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)

We have constructed a synthetic photoendonuclease composed of T7 RNA polymerase linked to rose bengal. The promoter-specific polymerase confers site-specific binding, and the photosensitizer rose bengal allows light-induced DNA cleavage. Using a gentle labeling procedure, we find that the polymerase can be labeled with 1-30 rose bengals. Polymerase labeled to about 8 rose bengals per molecule retains the same efficiency and specificity of binding to promoter-containing DNA as unlabeled polymerase. At this level of rose bengal substitution, the synthetic endonuclease, in the presence of visible light, specifically cleaves linear or supercoiled DNA containing a T7 promoter. It induces frank single-strand breaks, rather than labile sites convertible to breaks upon additional treatments. Neither the free rose bengal moiety not bonded to polymerase nor the free (not bound to DNA) rose bengal-substituted polymerase cleaves DNA. Although rose bengal is an efficient generator of singlet oxygen, depletion of oxygen from reaction mixtures increases the cleavage rate. This indicates that singlet oxygen cleavage is not a major mechanism of DNA nicking by the synthetic endonuclease. At higher levels of rose bengal substitution, the labeled polymerase shows decreased binding efficiency and increased nonspecific binding to DNA without a T7 promoter; the specificity of DNA cleavage also decreases. These results indicate that the site specificity of rose bengal photocleavage by the synthetic endonuclease results from specific binding of the polymerase, and thus rose bengal photonicking reflects polymerase binding.
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PMID:Promoter-specific synthetic photoendonuclease: rose bengal-labeled T7 RNA polymerase. 843 39

The crystal structure of EcoRV endonuclease has been determined at 2.5 A resolution and that of its complexes with the cognate DNA decamer GGGATATCCC (recognition sequence underlined) and the non-cognate DNA octamer CGAGCTCG at 3.0 A resolution. Two octamer duplexes of the non-cognate DNA, stacked end-to-end, are bound to the dimeric enzyme in B-DNA-like conformations. The protein--DNA interactions of this complex are prototypic for non-specific DNA binding. In contrast, only one cognate decamer duplex is bound and deviates considerably from canonical B-form DNA. Most notably, a kink of approximately 50 degrees is observed at the central TA step with a concomitant compression of the major groove. Base-specific hydrogen bonds between the enzyme and the recognition base pairs occur exclusively in the major groove. These interactions appear highly co-operative as they are all made through one short surface loop comprising residues 182-186. Numerous contacts with the sugar phosphate backbone extending beyond the recognition sequence are observed in both types of complex. However, the total surface area buried on complex formation is > 1800 A2 larger in the case of cognate DNA binding. Two acidic side chains, Asp74 and Asp90, are close to the reactive phosphodiester group in the cognate complex and most probably provide oxygen ligands for binding the essential cofactor Mg2+. An important role is also indicated for Lys92, which together with the two acidic functions appears to be conserved in the otherwise unrelated structure of EcoRI endonuclease. The structural results give new insight into the physical basis of the remarkable sequence specificity of this enzyme.
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PMID:The crystal structure of EcoRV endonuclease and of its complexes with cognate and non-cognate DNA fragments. 849 Nov 71

Primary lung fibroblasts were isolated from patients with idiopathic pulmonary fibrosis (HIPF), from normal human lung tissue (NH), from rats treated with 75% oxygen and paraquat (PA), and from normal adult rats (NR). Serum-free media conditioned by each fibroblast strain were tested on the human A549 cell line (HIPF and NH media) or on primary alveolar epithelial cells (AEC) isolated from normal adult rats (PA or NR media). Over 20-h incubation, HIPF- or PA-conditioned media induced DNA fragmentation and significant decreases in total recoverable DNA and cell number of A549 or AEC, respectively; NH or NR media had no significant effect relative to serum-free unconditioned media. Apoptosis of A549 and AEC was detected by altered nuclear morphology and was confirmed by terminal deoxynucleotidyl transferase-mediated bio-dUTP nick end labeling. The endonuclease inhibitors 10 microM aurintricarboxylic acid and 50 microM zinc inhibited HIPF-induced apoptosis of A549 cells by 68 and 71%, respectively. Both apoptosis and necrosis were induced by HIPF and PA media in a concentration-dependent manner. These results suggest that altered fibroblasts emerging during fibrotic lung injury release a soluble factor(s) capable of inducing cell death and net loss of AEC.
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PMID:Fibroblasts isolated after fibrotic lung injury induce apoptosis of alveolar epithelial cells in vitro. 857 43

Nitric oxide (NO) is a pathophysiological mediator with unique signal transducing properties. Signaling mechanisms are categorized as cGMP-dependent or cGMP-independent. Multiple interactions of NO with oxygen, superoxide, and transition metals determine the biological activity. Cyclic GMP-independent responses of NO account for the antimicrobial, the cytostatic, and the cytotoxic capacity of NO. Cytotoxicity is not only directed to harmful cells but also affects the NO-producing cell in a self-destructing loop. For macrophages and pancreatic beta-cells (RINm5F), we established NO-mediated apoptotic cell death. Endogenously generated or exogenously applied NO causes DNA cleavage after endonuclease activation. NO-mediated accumulation of the tumor suppressor p53 precedes apoptotic cell death.
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PMID:The role of nitric oxide in cell injury. 859 59

