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)

Both H2O2 (IC50 = 70 microM) and HOCl (IC50 = 8.5 microM) inhibited mitogen-induced MNL proliferation in a dose-dependent manner. This was found to be due to a depletion of intracellular ATP by at least two distinct mechanisms. HOCl and high concentrations (greater than 100 microM) of H2O2 inhibit ATP generation via sulfhydryl group oxidation on the active site of the glyceraldehyde-3-phosphate dehydrogenase (G3PDH) enzyme of the glycolytic pathway. On the other hand, low H2O2 concentrations cause ATP depletion by an activation of the DNA repair enzyme, poly(ADP-ribose)polymerase (pADPRP), leading to consumption of NAD+, an essential cofactor for G3PDH. The anti-oxidants ascorbate and cysteine protected MNL against the anti-proliferative effects of HOCl. Similar results were achieved with the HOCl-mediated inhibition of ATP production and G3PDH activity. However, ascorbate was unable to protect against H2O2-mediated inhibition of MNL functions, while cysteine protected against the inhibitory effects on ATP production and G3PDH activity, induced by this oxidant.
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PMID:Biochemical mechanisms of hydrogen peroxide- and hypochlorous acid-mediated inhibition of human mononuclear leukocyte functions in vitro: protection and reversal by anti-oxidants. 132 47

We surveyed the occurrence of unique restriction sites on the cDNAs of viroids, virusoids, and plant viral satellite RNAs that have a circular RNA as an intermediate of replication and found that four such sites would linearize their circular cDNAs. A rapid and simple method was then developed for cloning a naturally occurring viroid from Nematanthus wettsteinii plants. First-strand cDNA was synthesized using random hexanucleotide DNA primers and M-MuLV reverse transcriptase (Superscript RT). Second-strand DNA was synthesized by employing the replacement synthesis method using Escherichia coli RNase H, E. coli DNA polymerase I, E. coli DNA ligase, and beta-NAD+. The circular double-stranded DNA was analyzed for the presence of commonly available, unique restriction sites and subsequently linearized with a selected restriction enzyme. The linear cDNA was ligated to dephosphorylated plasmid vector pGEM 3Z f(+) and cloned in E. coli strain DH5 alpha. This cDNA cloning procedure is suitable for cloning sequence variants of well-characterized viroids, virusoids, certain plant viral satellite RNAs, and new such pathogens of unknown sequence.
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PMID:A rapid and versatile method for cloning viroids or other circular plant pathogenic RNAs. 138 86

Incubation of DNA polymerase alpha, DNA polymerase beta, terminal deoxynucleotidyl transferase, or DNA ligase II in a reconstituted poly(ADP-ribosyl)ating enzyme system markedly suppressed the activity of these enzymes. Components required for poly(ADP-ribose) synthesis including poly(ADP-ribose) polymerase, NAD+, DNA, and Mg2+ were all essential for the observed suppression. Purified poly(ADP-ribose) itself, however, was slightly inhibitory to all of these enzymes. Furthermore, the suppressed activities of DNA polymerase alpha, DNA polymerase beta, and terminal deoxynucleotidyl transferase were largely restored (3 to 4-fold stimulation was observed) by a mild alkaline treatment, a procedure known to hydrolyze alkaline-labile ester linkage between poly(ADP-ribose) and an acceptor protein. All of these results strongly suggest that the four nuclear enzymes were inhibited as a result of poly(ADP-ribosyl)ation of either the enzyme molecule itself or some regulatory proteins of these enzymes.
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PMID:Inhibition of DNA polymerase alpha, DNA polymerase beta, terminal deoxynucleotidyl transferase, and DNA ligase II by poly(ADP-ribosyl)ation reaction in vitro. 392 Oct 27

ADP-ribosyl transferase (ADP-RT) is a chromatin-bound nuclear enzyme catalysing the transfer of ADP-ribose from NAD+ to chromatin proteins. The enzyme is activated by DNA strand breaks and has been suggested to have roles in both DNA repair (via its effect on DNA ligase II) and in differentiation. We recently demonstrated that specific inhibitors of ADP-RT preferentially inhibit differentiation of human granulocyte-macrophage progenitor cells to the macrophage lineage and that the specific proliferation/differentiation stimulus granulocyte-macrophage colony stimulating activity (GM-CSA) activates ADP-RT in human marrow cells within 3 h of exposure. The purpose of this study was to investigate the role of ADP-RT in monocyte-macrophage differentiation. By altering the time of addition of ADP-RT inhibitor it was demonstrated that maximal inhibition of macrophage differentiation only occurs when the inhibitor is added within the first 24 h of culture. This suggests that it is an early event during the induced differentiation of granulocyte-macrophage progenitor cells which requires ADP-RT. Fluorometric assay of the level of DNA strand breaks showed that GM-CSA induces DNA strand breaks which are rapidly ligated only if ADP-RT is available. These data and those of our earlier studies suggest that DNA rearrangement may be involved in differentiation of granulocyte-macrophage progenitors to the monocyte-macrophage pathway. Such a DNA rearrangement could provide a molecular basis for commitment of multipotent progenitors to a single lineage.
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PMID:DNA strand breakage and ADP-ribosyl transferase mediated DNA ligation during stimulation of human bone marrow cells by granulocyte-macrophage colony stimulating activity. 608 35

