characteristics from restriction endonucleases R.EcoRI and R.EcoRII. Dependence of enzyme activity on pH, ionic strength, temperature, presence of divalent cations (Mn2+, Mg2+, Co2+, Zn2+, Ni2+ and Cd2+) and organic solvents (glycerol, dimethylsulfoxide, ethanol) has been studied. It was shown that under conditions of replacement of Mg2+ for Mn2+ or after addition of organic solvents relaxation of R.EcoRV specificity takes place. It was shown also that R.EcoRV is able to digest T-even bacteriophage DNAs with different types and extents of modification. DNA modified by the action of MR.EcoRV system in vivo is susceptible to R.EcoRV in vitro. Under conditions of relaxed specificity noncanonical sites are susceptible to R.EcoRV attack. The fragments resulted may be cloned in canonical pBR322 EcoRV site.
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PMID:[EcoRV restrictase: physical and catalytic properties of homogenous enzyme]. 620 Jul 65

A new procedure is developed for isolation of highly purified preparations of restrictional endonoucleases Bam HI and Eco RI by means of fractionation with isopropyl alcohol. Restrictional endonuclease Bam HI, practically free of unspecific nucleases, was isolated after ultrasonic destruction of cells, precipitation of the restrictases with isopropanol and chromatography on DEAE cellulose. Additional chromatography on hydroxyapatite enabled to obtain the homogenous preparation of Bam HI restrictase, as shown by polyacrylamide gel disc electrophoresis. Other organic solvents (acetone, ethanol) might be also used for purification of the restrictional endonucleases.
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PMID:[New means of isolating restriction endonuclease preparations using organic solvents]. 625 63

Purified DNA was modified in vitro by 3H-labelled O-acetyl or O,O'-diacetyl-4-hydroxyaminoquinoline-1-oxide (Ac4HAQO or di Ac-4HAQO). It was then subjected to the action of the single-stranded DNA specific nuclease S1 and the digested fractions were analysed. For both types of modified DNA, the release of non-modified nucleotides was faster than the release of modified nucleotides. This result is at variance with that obtained with acetoxy-acetylaminofluorene-modified DNA: in the latter case, the modified nucleotides were preferentially released. The results suggest that the S1 endonuclease can recognize different conformational changes in DNA, which depend on the carcinogen used. The enzymatic activity (or activities) present in Micrococcus luteus cell extracts released ethanol-soluble products from Ac-4HAQO modified DNA.
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PMID:In vitro enzymatic recognition of DNA modified by O,O'-diacetyl or O-acetyl derivatives of the carcinogen 4-hydroxyaminoquinoline-1-oxide. 628

A technique for conveniently radiolabeling DNA restriction endonuclease fragments to high specific activity is described. DNA fragments are purified from agarose gels directly by ethanol precipitation and are then denatured and labeled with the large fragment of DNA polymerase I, using random oligonucleotides as primers. Over 70% of the precursor triphosphate is routinely incorporated into complementary DNA, and specific activities of over 10(9) dpm/microgram of DNA can be obtained using relatively small amounts of precursor. These "oligolabeled" DNA fragments serve as efficient probes in filter hybridization experiments.
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PMID:A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. 631 38

Based on the results of a systematic study of factors affecting plasmid yield and purity, a procedure suitable for the rapid screening for and isolation of covalently closed circular DNA from Streptomyces lividans and Escherichia coli was developed. The method consists of lysis of lysozyme-treated bacteria combined with alkaline denaturation of DNA at high temperature. Renaturation of CCC DNA and precipitation of single-stranded DNA together with protein is achieved by the addition of a minimal amount of phenol/chloroform. The screening procedure uses only a single tube and the samples can be analyzed by agarose gel electrophoresis about 30 min after lysis. Removal of phenol and further purification of the plasmid preparation is achieved by consecutive precipitations with isopropanol and spermine, followed by extraction with ethanol, producing samples suitable for restriction endonuclease digestion, ligation, and transformation of S. lividans protoplasts or competent E. coli cells in about 2 h. All steps of the procedure are explained in detail with information about the effects of changing parameters. This should help the experimenter to obtain reproducible results and may be useful if the method has to be adapted to new strains or plasmids.
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PMID:Factors affecting the isolation of CCC DNA from Streptomyces lividans and Escherichia coli. 638 33

Poly (dG-dC) . poly(dG-dC) was modified by the reaction with N-acetoxy-N-acetyl-2-aminofluorene. The conformations of poly(dG-dC) . poly(dG-dC) and of poly d(G-C)AAF were studied by circular dichroism under various experimental conditions. In 95% ethanol, the two polynucleotides adopt the A-form. In 3.9 M LiCl, the transition B-form-C-form is observed with poly(dG-dC) . poly (dG-dC) but not with poly d(G-C)AAF. In 1 mM phosphate buffer, poly d(G-C)AAF behaves as a mixture of B- and Z-form, the relative percentages depending upon the amounts of modified bases. The percentage of Z-form is decreased by addition of EDTA and is increased by addition of Mg++. Spermine favors the Z-form in modified and unmodified polynucleotides. No defect in the double helix of poly d(G-C)AAF is detected by SI endonuclease.
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PMID:Conformational changes of poly(dG-dC) . poly(dG-dC) modified by the carcinogen N-acetoxy-N-acetyl-2-aminofluorene. 723 16