Human NK cells (with CD3-/56+ phenotype) acquired features characteristic of apoptosis after incubation with autologous monocytes, as revealed by apoptotic nuclear morphology, degradation of DNA into oligonucleosomal fragments, and reduced nuclear interchalation of propidium iodide. In contrast, T cells (CD3+/56-) remained non-apoptotic. The monocyte-induced apoptosis in NK cells was prevented by catalase, a scavenger of hydrogen peroxide; whereas superoxide dismutase (a scavenger of superoxide anion), hydroxyl radical scavengers such as mannitol and deferoxamine, or the hypochlorus acid scavenger taurine did not prevent apoptosis. Sodium azide, a myeloperoxidase inhibitor, substantially reduced the monocyte-induced apoptosis in NK cells. Exogenous hydrogen peroxide, at concentrations exceeding 1 microns, induced apoptosis in both NK and T cells. Apoptosis induced by hydrogen peroxide occurred independently of synthesis of protein or mRNA and was blocked by the endonuclease inhibitor aurin tricarboxylic acid. Furthermore, oxidatively induced apoptosis in NK cells was inhibited by herbimycin A, indicating that apoptosis was dependent on protein kinases. Two to five times more hydrogen peroxide was required to induce apoptosis in T cells compared with NK cells. Similarly, NK cells were considerably more susceptible to apoptosis induced by the topoisomerase II inhibitor etoposide or by gamma-irradiation than were T cells. We conclude that monocyte-derived reactive oxygen metabolites kill NK cells by apoptosis and that NK cells are unusually sensitive to oxidatively as well as non-oxidatively induced apoptosis.
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PMID:Induction of apoptosis in NK cells by monocyte-derived reactive oxygen metabolites. 859 91

A set of phosphorothioate-containing oligonucleotides based on pGACGATATCGTC, a self-complementary dodecamer that contains the EcoRV recognition sequence (GATATC), has been prepared. The phosphorothioate group has been individually introduced at the central nine phosphate positions and the two diastereomers produced at each site separated and purified. The Km and Vmax values found for each of these modified DNA molecules with the EcoRV restriction endonuclease have been determined and compared with those seen for the unmodified all-phosphate-containing dodecamer. This has enabled an evaluation of the roles that both of the non-esterified oxygen atoms in the individual phosphates play in DNA binding and hydrolysis by the endonuclease. The results have also been compared with crystal structures of the EcoRV endonuclease, complexed with an oligodeoxynucleotide, to allow further definition of phosphate group function during substrate binding and turnover. For further study, see the related article "Probing the Indirect Readout of the Restriction Enzyme EcoRV: Mutational Analysis of Contacts to the DNA Backbone" (Wenz, A., Jeltsch, A., and Pingoud, A. (1996) J. Biol. Chem. 271, 5565-5573).
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PMID:Influence of the phosphate backbone on the recognition and hydrolysis of DNA by the EcoRV restriction endonuclease. A study using oligodeoxynucleotide phosphorothioates. 862 26

Singlet molecular oxygen (1O2) has been implicated in several biological processes that may lead to genetic damage. The relevance of various repair pathways in plasmid inactivation mediated by 1O2 was investigated. Plasmid treated with 1O2, chemically generated, was transfected into Escherichia coli strains deficient in genes implicated in the DNA repair of oxidative damage. The ability to transform bacteria is significantly reduced in the double mutant xth,nfo, deficient in both exonuclease III and endonuclease IV, although it was similar to wild-type cells in single mutants. The products of these two genes are able to cleave DNA damaged by 1O2 and to remove DNA polymerization blocks from 3'-termini generated either directly by 1O2 treatment or after the action of the formamidopyrimidine-DNA-N-glycosylase (Fpg protein). The results indicate that the exonuclease III and endonuclease IV participate in the excision of lethal lesions induced in DNA by 1O2.
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PMID:Involvement of Escherichia coli exonuclease III and endonuclease IV in the repair of singlet oxygen-induced DNA damage. 864 Sep 34

