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)

The Bacillus subtilis phage phi 29 DNA polymerase, involved in protein-primed viral DNA replication, contains several amino acid consensus sequences common to other eukaryotic-type DNA polymerases. Using site-directed mutagenesis, we have studied the functional significance of a C-terminal conserved region, represented by the Lys-X-Tyr ("K-Y") motif. Single point mutants have been constructed and the corresponding proteins have been overproduced and characterized. Measurements of the activity of the mutant proteins indicated that the invariant Lys and Tyr residues play a critical role in DNA polymerization. Interestingly, substitution of the invariant Lys either by Arg or Thr, produced enzymes with an increased or a largely reduced, respectively, capability to use a protein as primer, an intrinsic property of TP-priming DNA polymerases. On the other hand, the viral protein p6, which stimulates initiation of phi 29 DNA replication by formation of a nucleoprotein complex at both DNA replication origins, increased (about 5-fold) the insertion fidelity of phi 29 DNA polymerase during the formation of the TP-dAMP initiation complex. We propose a model in which the special strategy to maintain the integrity of the phi 29 DNA ends, by means of a "sliding-back" mechanism, could also contribute to increase the fidelity of phi 29 DNA replication.
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PMID:Structural and functional studies on phi 29 DNA polymerase. 129 Dec 40

A molecular cDNA clone (P1 KIN) was isolated that encodes the human RNA-dependent P1/eIF-2 alpha protein kinase. The complete cDNA sequence of the P1 KIN cDNA was determined; the longest open reading frame (ORF) encoded a 551 amino acid protein with a deduced molecular weight of 62055 Da. Transcripts prepared from the P1 KIN cDNA by transcription in vitro with T7 RNA polymerase programmed the cell-free synthesis of a protein indistinguishable by immunoprecipitation and immunoblot gel analyses from the authentic 67-kDa P1 protein synthesized in human U cells treated with interferon (IFN). Furthermore, by use of a sensitive primer extension assay with T7 DNA polymerase, the major site of translation initiation within the deduced ORF of the P1 KIN cDNA was directly identified. Northern RNA gel-blot analysis revealed that the P1 KIN cDNA strongly hybridized to two IFN-induced mRNAs present in both human amnion U cells and HeLa cells; their sizes were 2.5 and 6 kb. Both transcripts were efficiently induced by IFN-alpha, but poorly by IFN-gamma. Polyclonal antibody was prepared against the product of the P1 KIN cDNA expressed in Escherichia coli. In Western blot analysis the antibody recognized a 67-kDa protein induced in human cells by IFN-alpha and, in addition, a 90-kDa protein whose level was not greatly altered by IFN treatment. The IFN-induced 67-kDa protein was found associated with the ribosomal salt-wash fraction of IFN-treated human cells, whereas the 90-kDa protein was predominantly in the S100 soluble fraction. The time course for the induction by IFN-alpha of RNA-dependent protein P1 kinase activity measured by immunoprecipitation was comparable to the time course for protein P1 induction measured by Western immunoblot analysis. The amino acid sequence of P1/eIF-2 alpha protein kinase deduced from the cDNA was 62% identical with the 518-residue murine TIK kinase and contained, within the carboxy-terminal half of the protein, the motifs commonly conserved among protein-serine/threonine kinases. The amino-terminal half of the P1 protein did not possess conserved kinase motifs, but did show extensive homology with vaccinia virus-predicted protein E3L.
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PMID:Mechanism of interferon action: cDNA structure, expression, and regulation of the interferon-induced, RNA-dependent P1/eIF-2 alpha protein kinase from human cells. 137 53

We have identified an amino-proximal sequence motif, Phe-Asp-Ile-Glu-Thr, in Saccharomyces cerevisiae DNA polymerase II that is almost identical to a sequence comprising part of the 3'----5' exonuclease active site of Escherichia coli DNA polymerase I. Similar motifs were identified by amino acid sequence alignment in related, aphidicolin-sensitive DNA polymerases possessing 3'----5' proofreading exonuclease activity. Substitution of Ala for the Asp and Glu residues in the motif reduced the exonuclease activity of partially purified DNA polymerase II at least 100-fold while preserving the polymerase activity. Yeast strains expressing the exonuclease-deficient DNA polymerase II had on average about a 22-fold increase in spontaneous mutation rate, consistent with a presumed proofreading role in vivo. In multiple amino acid sequence alignments of this and two other conserved motifs described previously, five residues of the 3'----5' exonuclease active site of E. coli DNA polymerase I appeared to be invariant in aphidicolin-sensitive DNA polymerases known to possess 3'----5' proofreading exonuclease activity. None of these residues, however, appeared to be identifiable in the catalytic subunits of human, yeast, or Drosophila alpha DNA polymerases.
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PMID:Eukaryotic DNA polymerase amino acid sequence required for 3'----5' exonuclease activity. 165 84