The recognition of 'regular' and 'oxidized' sites of base loss (AP sites) in DNA by various AP endonucleases was compared. Model substrates with regular AP sites (resulting from mere hydrolysis of the glycosylic bond) were produced by damaging bacteriophage PM2 DNA by exposure to low pH; those with AP sites oxidized at the C-4'- and C-1'-position of the sugar moiety by exposure to Fe(III)-bleomycin in the presence of H2O2 and to Cu(II)-phenanthroline in the presence of H2O2 and ethanol, respectively. The results confirmed that AP sites-together with single-strand breaks-are indeed the predominant type of DNA modification in all three cases. For the recognition of 4'-oxidized AP sites, a 400-fold higher concentration of Escherichia coli exonuclease III and between 5-fold and 50-fold higher concentrations of bacteriophage T4 endonuclease V, E. coli endonuclease III and E. coli FPG protein were required than for the recognition of regular AP sites. In contrast, the recognition of 4'-oxidized AP sites by E. coli endonuclease IV was effected by 4-fold lower concentrations than needed for regular AP sites. 1'-oxidized AP sites (generated by activated Cu(II)-phenanthroline) were recognized by endonuclease IV and exonuclease III only slightly (3-fold and 13-fold, respectively) less efficiently than regular AP sites. In contrast, there was virtually no recognition of 1'-oxidized AP sites by the enzymes which cleave at the 3' side of AP sites (T4 endonuclease V, endonuclease III and FPG protein). The described differences were exploited for the analysis of the DNA damage induced by hydroxyl radicals, generated by ionizing radiation or Fe(III)-nitrilotriacetate in the presence of H2O2. The results indicate that both regular and 1'-oxidized AP sites represent only minor fractions of the AP sites induced by hydroxyl radicals.
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PMID:Recognition of oxidized abasic sites by repair endonucleases. 751 77

We screened members of a new genus of grass-associated diazotrophs (Azoarcus spp.) for the presence of cellulolytic enzymes. Out of five Azoarcus strains representing different species, only in the endorhizosphere isolate BH72, which is also capable of invading grass roots, was significant endoglucanase activity, in addition to beta-glucosidase and cellobiohydrolase activity, present. Reducing sugars were readily released from medium-viscosity carboxymethylcellulose (CMC), but neither CMC, cellulose filter strips, Avicel, cellobiose, nor D-glucose served as the sole carbon source for growth of Azoarcus spp. Clones from a plasmid library of strain BH72 expressed all three enzymes in Escherichia coli, apparently not from their own promoter. According to restriction endonuclease mapping and subclone analysis, beta-glucosidase and cellobiohydrolase activities were localized on a single 2.6-kb fragment not physically linked to a 1.45-kb fragment from which endoglucanase (egl) was expressed. Two isoenzymes of endoglucanase probably resulting from proteolytic cleavage had pI values of 6.4 and 6.1 and an apparent molecular mass of approximately 36 kDa. Cellobiohydrolase and beta-glucosidase activity were conferred by one enzyme 41 kDa in size with a pI of 5.4, which we classified as an unspecific exoglycanase (exg) according to substrate utilization and specificity mapping; hydrolysis of various oligomeric substrates differentiated it from endoglucanase, which degraded substituted soluble cellulose derivatives but not microcrystalline cellulose. Both enzymes were not excreted but were associated with the surface of Azoarcus cells. Both activities were only slightly influenced by the presence of CMC or D-glucose in the growth medium but were enhanced by ethanol. egl was located on a large transcript approximately 15 kb in size, which was detectable only in cells grown under microaerobic conditions on N2. Surface-bound exo- and endoglucanases with some unusual regulatory features, detected in this study in a strain which is unable to metabolize cellulose or sugars, might assist Azoarcus sp. strain BH72 in infection of grass roots.
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PMID:Cloning, expression in Escherichia coli, and characterization of cellulolytic enzymes of Azoarcus sp., a root-invading diazotroph. 769 55

The conjugative transposon Tn916 was used for mutagenesis of Clostridium acetobutylicum ATCC 824. Tetracycline-resistant mutants were screened for loss of granulose synthesis and five classes of granulose mutants, that contained single transposon insertions, were identified on the basis of altered solvent production. Class 1 mutants did not make acetone or butanol, lacked activity of enzymes induced during solventogenesis, and did not sporulate, indicating that they are regulatory mutants. The class 2 mutant strains also did not produce acetone but did form small amounts of butanol and ethanol, while the class 3 mutants produced low amounts of all solvents. Class 4 and 5 mutants produced essentially the same or higher amounts of solvents than the parent strain. Transposon insertions in the class 1 mutants were used as markers for in vitro synthesis of flanking chromosomal DNA using Tn916-specific primers. The DNA fragments were labeled to produce specific probes. Transposon insertion sites in the chromosomes of 13 different class 1 regulatory mutants were compared by hybridization of the specific probes to Southern blots of restriction endonuclease-digested parental chromosomal DNA. Insertions in two mutants appeared to be in the same region of the chromosome. These results predict that multiple regulatory elements are required to induce solvent production and sporulation.
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PMID:Analysis of Tn916-induced mutants of Clostridium acetobutylicum altered in solventogenesis and sporulation. 776 50


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