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:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Microbial siderophores represent a class of iron chelators characterized by their high affinity (i.e., formation constants, greater than 10(40) M) for ferric iron. Previously, we demonstrated that the bacterial siderophores, N-[3-(2,3-dihydroxybenzamido)propyl]-N-[4-(2, 3-dihydroxybenzamino)butryl]-2-(2-hydroxyphenyl) trans-5-methyloxazoline-4-carboxamide (Parabactin) and N1,N8-bis(2,3-dihydroxybenzoyl)spermidine (Compound II), inhibit the growth of L1210 cells and the replication of DNA (but not RNA) viruses at low micromolar concentrations (Biochem. Biophys. Res. Commun., 121: 848-854, 1984). The basis for this antiproliferative effect on L1210 cells has now been investigated further. Onset of growth inhibition induced by 5 microM Parabactin occurs much earlier than with an equimolar concentration of Compound II but, once established by either chelator, inhibition appears to be irreversible. Growth inhibition was fully preventable with exogenous FeCl3 when given at the same time as the chelators. Flow cytometric analysis revealed a G1-S cycle block following treatment for 4 h with either 5 microM Parabactin or 30 microM Compound II. The block was readily reversed with exogenous FeCl3, allowing cells to progress to mid-S phase by 3 h and to G1 again by 9 h. Thereafter, cells accumulated at a second block located at S phase. The treatment conditions required for the initial cell cycle block (at 4 h) were adapted for subsequent studies. Clonogenicity of L1210 cells in soft agar following a 4-h exposure was reduced to 22% of control by 5 microM Parabactin and to 16% by 30 microM Compound II. Neither growth inhibition in suspension culture nor decreased clonogenicity in soft agar could be reversed with exogenous iron, following treatment with the chelators. Both chelators caused an early and significant decrease in [14C]thymidine incorporation over the 4-h period (50% inhibitory concentration at 4 h, 0.4 microM for Parabactin and 6.0 microM for Compound II). [3H]Uridine incorporation was inhibited later than [14C]thymidine and to a much lesser extent, while [3H]leucine incorporation was not significantly affected. Treatment of cells with 5 microM Parabactin or Compound II for 4 h decreased deoxy-adenosine triphosphate pools by 38 and 70%, respectively, and increased deoxythymidine triphosphate pools by 67 and 36%, respectively, suggesting interference with ribonucleotide reductase. Indeed, extracts of cells treated for 4 h with either 5 microM Parabactin or 30 microM Compound II exhibit a 97 to 98% decrease in cytidine-5'-diphosphate reductase activity compared to control, whereas DNA polymerase was elevated slightly.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Characterization of L1210 cell growth inhibition by the bacterial iron chelators parabactin and compound II. 402 62

Infection of monkey kidney (BSC-40) cells with vaccinia virus strain WR resulted in a marked increase in ribonucleoside diphosphate reductase (EC 1.17.4.1) activity as measured by CDP reduction in cell-free extracts. After a synchronous infection, increased activity was detected at 2 h, peaked at 4 to 5 h, and then declined between 6 and 8 h to the endogenous cellular level. The induction, detectable at 0.5 PFU/cell, correlated strongly with multiplicity of infection to 10 PFU/cell and continued to increase to 50 PFU/cell. It paralleled the previously described induction of viral DNA polymerase and thymidine kinase, suggesting that the reductase may also be a product of early transcription of the viral genome. The inhibition of DNA synthesis throughout infection resulted in prolonged accumulation of reductase activity and delayed and incomplete down-regulation at 8 h, suggesting that repression involves late functions. Rescue of fluorodeoxyuridine-inhibited DNA synthesis with exogenous thymidine restored the normal pattern. Preferential association of the induced reductase with the cytoplasmic sites of vaccinia virus DNA replication (virosomes) was not detected. The induced enzyme is similar in several respects to other eucaryotic ribonucleotide reductases, but is distinct from host cell reductase in response to certain modulators of reductase activity (M. B. Slabaugh and Christopher K. Mathews, J. Virol. 52:501-506, 1984). Full activity required an activator, exogenous reducing equivalents, and iron. Hydroxyurea, EDTA, dATP, and dTTP inhibited CDP reduction, setting this reductase apart from T4 reductase, which is not inhibited by dATP, and from herpesvirus reductase, which requires no activation and is insensitive to deoxyribonucleoside triphosphate inhibition.
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PMID:Vaccinia virus induces ribonucleotide reductase in primate cells. 638 75

