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:6.5.1.2 (DNA ligase)
2,749 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Nitric oxide (NO.) is produced as a cytotoxic free radical through enzymatic oxidation of L-arginine in activated macrophages. Pure NO. gas was previously found to induce the Escherichia coli soxRS oxidative stress regulon, which is readily monitored by using a soxS'::lac fusion. The soxRS system includes antioxidant defenses, such as a superoxide dismutase and a DNA repair enzyme for oxidative damage, and protects E. coli from the cytotoxicity of NO.-generating macrophages. Previous experiments involved exposing E. coli to a bolus of NO. rather than the steadily generated gas expected of activated macrophages. We show here detectable induction of soxS transcription by NO. delivered at rates as low as 25 microM/h. Maximal induction was observed at 25 microM NO. per h under anaerobic conditions but at 125 microM/h aerobically. After incubation with murine macrophages, soxS expression was induced in the phagocytosed bacteria up to approximately 30-fold after an 8-h exposure. This in vivo induction was almost completely eliminated by the NO. synthase inhibitor NG-monomethyl-L-arginine. The inhibitor increased the survival of a delta soxRS strain but not that of wild-type E. coli after phagocytosis, which suggests that induction of the soxRS regulon by NO. can counteract most of the cytotoxic effects of NO. production by the macrophages. We show that the soxRS-regulated enzyme glucose-6-phosphate dehydrogenase is an important element of the defense against macrophages.
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PMID:Roles of nitric oxide in inducible resistance of Escherichia coli to activated murine macrophages. 753 26

Nitric oxide has been shown to be a mediator molecule in the regulation of many physiological functions. However, this small diatomic molecule in the presence of O2 generates reactive intermediates which modify DNA bases and inactive enzymes at high concentrations (100 microM). We report that NO generated by 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEA/NO, Et2NN(O)NO-Na+), a compound known to release NO in a predictable manner, caused irreversible damage at physiological concentrations to the zinc finger-containing DNA repair enzyme formamidopyrimidine-DNA glycolyase (Fpg protein). The inhibition of the enzyme activity was DEA/NO dose and time dependent with IC50s with respect to total NO released from this compound of approximately 110 and approximately 120 mumol/l respectively. This inhibitory effect by P3 was not reversible over time in the presence of reducing agents and/or Zn2+. Nitrite and diethylamine, the nitrogenous products of the decomposition of DEA/NO, did not inhibit the enzyme. The presence of 500 micrograms/ml bovine serum albumin did not protect the protein from the inhibitory effects of DEA/NO, however, the presence of 10 mM cysteine did dramatically abate the inhibition of the Fpg protein by DEA/NO. Other DNA glycosylases tested were not inhibited by exposure to these concentrations of NO. These results, together with reports of site-directed mutagenesis of this protein, suggest that the cysteine residues contained within the zinc finger motif of the Fpg protein are the primary sites of NO interaction. Our studies were then extended to intact cells. The Fpg protein activity was decreased following treatment in vivo when Escherichia coli MH321 (acr A-) cells were treated with DEA/NO. Furthermore, the Fapy-DNA glycosylase activity in H4 cells, a rat hepatoma line, was decreased when intact cells were incubated with DEA/NO.
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PMID:The Fpg protein, a DNA repair enzyme, is inhibited by the biomediator nitric oxide in vitro and in vivo. 795 43