This review discusses the potential relationships between ADP-ribosylation reactions, DNA repair, cell differentiation, and cancer. ADP-ribosylation of chromatin proteins has been shown to participate in DNA excision repair in all nucleated cells. ADP-ribosylation of chromatin proteins is catalysed by nuclear ADP-ribosyl transferase (ADPRT). This enzyme is entirely dependent on DNA for its activity because it has an absolute requirement for ends or nicks in double-stranded DNA. Exposure of cells to small alkylating agents or to radiation causes a fall in cellular NAD+ levels due to a transient activation of ADPRT and a consequent ADP-ribosylation of chromatin proteins. Inhibitors of ADPRT retard DNA strand-rejoining induced by radiation or by small alkylating agents; such inhibition has at least two biological consequences; a synergistic potentiation of cytotoxicity and an enhancement of sister chromatid exchanges and chromosomal aberrations. No species differences have yet been reported; there are variations between cell types and between different damaging agents. The enzyme inhibitors do not block early steps in DNA repair, and repair synthesis does not require ADPRT activity. DNA damage increases the activity of both DNA polymerase beta and DNA ligase II. The activation of DNA ligase II can be blocked by ADPRT inhibitors; presumably ADPRT activity is required for the activation of DNA ligase II. A plausible molecular explanation for the function of ADPRT in DNA repair is that ADPRT regulates the activity of DNA ligase II, the "non-replicative" ligase. In addition to its function in DNA repair, ADPRT is an obligatory requirement in certain categories of cell differentiation. Inhibitors of ADPRT and nicotinamide starvation both reversibly block cell differentiation. We suggest that a similar mechanism to that of DNA repair may be involved because we observe 100 to 300 single-strand DNA breaks during the cytodifferentiation of primary chick myoblasts. These breaks are not due to a general deficiency in DNA repair. I suggest that in certain categories of cell differentiation there are rearrangements or transpositions within the mammalian genome, and that ADP-ribosylation reactions have a general function to be sensitive to DNA breaks and to regulate subsequent DNA ligation in DNA repair, in DNA recombination, in sister chromatid exchanges, in chromosome aberrations, in gene rearrangements, in transpositions and in certain categories of cell differentiation. The relevance of these observations and ideas to cancer is discussed.
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PMID:ADP-ribosylation, DNA repair, cell differentiation and cancer. 631 41

Rejoining of DNA single-strand breaks generated by treatment of plasmids with gamma-rays, neocarzinostatin, or bleomycin was catalyzed inefficiently by human cell extracts. The reaction was strongly promoted by the addition of NAD+, which was employed for rapid and transient synthesis of poly(ADP-ribose). The DNA rejoining reaction was accompanied by DNA repair replication, apparently due to replacement of damaged residues at termini. Selective depletion of poly(ADP-ribose) polymerase from cell extracts improved the repair of DNA exposed to a variety of DNA-damaging agents by removing the NAD+ dependence of the repair reaction. NAD(+)-promoted DNA repair by soluble cell extracts also occurred with alkylated DNA as substrate and was suppressed by 3-aminobenzamide. A similar stimulatory effect by NAD+ was observed for repair of ultraviolet-irradiated DNA, and this could be ascribed to the presence of pyrimidine hydrates as minor radiation-induced DNA lesions. No effect was observed on the sealing of gamma-irradiated DNA by supplementation of cell extracts with purified mammalian DNA ligase I or DNA ligase II. The results indicate that poly(ADP-ribose) polymerase interferes with base excision-repair processes because bound enzyme molecules block DNA strand interruptions. Release of bound poly-(ADP-ribose) polymerase following automodification, or physical removal of the protein from reaction mixtures, facilitates DNA repair.
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PMID:NAD(+)-dependent repair of damaged DNA by human cell extracts. 768 Jun 46

