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)

Synthesis of the oligonucleotides that prime replication of phiX174 single-stranded DNA employs complex protein machinery of the host cell which is probably used by the cell to replicate its own chromosome. Primer synthesis depends on at least five proteins (DNA binding protein, dnaB and dnaC proteins, protein i, and protein n) and ATP to form a replication intermediate and another protein, primase (dnaG protein), to assemble the oligonucleotide by template transcription. The data in this paper show that ribo- and deoxyribonucleoside triphosphates can serve as substrates and form hybrid primers when present together. Both RNA and DNA primers were initiated with ATP. At least three of the four base-pairing nucleoside triphosphates were required for the transcription that generates effective primers. Over 90% of the RNA and DNA transcripts were extended into complementary strands by DNA polymerase III holoenzyme. At optimal triphosphate concentrations, the rate and extent of primer formation were greater from ribonucleoside triphosphates than from deoxyribonucleoside triphosphates. Uncoupled from DNA replication, the length of RNA primers was 14 to 50 residues, the DNA primers 4 to 20 residues. The fingerprint pattern of an RNase digest of RNA primers has a complexity suggestive of transcription from many sites on the phiX174 template. The multienzyme priming system is highly specific for phiX174 DNA as template.
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PMID:A multienzyme system for priming the replication of phiX174 viral DNA. 34 90

Adenosine 2',3'-riboepoxide 5'-triphosphate (epoxyATP) has been found to be a suicidal inactivator of DNA polymerase I from Escherichia coli by the following criteria. Inactivation is complete, is first order in enzyme activity, and shows saturation kinetics with an apparent KD of 30 +/- 10 micron for epoxy ATP. This KD is comparable to the KM of the substrate dATP. The t1/2 for inactivation is 1.3 min. Inactivation requires Mg2+ and the complementary template. The enzyme is protected by dATP but not by an excess of template. Gel filtration of the reaction mixture after inactivation with [3H]epoxy ATP results in the comigration of E. coli DNA polymerase I, the tritium-labeled inactivator, and the DNA template. The stoichiometry of binding approaches 1 mol of [3H]epoxy nucleotide per mol of inactivated enzyme. These results are consistent with the hypothesis that epoxy ATP initially serves as a substrate for the polymerase reaction, elongating the DNA chain by a nucleotidyl unit, and subsequently alkylates an essential base at the primer terminus binding site of the enzyme. Epoxy ATP also inactivates human and viral DNA polymerases but not E. coli RNA polymerase or rabbit muscle pyruvate kinase. Hence epoxy ATP may be a specific suicide reagent for DNA polymerases.
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PMID:Apparent suicidal inactivation of DNA polymerase by adenosine 2',3'-riboepoxide 5'-triphosphate. 34 91

2'-Deoxy-2'-azidocytidine-5'-triphosphate was investigated as an inhibitor in two reconstructed enzyme systems which catalyze the replication of two viral DNAs. During replication of the duplex replicative form of phiX174 DNA, DNA polymerase III holoenzyme was weakly inhibited and inhibition was reversed by dCTP. A more pronounced inhibition, not reversed by either dCTP or CTP, was observed during replication of the single-stranded DNA of the bacteriophage G4, a close relative of phiX174. This effect depended on the incorporation of 2'-deoxy-2'-azidocytidine-5'-triphosphate by primase (dnaG protein) which synthesizes a 29-residue RNA primer at the unique origin of bacteriophage G4 DNA replication. Extension of the primer strand, terminated by 2'-deoxy-2'-azidocytidine-5'-triphosphate is then severely inhibited. Primase was also inhibited by the 2'-deoxy-2'-azido derivatives of ATP, GTP, and UTP.
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PMID:Inhibition of primase, the dnaG protein of Escherichia coli by 2'-deoxy-2'-azidocytidine triphosphate. 35 34