Ferric nitrilotriacetate (Fe(3+)-NTA) catalyzes hydrogen peroxide-derived production of hydroxyl radicals, which are known to cause DNA damage. In the present work, Fe(3+)-NTA plus hydrogen peroxide-induced single-strand DNA breaks and repair of the DNA damage were studied in vitro by monitoring DNA damage- and DNA repair-dependent conformational changes of pUC18 plasmid DNA. Single-strand DNA breaks were induced in the pUC18 DNA by Fe(3+)-NTA plus hydrogen peroxide in a dose-dependent fashion. Induction of the DNA damage was inhibited by deferoxamine mesylate (an iron chelator) and by hydroxyl radical scavengers such as dimethyl sulfoxide (DMSO), D-mannitol and ethanol indicating that the DNA damage was caused by hydroxyl radicals which were generated by reaction of Fe(3+)-NTA with hydrogen peroxide. The oxygen radical-induced single-strand DNA breaks were repaired partly (more than 50%) by incubating the damaged DNA at 37 degrees C for 3 h with a partially purified preparation of APEX nuclease (a multifunctional DNA repair enzyme), DNA polymerase beta, four deoxyribonucleoside triphosphates, T4 DNA ligase and ATP. Analyses of the partially purified preparation of APEX nuclease revealed that a 45-kDa protein as well as APEX nuclease in the preparation were involved in the repair of the single-strand DNA breaks. APEX nuclease was suggested to initiate the repair by removing 3' termini blocked by the nucleotide fragments and also by incising the 5' side of AP sites. The 45-kDa protein was suggested to be required for removal of the 5' tags such as 5'-terminal deoxyribose phosphate residues produced by the action of APEX nuclease on AP sites.
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PMID:Oxygen radical-induced single-strand DNA breaks and repair of the damage in a cell-free system. 756 64

In recent years two mechanisms have been proposed for the production of DNA strand breaks in cells undergoing oxidative stress: (i) DNA attack by OH radical, produced by Fenton reaction catalyzed by DNA-bound iron; and (ii) DNA attack by calcium-activated nucleases, due to the increase of cytosolic and nuclear calcium induced by oxidative stress. We set out to investigate the participation of the former mechanism by detecting and quantifying 3'-phosphoglycolate, a 3' DNA terminus known to be formed by OH radical attack to the deoxyribose moiety, followed by sugar ring rupture and DNA strand rupture. These structures were found in DNA of monkey kidney cells exposed to hydrogen peroxide, iron nitrilotriacetate or ascorbate, all species known to favor a cellular pro-oxidant status. The method employed to measure 3' phosphoglycolate was the 32P-postlabeling assay. Repair time course experiments showed that it takes 10 h for 3'-phosphoglycolate to be removed from DNA. It was found that the DNA of both control cells and cells exposed to hydrogen peroxide had a very poor capacity of supporting in vitro DNA synthesis, catalyzed by DNA polymerase I. If the DNA was previously incubated with exonuclease III, an enzyme able to expose 3'-OH primers by removal of 3'-phosphoglycolate and 3'-phosphate termini the in vitro synthesis was substantially increased. This result shows that either of these termini are present at the break and that 3'-hydroxyl termini are virtually absent. At least 25% of the strand breaks exhibited 3'-phosphoglycolate termini as determined by the 32P-postlabeling assay, but due to the characteristic of the method this percentage is likely to be higher. These results favor the hypothesis that an oxidative agent generated by Fenton reaction is responsible for DNA strand breakage in cells undergoing oxidative stress.
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PMID:DNA strand breaks produced by oxidative stress in mammalian cells exhibit 3'-phosphoglycolate termini. 765 23

