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)

HIV-IN protein, tagged with a hexahistidine tail was expressed in Escherichia coli and purified by a one-step nickel chelate affinity chromatography procedure. The purified IN protein was characterized in terms of its endonuclease and integrase properties in vitro. Specific cleavage and integration of HIV U5 LTR ends were observed in the presence of 2-5 mM Mg2+ or Ca2+. In the presence of 2 mM Mn2+, cleavage and integration occurred at additional sites indicating a decreased specificity. The properties of mutant IN proteins were examined in vitro. Deletion of 39 amino acids from the N-terminus and a minimum of 25 residues from the C-terminus impaired IN-mediated cleavage and integration activities. The results of site-directed mutagenesis experiments showed that residues critical for IN function are highly conserved. Mutations of conserved residues Asp64, Pro109, Asp116, and Glu152 adversely affected IN function in vitro. Mutations of nonconserved residues Gly189 and Thr112 had no effect. Mutation of a conserved Thr115 to Ala caused a near complete loss of Mg(2+)-dependent integration activity, but only partially effected endonucleolytic cleavage activity of IN. These results suggest that not all conserved residues are involved in both endonucleolytic cleavage and integration activities of HIV-IN.
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PMID:Identification of amino acid residues critical for endonuclease and integration activities of HIV-1 IN protein in vitro. 158 29

Treatment of rat thymocytes with micromolar concentrations of tributyltin caused a rapid increase in the cytosolic free Ca2+ concentration that was inhibited by Ni2+, which blocks Ca2+ influx through membrane channels. The elevation of cytosolic Ca2+ was associated with extensive DNA fragmentation, which was prevented by pretreatment of the cells with either of the intracellular Ca2+ chelators quin-2 or 1,2-bis(2-amino-phenoxy)ethane-N',N',N',N',-tetraacetic acid. Loss of thymocyte viability, which followed DNA fragmentation, was also prevented by the two Ca2+ chelators or by removing extracellular Ca2+ with ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid. The pattern of DNA fragmentation was characteristic of that produced by agents which activate a Ca2(+)- and Mg2(+)-dependent endogenous endonuclease during apoptosis or programmed cell death. Additional studies showed that other organotin compounds, including trimethyltin, triphenyltin, and dibutyltin had minimal effects on cytosolic Ca2+, DNA fragmentation, and cell viability. These results are consistent with a greater susceptibility of thymocytes to tributyltin and provide a basis for understanding its selective immunotoxicity in vivo.
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PMID:Tributyltin stimulates apoptosis in rat thymocytes. 224 Nov 74

The 163-kilobase-pair (kb) plasmid pMOL28, which determines inducible resistance to nickel, cobalt, chromate, and mercury salts in its native host Alcaligenes eutrophus CH34, was transferred to a derivative of A. eutrophus H16 and subjected to cloning procedures. After Tn5 transposon mutagenesis, restriction endonuclease analysis, and DNA-DNA hybridization, two DNA fragments, a 9.5-kb KpnI fragment and a 13.5-kb HindIII fragment (HKI), were isolated. HKI contained EK1, the KpnI fragment, as a subfragment flanked on both sides by short regions. Both fragments were ligated into the suicide vector pSUP202, the broad-host-range vector pVK101, and pUC19. Both fragments restored a nickel-sensitive Tn5 mutant to full nickel and cobalt resistance. The hybrid plasmid pVK101::HKI expressed full nickel resistance in all nickel-sensitive derivatives, either pMOL28-deficient or -defective, of the native host CH34. The hybrid plasmid pVK101::HKI also conferred nickel and cobalt resistance to A. eutrophus strains H16 and JMP222, Alcaligenes hydrogenophilus, Pseudomonas putida, and Pseudomonas oleovorans, but to a lower level of resistance. In all transconjugants the metal resistances coded by pVK101::HKI were expressed constitutively rather than inducibly. The hybrid plasmid metal resistance was not expressed in Escherichia coli. DNA sequences responsible for nickel resistance in newly isolated strains showed homology to the cloned pMOL28-encoded nickel and cobalt resistance determinant.
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PMID:Cloning of pMOL28-encoded nickel resistance genes and expression of the genes in Alcaligenes eutrophus and Pseudomonas spp. 254 12

The restriction endonuclease FokI from Flavobacterium okeanokoites was purified to homogeneity. Based on gel filtration, sedimentation and sodium dodecyl sulfate-polyacrylamide-gel electrophoresis, the following properties of the enzyme were determined: FokI exists in one active monomeric form, and has an Mr of 64-65.4 x 10(3).FokI is a strongly basic protein with an isoelectric point of 9.4. The enzyme exhibits restriction activity in the pH range 5.0 to 10.5 (maximum level at pH 7.0-8.5) and its divalent cation requirement is satisfied not only by Mg2+, but also by Co2+, Mn2+, Ni2+, Cd2+, Zn2+ and Fe2+.
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PMID:Purification and characterization of the FokI restriction endonuclease. 258 11

The synthesis of chelating sorbents for ligand-exchange chromatography of enzymes is described. An inorganic support "Silochrom" and organic "Spheron", TSK-Gel HW 55 and cellulose were used as initial supports. The chelating sorbents contained iminodiacetic acid and iminodimethylphosphonic acid as stationary ligands. In order to obtain monofunctional sorbents, iminodiacetic acid was added in the form of its dimethyl ester. The concentration of stationary ligands on the sorbents varied from 10 to 100 mumol per ml sorbent. A chelating sorbent (in nickel form) was shown to be effective in the purification of exonuclease A5 from actinomyces. Electrophoretically homogeneous exonuclease A5 was obtained with a 25% yield. A chelating sorbent with iminodiacetic groups (in copper form) was applied to the isolation of endonuclease from Serratia marcescens directly from the culture medium. The capacity of the chelating sorbents for the endonuclease was studied as a function of the stationary ligand concentration. After one stage of purification, more than 70% pure enzyme was obtained with a yield exceeding 80%.
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PMID:Ligand-exchange chromatography of nucleases. 302 93