The free radicals nitric oxide and superoxide anion react to form peroxynitrite (ONOO-), a highly toxic oxidant species. In vivo formation of ONOO- has been demonstrated in shock and inflammation. Herein we provide evidence that cytotoxicity in cells exposed to ONOO- is mediated by DNA strand breakage and the subsequent activation of the DNA repair enzyme poly(ADP ribose) synthetase (PARS). Exposure to ONOO- (100 microM to 1 mM) inhibited mitochondrial respiration in cultured J774 macrophages and in rat aortic smooth muscle cells. The loss of cellular respiration was rapid, peaking 1-3 h after ONOO- exposure, and reversible, with recovery after a period of 6-24 h. The inhibition of mitochondrial respiration was paralleled by a dose-dependent increase in DNA strand breakage, reaching its maximum at 20-30 min after exposure to ONOO-. We observed a dose-dependent increase in the activity of PARS in cells exposed to ONOO-. Inhibitors of PARS such as 3-aminobenzamide (1 mM) prevented the inhibition of cellular respiration in cells exposed to ONOO-. Activation of PARS by ONOO--mediated DNA strand breakage resulted in a significant decrease in intracellular energy stores, as reflected by a decline of intracellular NAD+ and ATP content. 3-Aminobenzamide prevented the loss of NAD+ and ATP in cells exposed to ONOO-. In contrast, impairment of cellular respiration by the addition of the nitric oxide donors S-nitroso-N-acetyl-DL-penicillamine or diethyltriamine nitric oxide complex, was not associated with the development of DNA strand breaks, in concentrations up to 1 mM, and was largely refractory to PARS inhibition. Our results suggest that DNA damage and activation of PARS, an energy-consuming futile repair cycle, play a central role in ONOO--mediated cellular injury.
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PMID:DNA strand breakage, activation of poly (ADP-ribose) synthetase, and cellular energy depletion are involved in the cytotoxicity of macrophages and smooth muscle cells exposed to peroxynitrite. 870 Aug 30

All organisms have adapted to environmental changes by acquiring various functions controlled by gene regulation. In bacteria, a number of specific responses have been found to confer cell survival in various nutrient-limited conditions, and under physiological stresses such as high or low temperature, extreme pH, radiation, and oxidation (for review, see Neidhardt et al., 1987). In this article, I introduce an Escherichia coli (E. coli) global response induced by superoxide stress, the soxRS regulon. The functions controlled by this system consist of a wide variety of enzymes such as manganese-containing SOD (Mn-SOD); glucose 6-phosphate dehydrogenase (G6PD), the DNA repair enzyme endonuclease IV, fumarase C, NADPH:ferredoxin oxidoreductase, and aconitase. This response is positively regulated by a two-stage control system in which SoxR iron-sulfur protein senses exposure to superoxide and nitric oxide, and then activates transcription of the soxS gene, whose product stimulates the expression of the regulon genes. Our recent finding indicates that soxS transcription is initiated in a manner dependent on the rpoS gene encoding RNA polymerase sigma factor, theta s, in response to entering the stationary phase of growth. With this information, mechanisms for prokaryotic coordinating gene expression in response to superoxide stress and in stationary phase are discussed.
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PMID:Two-stage gene regulation of the superoxide stress response soxRS system in Escherichia coli. 895 73

Nitric oxide-induced modifications of DNA occur either by directly altering DNA chemically through reactive nitrogen oxide species (RNOS) or indirectly by inhibiting various repair processes. DNA ligases are enzymes which rejoin single-strand breaks and are critical for DNA integrity during processes such as gene transcription and repair. The eukaryotic and T4 DNA ligases are active in the presence of ATP and act in two steps: the formation of protein-AMP intermediates, then the ligation of DNA breaks. When T4 DNA ligase was exposed to the NO generator DEA/NO (Et2N[NO(NO)]Na), a concentration- and time-dependent inhibition of these two steps, adenylylation of the protein and ligation of the substrate, was observed. This inhibition was abated by the presence of cysteine, suggesting that RNOS, rather than NO, mediated the inhibition of the ligase activity. As mammalian and T4 DNA ligases act by the same mechanism, the inhibition of DNA ligase may explain the increase in single-strand breaks reported for cells exposed to NO and provides a mechanism to increase DNA lesions without direct chemical modification of DNA by NO or RNOS.
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PMID:Nitric oxide inhibits DNA ligase activity: potential mechanisms for NO-mediated DNA damage. 896 69