Salmonella typhimurium exhibits a low-pH-inducible acid tolerance response (ATR) that can protect the adapted cell from severe acid challenge (pH 3.3). It is a two-stage system, with some proteins induced at pH 5.8 (pre-acid shock) and others induced below pH 4.5 (acid shock). The genetics of acid resistance was investigated through the use of a new screening medium. The medium contained 200 microM dinitrophenol (DNP) and was adjusted to pH 4.7 to 4.8. The medium will lower the internal pH of cells to a lethal level. However, cells capable of mounting an ATR will survive longer on this medium than acid-intolerant cells. Using this DNP lethal screening strategy, we isolated several acid-sensitive insertion mutants. Some mutants were defective in the pre-acid shock ATR stage but exhibited a normal or nearly normal post-acid shock-induced acid tolerance (atrB and atrC). Others could not induce acid tolerance by using either pre- or post-acid shock strategies (atrD, atrF, and atrG). The atrB locus was found to be part of a regulon under the control of a trans-acting regulator, atbR. An insertion in atbR caused constitutive acid tolerance because of overexpression of the regulon. Mutations in atrD and atrF affected iron metabolism and, in a manner analogous to ferric uptake regulator (fur) mutations, diminished acid resistance. The atrF mutation mapped within the ent cluster, probably in a fep uptake locus. The atrD locus mapped near metC and may represent an insertion into the S. typhimurium homolog of the Escherichia coli exbB or exbD locus. The mutation in atrC caused extreme UV light sensitivity and proved to occur within the polA (DNA polymerase I) locus. The results support the concept of overlapping acid protection systems in S. typhimurium.
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PMID:Acid-sensitive mutants of Salmonella typhimurium identified through a dinitrophenol lethal screening strategy. 816 7

ACH-2 cells, an immortalized human T-cell line, contain a single integrated copy of the HIV-1 provirus. Here, the structure of HIV-1 chromatin was probed using a DNA cleavage reagent. Nuclei were isolated from ACH-2 cells and treated with methidiumpropyl-EDTA (MPE)-iron(II) to produce limited DNA cleavage. Primers were selected at approximately 300 bp intervals along the HIV-1 DNA, and sites of preferential cleavage were mapped by carrying out 50 cycles of primer extension using a thermo-stable DNA polymerase in the presence of [32P]dATP. By comparing the resulting cleavage pattern with patterns derived from human cell lines not containing HIV-1 sequences, it was possible to map the arrangement of nucleosomes across the integrated HIV-1 genome. Particularly regular spacing was seen in the 3' end of the pol and env coding regions, and several extended blocks spared of nucleosomes were found in gag and pol, the largest being an approximately 450 bp region in gag. For comparison, and to examine nucleosome placement on HIV-1 DNA when it is not integrated, overlapping segments of HIV-1 DNA were cloned into an EBV-oriP plasmid, grown as stable episomes in a human B lymphoblastoid cell line, and the same analysis using MPE-iron(II) cleavage and primer extension carried out. The major features of nucleosome placement on these EBV/HIV minichromosomes was very similar to that observed in the integrated HIV-1 genome arguing for a strong sequence dependence for nucleosome placement along HIV-1 DNA.
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PMID:Nucleosomal arrangement of HIV-1 DNA: maps generated from an integrated genome and an EBV-based episomal model. 860 34

Oxidative DNA damage and its repair in primary rat hepatocyte cultures was investigated following 4 h of incubation with the toxic iron chelate, ferric nitrilotriacetate (Fe-NTA), in the presence or absence of the potent protective flavonoid myricetin (25-50-100 microM). Seven DNA base oxidation products were quantified in DNA extracts by gas chromatography-mass spectrometry (GC-MS) in selected ion monitoring mode. Concomitantly, DNA repair capacity of hepatocytes was estimated by the release of oxidized-base products into culture media, using the same GC-MS method. A genotoxic effect of Fe-NTA (100 microM) in hepatocytes was evidenced by a severe increase in DNA oxidation over basal levels, with accumulation in cellular DNA of five oxidation products derived from both purines and pyrimidines. This prooxidant effect of iron was also noted by an induction of lipid peroxidation, estimated by free malondialdehyde production. Addition of increasing concentrations of myricetin (25-50-100 microM) simultaneously with iron prevented both lipid peroxidation and accumulation of oxidation products in DNA. Moreover, as an activation of DNA repair pathways, myricetin stimulated the release of DNA oxidation bases into culture media, especially of purine-derived oxidation products. This removal of highly mutagenic oxidation products from DNA of hepatocytes might correspond to an activation of DNA excision-repair enzymes by myricetin. This was verified by RNA blot analysis of DNA polymerase beta gene expression which was induced by myricetin in a dose-dependent manner. This represented a novel and original mechanism of cytoprotection by myricetin against iron-induced genotoxicity via stimulation of DNA repair processes. Since iron-induced DNA damage and inefficient repair in hepatocytes could be related to genotoxicity and most probably to hepatocarcinogenesis, modulation of these processes in vitro by myricetin might be relevant in further prevention of liver cancer derived from iron overload pathologies.
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PMID:Repair of iron-induced DNA oxidation by the flavonoid myricetin in primary rat hepatocyte cultures. 1040 9