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 specific type-II restriction endonuclease BcnI from Bacillus centrosporus has been purified to electrophoretic homogeneity in three chromatographic steps. Around 15 micrograms of such a preparation can be isolated from 1 g of the cell paste. The yield of the enzyme is higher than that of any type-II restriction endonuclease so far reported. The molecular weight of the enzyme determined by gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate equals 27 500 and 28 000 respectively. The activity of the restriction endonuclease is maximal at pH 9.2 and 40--45 degrees C. The optimal magnesium concentration was estimated to be 7.5 mM. The activity of BcnI may also be observed in the presence of Co2+, Mn2+, Ni2+ and Zn2+ but it is markedly less than in the presence of Mg2+.
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PMID:Isolation and some properties of the restriction endonuclease BcnI from Bacillus centrosporus. 627 27

The bacterial expression plasmids, pET3b and pET16b, that contain the integrase domain of the human foamy virus (HFV) reverse transcriptase were constructed and expressed in Escherichia coli. The histidine-tagged HFV IN protein was purified to near homogeneity by single-step Ni2+ chelate affinity chromatography. HFV-specific proteins of 39 and 120 kDa from virus-infected cells reacted with antisera raised against the recombinant IN protein. Purified recombinant HFV IN protein was active as an endonuclease specifically cleaving two nucleotides from a 20-bp oligodeoxynucleotide substrate that mimics the authentic 5' ends of HFV DNA. Substrates with mutations relatively close to the cleavage site were less efficiently cleaved or not cleaved at all compared with the HFV U5 DNA end. The purified recombinant protein was active as integrase with double-stranded oligodeoxynucleotide substrates. The reverse reaction of DNA strand transfer, the disintegration activity, was shown by efficient cleavage of an intermediate Y-shaped oligodeoxynucleotide. In the presence of Mn2+ as the preferred divalent cation, oligodeoxynucleotides were specifically and efficiently cleaved. In contrast, endonucleolytic cleavages in the presence of Mg2+ ions led to a broad range of reaction products with the His-tagged HFV IN protein. After further purification of the HFV IN by cation-exchange chromatography, the unspecific degradation of oligonucleotide substrate in the presence of Mg2+ was not detectable.
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PMID:Endonucleolytic cleavages and DNA-joining activities of the integration protein of human foamy virus. 768 24

Spontaneous mutants that were resistant to zinc were isolated from Alcaligenes eutrophus CH34 containing either the native plasmid pMOL28 or a derivative derepressed for its self-transfer, pMOL50. With the cured plasmid-free derivative of CH34, strain AE104, such mutants were not detected. The mutations, which were shown to be located in the plasmid, increased the level of the nickel and cobalt resistance determined by the cnr locus. The chromate resistance closely linked to the cnr locus was not affected by these mutations. In the Znr mutants, the resistance to zinc and nickel was constitutively expressed. Uptake studies showed that the zinc resistance in a Znr mutant resulted from reduced accumulation of zinc ions in comparison with that in the plasmid-free strain. Reduced accumulation of zinc was also observed to a lesser degree in the parental strain induced with nickel, suggesting that zinc interferes with the Ni2+ and Co2+ efflux system. A 12.2-kb EcoRI-XbaI restriction endonuclease fragment containing the cnr locus was cloned from plasmid pMOL28 harboring the mutation and shortened to an 8.5-kb EcoRI-PstI-PstI fragment conferring resistance to zinc, nickel, and cobalt. The 12.2-kb EcoRI-XbaI fragment was also reduced to a 9.7-kb BamHI fragment still encoding weak resistance to nickel and cobalt but not to zinc. Complementation studies demonstrated the recessivity of the cnr mutations with a Znr phenotype. Such mutations thus allow positive selection of mutants affected in the expression of the cnr operon.
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PMID:A new type of Alcaligenes eutrophus CH34 zinc resistance generated by mutations affecting regulation of the cnr cobalt-nickel resistance system. 842 50

Intron-encoded endonucleases are distinguished by their ability to catalyze the cleavage of double-stranded DNA with high specificity. I-PpoI endonuclease, an intron-encoded endonuclease from the slime mold Physarum polycephalum, is a small enzyme (2 x 20 kDa) that catalyzes the cleavage of a large asymmetric DNA sequence (15 base pairs). Here, the interactions of I-PpoI with its substrate were examined during both binding (in the absence of Mg2+) and catalysis (in the presence of Mg2+). Using circular permutation assays, I-PpoI was shown to bend its substrate by 38 +/- 4 degrees upon binding. Two independent methods, gel mobility shift assays and fluorescence polarization assays, revealed that I-PpoI binds tightly to its substrate. Values of Kd range from 3.3 to 112 nM, increasing with increasing NaCl concentration. Similar salt effects on the values of Km were observed during steady-state catalysis. At low salt concentrations, the value of kcat/Km for the cleavage of an oligonucleotide duplex approaches 10(8) M-1 s-1. Although other divalent cations can replace Mg2+, catalysis by I-PpoI is most efficient in the presence of an oxophilic metal ion that prefers an octahedral geometry: Mg2+ > Mn2+ > Ca2+ = Co2+ > Ni2+ > Zn2+. Together, these results provide the first chemical insight into substrate binding and turnover by an intron-encoded endonuclease.
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PMID:Substrate binding and turnover by the highly specific I-PpoI endonuclease. 854 43


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