A model for eukaryotic DNA damage repair is proposed in which poly(ADP-ribose) polymerase(NAD+ ADP-ribosyl transferase, EC 2,4,2,30) plays an important role. In this model, poly(ADP-ribose) polymerase regulates transcription of genes that are induced by DNA-damaging agents. This transcriptional regulation results from poly(ADP-ribosyl)ation and inactivation of DNA sequence-specific regulatory proteins such as silencer element-binding proteins, thereby inducing transcription of DNA polymerase beta, which is a DNA repair enzyme in higher eukaryotes. Poly(ADP-ribose) polymerase has a number of similarities to RecA in Escherichia coli. Therefore, the genes related to DNA damage repair in higher eukaryotes are proposed to form a "poly(ADP-ribose) polymerase regulatory network" similar to the "SOS regulatory network" in E. coli.
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PMID:Speculations on the roles of ADP-ribosyl transferase based on analogies between RecA and poly(ADP-ribose) polymerase. 824 22

Studies on human patients and experimental animals indicate that hyperbaric O2 can opacify the lens nucleus and damage the lens epithelium in vivo. Here we investigate the effects of hyperbaric O2 on cultured rabbit lens epithelial cells (LECs). When the cells were exposed to 50 atm O2 (99% O2 + 1% CO2) for 3 hr there were no immediate effects on morphology, viability and transport processes (uptake of 86Rb and 14C-alpha AIB). In addition, the O2 treatment did not lower the high level of reduced glutathione or increase the low level of oxidized glutathione. However, 50 atm O2 did produce a near doubling in the glycolytic rate which maintained ATP at levels only slightly lower than normal. Although the 3-hr O2 treatment was not lethal, it completely inhibited cell division for 2 days. After 2 days, growth was initiated and, at day 7 the rate of growth was faster than the controls (control cells were treated with ambient air or 50 atm N2 for 3 hr). Cells treated with 8 atm O2 for 3 hr exhibited a slowed rate of growth, relative to controls, while exposure to 2 atm O2, did not inhibit mitosis. Changes in morphology (multilayering and elongation) of cells exposed to 50 atm O2, but not the controls, were evident 7 days after the 3-hr exposure. The incorporation of [35S]methionine into individual polypeptides and [3H]thymidine into DNA was significantly inhibited immediately following a 3-hr treatment with 50 atm O2, but both parameters recovered within 2 days. DNA strand breaks were observed in LECs following hyperbaric O2 treatment as low as 4 atm O2 for 3 hr and increased with higher pressures of O2, but not N2. Treatment with 50 atm O2 nearly doubled the activity of the DNA repair enzyme, poly-ADP-ribose polymerase, and decreased the level of its substrate NAD+; the latter effect was reduced by 3-aminobenzamide, an inhibitor of the enzyme. Thus, although LECs tolerated brief exposures to high pressures of O2 without cell death, DNA damage occurred at relatively low pressures of O2. All of the effects of hyperbaric O2 on LECs occurred without any alteration of the normal levels of reduced and oxidized glutathione. It appears that GSH is important in maintaining cell viability during exposure to an elevated level of O2, but that it is incapable of preventing O2-induced effects on growth and DNA.
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PMID:Hyperbaric oxygen inhibits the growth of cultured rabbit lens epithelial cells without affecting glutathione level. 850 May 57

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

DNA ligases play a pivotal role in DNA replication, repair and recombination. Reactions catalyzed by DNA ligases consist of three steps: adenylation of the ligase in the presence of ATP or NAD+, transferring the adenylate moiety to the 5'-phosphate of the nicked DNA substrate (deadenylation) and sealing the nick through the formation of a phosphodiester bond. Thermus thermophilus HB8 DNA ligase (Tth DNA ligase) differs from mesophilic ATP-dependent DNA ligases in three ways: (i) it is NAD+ dependent; (ii) its optimal temperature is 65 instead of 37 degrees C; (iii) it has higher fidelity than T4 DNA ligase. In order to understand the structural basis underlying the reaction mechanism of Tth DNA ligase, we performed site-directed mutagenesis studies on nine selected amino acid residues that are highly conserved in bacterial DNA ligases. Examination of these site-specific mutants revealed that: residue K118 plays an essential role in the adenylation step; residue D120 may facilitate the deadenylation step; residues G339 and C433 may be involved in formation of the phosphodiester bond. This evidence indicates that a previously identified KXDG motif for adenylation of eukaryotic DNA ligases [Tomkinson, A.E., Totty, N.F., Ginsburg, M. and Lindahl, T. (1991) Proc. Natl. Acad. Sci. USA, 88, 400-404] is also the adenylation site for NAD+-dependent bacterial DNA ligases. In a companion paper, we demonstrate that mutations at a different Lys residue, K294, may modulate the fidelity of Tth DNA ligase.
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PMID:Identification of essential residues in Thermus thermophilus DNA ligase. 876 Aug 97


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