V79mut1 cells are resistant to the toxic effects of 5-hydroxymethyl-2'-deoxyuridine (hmdUrd) and are deficient in the DNA repair enzyme hydroxymethyluracil-DNA glycosylase (hmUDG). We have therefore proposed that the toxicity of hmdUrd results from the repair of the lesion from DNA. In order to clarify the biological role of hmUDG, we have determined whether the repair-deficient cells showed resistance or sensitivity to the toxic or mutagenic effects of other DNA-damaging agents. Cells were exposed to hmdUrd, ionizing or ultraviolet radiation, to the alkylating agent MNNG, and to oxidative stress produced by hypoxanthine/xanthine oxidase, glucose/glucose oxidase, nitric oxide donor SNAP, or to H2O2. The V79mut1 cells did not show increased mutagenesis in response to hmdUrd. Relative to the V79 parent cells, the V79mut1 cells were not markedly altered in sensitivity to oxidizing agents and ionizing radiation (which produce hmdUra in DNA). The repair-deficient cells wee equally sensitive as the parent V79 cells to DNA damage induced by ultraviolet radiation or by MNNG. No significant differences were seen between the parent and the repair-deficient cells in terms of synthesis of poly(ADP-ribose) in response to damage or in their sensitization to 3-aminobenzamide. Thus, the loss of the 5-hydroxymethyluracil (hmUra)-DNA glycosylase activity in mammalian cells in culture confers no obvious deleterious effect on cell survival or mutagenicity in response to a wide range of DNA damage. These studies indicate that the major lesion known to be repaired by hmUra-DNA glycosylase, an hmUra residue replacing thymine, is produced in cells only in small quantities as the result of exposure to common DNA-damaging agents. These results raise the possibility that hmUra-DNA glycosylase may have evolved to respond to other lesions than hmUra residues formed from the oxidation of thymine.
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PMID:Lack of phenotypic alteration of hmUra-DNA glycosylase-deficient hamster cells exposed to DNA-damaging agents. 910 Aug 52

Crocidolite asbestos is known to cause cellular damage, leading to asbestosis, bronchogenic carcinoma, and mesothelioma in humans. The mechanism responsible for the carcinogenicity of asbestos is not known. Iron associated with asbestos is thought to play a role by catalyzing the formation of reactive oxygen species, which may cause DNA damage, leading to mutations and cancer. Here, we examined whether asbestos can induce mutations in Chinese hamster hgprt+ V79 cells or transgenic hgprt-, gpt+ V79 cells (G12). Treatment with 6 microg/cm2 crocidolite for 24 h caused a 2-fold increase in the mutation frequency at the gpt locus of G12 cells, but no increase at the hgprt locus of V79 cells. The mutation frequency at the gpt locus of G12 cells increased with increasing treatment dose of crocidolite. The mutations induced by crocidolite appeared to be due to the generation of reactive oxygen species catalyzed by iron associated with the fibers, because treatment of G12 cells in iron-free medium with fibers from which redox active iron had been removed with desferrioxamine B prevented all of the gpt- mutations above untreated control levels. In addition, treatment of cells with a soluble form of iron, 1.5 mM ferric ammonium citrate, resulted in an increase in mutation frequency at the gpt locus of approximately 1.5 fold above that of untreated G12 cells with no increase in mutations at the hgprt locus of V79 cells with ferric ammonium citrate. We also investigated the effect of nitric oxide on the mutagenicity of crocidolite in G12 cells. When G12 cells were treated with 3 microg/cm2 of crocidolite in the presence of nitric oxide-generating compound, 200 microM diethyltriamine/NO, the mutation frequency increased to a level that was more than additive for crocidolite or diethyltriamine/NO treatment alone. These results strongly suggest that the presence of iron and nitric oxide may either lead to the generation of another reactive, mutagenic species, such as peroxynitrite, or that nitric oxide inhibits a DNA repair enzyme(s), leading to an increase in mutations.
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PMID:Participation of iron and nitric oxide in the mutagenicity of asbestos in hgprt-, gpt+ Chinese hamster V79 cells. 951 98