Ionizing radiation and normal cellular respiration form reactive oxygen species that damage DNA and contribute to a variety of human disorders including tumor promotion and carcinogenesis. A major product of free radical DNA damage is the formation of 8-oxoguanine, which is a highly mutagenic base modification produced by oxidative stress. Here, Drosophila ribosomal protein S3 is shown to cleave DNA containing 8-oxoguanine residues efficiently, The ribosomal protein also contains an associated apurinic/apyrimidinic (AP) lyase activity, cleaving phosphodiester bonds via a beta,delta elimination reaction. The significance of this DNA repair activity acting on 8-oxoguanine is shown by the ability of S3 to rescue the H2O2 sensitivity of an Escherichia coli mutM strain (defective for the repair of 8-oxoguanine) and to abolish completely the mutator phenotype of mutM caused by 8-oxoguanine-mediated G-->T transversions. The ribosomal protein is also able to rescue the alkylation sensitivity of an E.coli mutant deficient for the AP endonuclease activities associated with exonuclease III (xth) and endonuclease IV (nfo), indicating for the first time that an AP lyase can represent a significant source of DNA repair activity for the repair of AP sites. These results raise the possibility that DNA repair may be associated with protein translation.
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PMID:A Drosophila ribosomal protein contains 8-oxoguanine and abasic site DNA repair activities. 864 Dec 96

N-Hydroxypyridine-2-thione (2-HPT), known to release hydroxyl radicals on irradiation with visible light, and two related compounds, viz. N-hydroxypyridine-4-thione (4-HPT) and N-hydroxyacridine-9-thione (HAT), were tested for their potency to induce DNA damage in L1210 mouse leukemia cells and in isolated DNA from bacteriophage PM2. DNA single-strand breaks and modifications sensitive to various repair endonucleases (Fpg protein, endonuclease III, exonuclease III, T4 endonuclease V) were quantified. Illumination of cell-free DNA in the presence of 2-HPT and 4-HPT gave rise to damage profiles characteristic for hydroxyl radicals, i.e. single-strand breaks and the various endonuclease-sensitive modifications were formed in the same ratios as after exposure to established hydroxyl radical sources. In contrast, HAT plus light gave rise to a completely different DNA damage profile, namely that characteristic for singlet oxygen. Experiments with various scavengers (t-butanol, catalase, superoxide dismutase) and in D2O as solvent confirmed that hydroxyl radicals are directly responsible for the DNA damage caused by photoexcited 2-HPT and 4-HPT, while the damage by HAT plus light is mediated by singlet oxygen and type I reactions. The type of DNA damage characteristic of hydroxyl radicals was also observed in L1210 mouse leukemia cells when treated with 2-HPT plus light or with H2O2 at 0 degrees C. t-Butanol (2%) inhibited the cellular DNA damage by approximately 50%. A dose of 2-HPT plus light that generated single-strand breaks at a frequency of 5 x 10(-7)/bp was associated with 50% cell survival. No DNA damage and cytotoxicity was observed after treatment with 2-HPT in the dark. We propose that 2-HTP and 4-HTP may serve as new agents to study the consequences of DNA damage induced by hydroxyl radicals in cells. In addition, the data provide direct evidence that hydroxyl radicals are ultimately responsible for the genotoxic effects caused by H2O2 in the dark.
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PMID:Photolysis of N-hydroxpyridinethiones: a new source of hydroxyl radicals for the direct damage of cell-free and cellular DNA. 864 78

In the current study, the role of endonuclease activity in calcium ionophore A23187-induced gastric mucosal cellular disruption was examined using rabbit gastric mucosal cells. Cell integrity was assessed using trypan blue dye exclusion and Alamar blue dye absorbance. Ionophore A23187 (1.6-25 microM) induced a concentration-dependent decrease in dye exclusion and cell metabolism in cells suspended in a medium containing Ca2+ (2 mM), while no such effect was observed in cells incubated in the absence of extracellular Ca2+. Cells that were pretreated with the endonuclease inhibitors aurintricarboxylic acid (ATCA; 0.2 or 0.5 mM or Zn2+; 0.01 and 0.1 mM) exhibited significant reduction in the total extent of cell injury when incubated with A23187 in the presence of Ca2+. DNA fragmentation as assessed by measurement of [3H]thymidine liberation or gel electrophoresis was increased in response to ionophore A23187 (12.5 or 25 microM) treatment. A minimal degree of fragmentation was observed when cells were suspended in a Ca(2+)-free medium or incubated in the presence of ATCA or Zn2+. Addition of ethanol (8% w/v) induced a significant increase in cell injury, which was not affected by either removal of extracellular Ca2+ or ATCA pretreatment. Furthermore, treatment with the antioxidants catalase (50 micrograms/ml) or 2',2'-dipyridyl (2 mM) reduced ionophore-induced cell injury but did not reduce the extent of DNA fragmentation. These data suggest that sustained increases in intracellular Ca2+ result in increased endonuclease activity in gastric mucosal cells, leading to extensive DNA lysis and cell damage. Ethanol-induced cell damage does not involve Ca2+ influx and therefore is not mediated by endonuclease activation. Furthermore, sustained increases in cellular Ca2+ may also mediate their effects via formation of reactive oxygen metabolites, but this mechanism of cell damage does not appear to involve DNA fragmentation.
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PMID:Role of endonuclease activity and DNA fragmentation in Ca2+ ionophore A23187-mediated injury to rabbit isolated gastric mucosal cells. 865 49


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