We have examined the role of the uvrC gene in UV excision repair by studying incision, excision, repair synthesis, and DNA strand reformation in Escherichia coli mutants made permeable to nucleoside triphosphates by toluene treatment. After irradiation, incisions occur normally in uvrC cells in the presence of nicotinamide mononucleotide (NMN), a ligase-blocking agent, but cannot be detected otherwise. We conclude that repair incisions are followed by a ligation event in uvrC mutants, masking incision. However, a uvrC polA12 mutant accumulates incisions only slightly less efficiently than a polA12 strain without NMN. Excision of pyrimidine dimers is defective in uvrC mutants (polA(+) or polA12) irrespective of the presence or absence of NMN. DNA polymerase I-dependent, NMN-stimulated repair synthesis, which is demonstrable in wild-type cells, is absent in uvrC polA(+) cells, but the uvrC polA12 mutant exhibits a UV-specific, ATP-dependent repair synthesis like parental polA12 strains. A DNA polymerase I-mediated reformation of high-molecular-weight DNA takes place efficiently in uvrC polA(+) mutants after incision accumulation, and the uvrC polA12 mutant shows more reformation than the polA12 strain after incision. These results indicate that normal incision occurs in uvrC mutants, but there appears to be a defect in the excision of pyrimidine dimers, allowing resealing via ligation at the site of the incision. The lack of NMN-stimulated repair synthesis in uvrC polA(+) cells indicates that incision is not the only requirement for repair synthesis.
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PMID:uvrC gene function in excision repair in toluene-treated Escherichia coli. 36 20

An in vitro system was used to study DNA synthesis in lysates of Escherichia coli cells which had been grown in the presence of ethionine. Such lysates showed a reduced capacity to incorporate [3H]TTP into high-molecular-weight material. Activity could be restored by incubation with S-adenosyl methionine and ATP. S-adenosyl methionine-reactivated TTP incorporation required the presence of DNA polymerase I, ATP, and all four deoxyribonucleotide triphosphates. DNA polymerase III was not required.
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PMID:Methylation-dependent DNA synthesis in Escherichia coli mediated by DNA polymerase I. 36 22

RNA-linked DNA fragments of T7-infected Escherichiacoli were labeled with [(32)P]orthophosphate invivo. The RNA segments of the labeled fragments were isolated by degrading the DNA portion with the 3'--> 5' exonuclease intrinsic to bacteriophage T4 DNA polymerase and fractionated according to net charge by a DEAE-Sephadex A-25 column chromatography in the presence of 7 M urea. Tri-, tetra- and pentanucleotides were obtained which have ATP residues at their 5' ends. Most of the pentanucleotides had a single deoxynucleotide at the 3' end but a minor portion was totally an oligoribonucleotide. In the light of prior results, the former is a cooligomer of an intact tetraribonucleotide primer and a monodeoxynucleotide and the latter is an intact pentaribonucleotide primer. Tri- and tetraribonucleotides with ATP at the 5' ends had no deoxynucleotide at the 3' ends, therefore it is not clear if intact triribonucleotide primers are present. The 5'-terminal dinucleotides of the tetra- and pentanucleotides were mostly pppApC and a trace amount of pppApA was present.Images
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PMID:RNA-linked nascent DNA pieces in phage T7-infected Escherichia coli. III. Detection of intact primer RNA. 38 59

The optimal conditions and the effect of deoxyribonucleoside triphosphates were determined for CDP reductase activity in PHA-stimulated lymphocytes. The enzymatic reaction showed an absolute requirement for ATP. In the absence of ATP, only dATP showed a minor stimulation of the reduction of CDP to dCDP. During transformation the CDP reductase activity reached a maximum at the same time as the four deoxyribonucleoside triphosphate pools, corresponding to mid S-phase at about 50 h after PHA addition. The DNA polymerase activity reached a maximum at 57 h.
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PMID:Cytidine 5'-diphosphate reductase activity in phytohemagglutinin stimulated human lymphocytes. 42 94