Cerebral ischemia and the aftermath of reperfusion form a hypoxic/hyperoxic sequence of events that can trigger oxidative stress response cascades in neurons of the central nervous system. After transient ischemia there is an increase in intracellular Ca2+ release, extracellular glutamate, reactive oxygen species (ROS) and nitric oxide, genotoxic events that stimulate DNA repair. Increased oxidative stress and interrupted blood flow in ischemia, like DNA repair, also deplete cellular ATP and commit neurons to apoptosis. We report that levels of the DNA repair enzyme apurinic/apyrimidinic endonuclease (APE/Ref-1) decreased significantly in the hippocampus but not other brain areas after 6 h of reperfusion following an induced ischemic insult. This specific inhibition of APE/Ref-1 expression may affect the extent of apoptosis after ischemia.
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PMID:APE/Ref-1 responses to ischemia in rat brain. 992 39

Neuronal injury may be dependent upon the generation of the free radical nitric oxide (NO) and the subsequent induction of programmed cell death (PCD). Although the nature of this injury may be both preventable and reversible, the underlying mechanisms that mediate PCD are not well understood. Using the agent nicotinamide as an investigative tool in primary rat hippocampal neurons, the authors examined the ability to modulate two independent components of PCD, namely the degradation of genomic DNA and the early exposure of membrane phosphatidylserine (PS) residues. Neuronal injury was determined through trypan blue dye exclusion, DNA fragmentation, externalization of membrane PS residues, cysteine protease activation, and the measurement of intracellular pH (pHi). Exposure to the NO donors SIN-1 and NOC-9 (300 micromol/L) alone rapidly increased genomic DNA fragmentation from 20 +/- 4% to 71 +/- 5% and membrane PS exposure from 14 +/- 3% to 76 +/- 9% over a 24-hour period. Administration of a neuroprotective concentration of nicotinamide (12.5 mmol/L) consistently maintained DNA integrity and prevented the progression of membrane PS exposure. Posttreatment paradigms with nicotinamide at 2, 4, and 6 hours after NO exposure further demonstrated the ability of this agent to prevent and reverse neuronal PCD. Although not dependent upon pHi, neuroprotection by nicotinamide was linked to the modulation of two independent components of neuronal PCD through the regulation of caspase 1 and caspase 3-like activities and the DNA repair enzyme poly(ADP-ribose) polymerase. The current work lays the foundation for the development of therapeutic strategies that may not only prevent the course of PCD, but may also offer the ability for the repair of neurons that have been identified through the loss of membrane asymmetry for subsequent destruction.
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PMID:Prevention of nitric oxide-induced neuronal injury through the modulation of independent pathways of programmed cell death. 1099 60

Microvascular endothelial cell (EC) apoptosis or programmed cell death (PCD) during free radical injury may be involved in the development of cerebral ischemic and degenerative diseases. Yet, the cellular mechanisms that mediate cerebral EC injury require further definition. We therefore used the agent nicotinamide as an investigative tool in EC cultures to examine the role of free radical nitric oxide (NO)-induced PCD. EC injury was evaluated by the trypan blue dye exclusion method, DNA fragmentation, membrane phosphatidylserine (PS) exposure, cysteine protease activity, mitochondrial membrane potential, and mitogen-activated protein kinase phosphorylation. We demonstrate that cerebrovascular PCD consists of two distinct pathways that involve the degradation of genomic DNA and the exposure of membrane PS residues. Each of these pathways is reversible in nature and is controlled independently by caspase 8, caspase 1, and caspase 3. As a cytoprotectant, nicotinamide is novel in the vascular system and functions at two levels. Nicotinamide not only maintains the mitochondrial membrane potential and the prevention of cytochrome c release, but also prevents the induction of caspase-8-, caspase-1- and caspase-3-like activities linked to the DNA repair enzyme poly(ADP-ribose) polymerase through mechanisms that are independent from the MAP kinase systems of p38 and JNK. The work begins to identify therapeutic strategies for the protection of the cerebral vasculature during both acute and chronic degenerative disorders.
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PMID:Nicotinamide modulates mitochondrial membrane potential and cysteine protease activity during cerebral vascular endothelial cell injury. 1201 85


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