Vibrio ordalii is a major cause of vibriosis in wild and cultured marine salmonids and carries pMJ101, a 30-kb cryptic plasmid that replicates in the absence of DNA polymerase I without producing single-stranded intermediates. A recombinant derivative harboring the pMJ101 replication region proved to be compatible with pJM1, a plasmid containing the iron acquisition system required for the virulence of V. anguillarum 775, another important pathogen that causes vibriosis. Sequence analysis of a 1.56-kb fragment harboring the pMJ101 replication region revealed the presence of typical features found in DNA origins including an AT-rich region, 11 dam-methylation sites of which 5 are within the putative ori region, and five copies of the 9-bp consensus sequence for DnaA binding. Gel retardation assays demonstrated that the latter replication element indeed binds DnaA purified from Escherichia coli. A potential open reading frame encoding a hydrophilic protein with a predicted pI of 10.3 and an M(r) of 33,826 was found adjacent to the ori region. Although these properties are typical of DNA-binding proteins, no significant homology was found between this predicted protein, named RepM, and other previously characterized proteins. Reverse transcriptase-polymerase chain reaction analysis of total RNA demonstrated the presence of repM mRNA in V. ordalii. The major initiation site of this mRNA was located 187 nucleotides upstream of the GTG initiation codon as determined by nuclease S1 protection assays. This transcription initiation site is preceded by putative -10 and -35 promoter sequences that control the expression of the repM replication gene. These results demonstrate that the replication region of pMJ101 shares some structural and sequence similarities with other DNA replication regions, which include DnaA binding and methylation sites and an open reading frame encoding a distinct protein required for its replication.
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PMID:Analysis of the replication elements of the pMJ101 plasmid from the fish pathogen Vibrio ordalii. 1041 62

Hyperbaric oxygen (HBO) treatment of human subjects (i.e. exposure to 100% oxygen at a pressure of 2.5 ATA for a total period of 3 x 20 min) caused clear and reproducible DNA damage in lymphocytes, as detected with the comet assay (single cell gel electrophoresis). Induction of DNA damage was found only after the first HBO exposure and not after further treatments of the same individuals. Furthermore, blood taken 24 h after HBO treatment was significantly protected against the induction of DNA damage by hydrogen peroxide (H(2)O(2)) in vitro, indicating that adaptation occurred due to induction of antioxidant defenses. The cells were not significantly protected against the genotoxic effects of gamma-irradiation, suggesting increased scavenging of reactive oxygen species distant from nuclear DNA or an inducible change in the levels of free transition metals. We now demonstrate increased levels of heme oxygenase-1 (HO-1) in lymphocytes 24 h after HBO treatment of volunteers. Under the same conditions, superoxide dismutase, catalase and the DNA repair enzymes apurinic endonuclease and DNA polymerase beta were not enhanced in expression. We also show that protection against the induction of DNA damage by H(2)O(2) in lymphocytes even occurs with a shortened HBO treatment which did not induce significant DNA damage by itself. Our results suggest that increased sequestration of iron as a consequence of induced HO-1 might be involved in the adaptive protection after HBO treatment and that the induction of DNA damage is not the trigger for adaptive protection.
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PMID:Induction of heme oxygenase-1 and adaptive protection against the induction of DNA damage after hyperbaric oxygen treatment. 1102 35

Poly(A)-specific ribonuclease (PARN) is the only mammalian exoribonuclease characterized thus far with high specificity for degrading the mRNA poly(A) tail. PARN belongs to the RNase D family of nucleases, a family characterized by the presence of four conserved acidic amino acid residues. Here, we show by site-directed mutagenesis that these residues of human PARN, i.e. Asp(28), Glu(30), Asp(292), and Asp(382), are essential for catalysis but are not required for stabilization of the PARN x RNA substrate complex. We have used iron(II)-induced hydroxyl radical cleavage to map Fe(2+) binding sites in PARN. Two Fe(2+) binding sites were identified, and three of the conserved acidic amino acid residues were important for Fe(2+) binding at these sites. Furthermore, we show that the apparent dissociation constant ((app)K(d)) values for Fe(2+) binding at both sites were affected in PARN polypeptides in which the conserved acidic amino acid residues were substituted to alanine. This suggests that these residues coordinate divalent metal ions. We conclude that the four conserved acidic amino acids are essential residues of the PARN active site and that the active site of PARN functionally and structurally resembles the active site for 3'-exonuclease domain of Escherichia coli DNA polymerase I.
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PMID:Identification of the active site of poly(A)-specific ribonuclease by site-directed mutagenesis and Fe(2+)-mediated cleavage. 1174 7


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