DNA synthesis starts about 12 h after water imbibition in wheat embryos. We have determined that noticeable amounts of labelled thymidine are found inside the embryo only after 6 hr of germination. DNA polymerase C from ungerminated wheat embryos decreased markedly in activity during the first hours of germination, whereas the activities of DNA polymerases A and B increased, having a maximum at about 15 h or germination. Serological evidence has suggested a clear antigenic relationship between DNA polymerases A and C. Although the pool of ATP increases rapidly after water imbibition, the increase in the pool of dNTP species was much slower.
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PMID:Factors affecting the onset of deoxyribonucleic acid synthesis during wheat embryo germination. Study of the changes in DNA polymerases A, B and C and the pool of DNA precursors. 48 51

Bacteriophage T7 DNA primase (gene-4 protein, 66,000 daltons) enables T7 DNA polymerase to initiate the synthesis of DNA chains on single-stranded templates. An initial step in the process of chain initiation is the formation of an oligoribonucleotide primer by T7 primase. The enzyme, in the presence of natural SS DNA, Mg++ (or Mn++), ATP and CTP (or a mixture of all 4 rNTPs), catalyzes the synthesis of di-, tri-, and tetraribonucleotides all starting at the 5' terminus with pppA. In a subsequent step requiring both T7 DNA polymerase and primase, the short oligoribonucleotides (predominantly pppA-C-C-AOH) are extended by covalent addition of deoxyribonucleotides. With the aid of primase, T7 DNA polymerase can also utilize efficiently a variety of synthetic tri-, tetra-, or pentanucleotides as chain initiators. T7 primase apparently plays an active role in primer extension by stabilizing the short primer segments in a duplex state on the template DNA.
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PMID:Role of bacteriophage T7 DNA primase in the initiation of DNA strand synthesis. 60 Jul 93

Upon exposure to the carcinogens N-acetoxy-N-2-acetylaminofluorene and 7-bromomethyl-benz[a]anthracene, which bind covalently to DNA, ether-permeabilized (nucleotide-permeable) Escherichia coli wild-type cells responded with DNA excision repair. This repair was missing in mutants carrying defects in genes uvrA, uvrB and uvrC, whereas it was present in uvrD and several rec mutants. Enzymic activities involved were identified by measuring repair polymerization and size reduction of denatured DNA. 1. An easily measurable effect in E. coli wild-type cells was carcinogen-induced repair polymerization. When initiated by N-acetoxy-N-2-acetylaminofluorene or 7-bromomethyl-benz[a]anthracene, it depended upon an ATP-requiring step; CTP, GTP or UTP did not substitute for ATP. DNA repair synthesis was inhibited by p-chloromercuribenzoate and quinacrine. In uvrA, uvrB and uvrC mutants no carcinogen-stimulated DNA synthesis could be detected, indicating that steps involved in pyrimidine dimer excision are also involved in chemorepair. In recA, recB and recC mutant cells, repair synthesis was stimulated by the carcinogens to a normal extent. This evidence excludes the ATP-dependent recB,C deoxyribonuclease and recA gene products as playing an important role in carcinogen-induced excision repair. polA1 cells showed drastically reduced levels of rapair polymerization, indicating that DNA polymerase I is the main polymerizing enzyme. 2. As determined by DNA size reduction in alkaline sucrose gradients, the arylalkylating carcinogens caused endonucleolytic cleavage of endogenous DNA in wild-type cells. This incision step was most effectively performed in the presence of ATP; UTP, CTP and GTP were only slightly effective. Incision was inhibited by p-chloromercuribenzoate and quinacrine. When exposed to the arylalkylating carcinogens, uvrA, uvrB and uvrC mutant cells did not perform the incision step in the presence of ATP, suggesting the involvement of the respective gene products in the initiation of chemorepair.
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PMID:Carcinogen-induced DNA repair in nucleotide-permeable Escherichia coli cells. Analysis of DNA repair induced by the carcinogens N-acetoxy-N-2-acetylaminofluorene and 7-bromomethyl-benz(a)anthracene. 76 31


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