We determined that 85 microM aphidicolin was sufficient to block macroscopic plaque formation by vaccinia virus and to cause a 10(4)-fold reduction in viral yield from a wild-type infection. A chemically mutagenized viral stock was passaged sequentially in the presence of drug, and plaque-purified viral stocks resistant to aphidicolin were isolated and characterized. By use of a marker rescue protocol, the lesion in each mutant was found to map within the same 500-bp fragment within the DNA polymerase gene. All of the mutants were found to contain a single nucleotide change in the same codon. In nine of these mutants, the alanine residue at position 498 was changed to a threonine, whereas a 10th mutant sustained a valine substitution at this position. Congenic viral strains which carried the Aphr lesion in an unmutagenized wild-type background were isolated. The Thr and Val mutations were found to confer equivalent levels of drug resistance. In the presence of drug, viral yields were 25% of control levels, and the levels of viral DNA synthesized were 30 to 50% of those seen in control infections. The two mutations also conferred an equivalent hypersensitivity to the cytosine analog 1-beta-D-arabinofuranosylcytosine (araC); strains carrying the Thr mutation were moderately hypersensitive to the pyrophosphate analog phosphonoacetic acid and the adenosine analog araA, whereas the Val mutation conferred acute hypersensitivity to these inhibitors. The Val mutation also conferred a mutator phenotype, leading to a 20- to 40-fold increase in the frequency of spontaneous mutations within the viral stock.
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PMID:Genetic characterization of the vaccinia virus DNA polymerase: identification of point mutations conferring altered drug sensitivities and reduced fidelity. 189 73

The sequence Gly-Asp-Met-Asp, spanning positions 189-192 of rat DNA polymerase beta, is similar to the sequence motif Gly-Asp-Thr-Asp that is highly conserved in a number of replicative DNA polymerases from eukaryotic cells, viruses, and phages. The role of this sequence in the catalytic function of rat DNA polymerase beta was investigated by individually changing each amino acid in this region by site-directed mutagenesis. The mutant enzymes DE190 and DE192, in which aspartic acid residues at positions 190 and 192, respectively, were replaced by glutamic acid, showed about 0.1% activity of the wild-type enzyme. On the other hand, the replacement of Gly-189 by alanine or Met-191 by isoleucine or threonine only slightly affected the enzyme activity. A gel mobility shift assay showed that DNA complexes with enzyme DE190 and especially with DE192 were less stable than the corresponding complex with the wild-type enzyme. Kinetic analysis with these mutant enzymes indicate that their Km's for primer DNA were about 10-fold higher than that of the wild type, while Km's for deoxyribonucleoside triphosphate were not changed. Since neither DE190 nor DE192 had any significant alteration in secondary structure, our results suggest that both Asp-190 and Asp-192 are located in the active site and are involved in the interaction of DNA polymerase beta with primer.
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PMID:Aspartic acid residues at positions 190 and 192 of rat DNA polymerase beta are involved in primer binding. 203 95

The alpha-like DNA polymerases from bacteriophage phi 29 and other viruses, prokaryotes and eukaryotes contain an amino acid consensus sequence that has been proposed to form part of the dNTP binding site. We have used site-directed mutants to study five of the six highly conserved consecutive amino acids corresponding to the most conserved C-terminal segment (Tyr-Gly-Asp-Thr-Asp-Ser). Our results indicate that in phi 29 DNA polymerase this consensus sequence, although irrelevant for the 3'----5' exonuclease activity, is essential for initiation and elongation. Based on these results and on its homology with known or putative metal-binding amino acid sequences, we propose that in phi 29 DNA polymerase the Tyr-Gly-Asp-Thr-Asp-Ser consensus motif is part of the dNTP binding site, involved in the synthetic activities of the polymerase (i.e., initiation and polymerization), and that it is involved particularly in the metal binding associated with the dNTP site.
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PMID:The highly conserved amino acid sequence motif Tyr-Gly-Asp-Thr-Asp-Ser in alpha-like DNA polymerases is required by phage phi 29 DNA polymerase for protein-primed initiation and polymerization. 219 Dec 96

We describe the polypeptide structure and some of the catalytic properties of a DNA polymerase alpha.DNA primase complex that can be prepared from KB cells by immunoaffinity purification. The procedure is based on monoclonal antibodies that were raised against a biochemically purified, catalytically active core protomer of the polymerase. In all respects tested, the basic mechanism of substrate recognition and binding by the immunoaffinity-purified polymerase is qualitatively identical to that of the core protomer. The immunoaffinity-purified KB cell polymerase alpha X DNA primase is structurally complex. On the basis of extensive immunochemical analyses with five independent monoclonal antibodies, three of which are potent neutralizers of polymerase alpha activity, peptide mapping studies, and the application of a sensitive immunoassay that permits detection of polymerase alpha antigens in crude cell lysates, we have established that the principal form of catalytically active DNA polymerase alpha in KB cells is a phosphoprotein with a molecular mass of 180 kilodaltons. This protein is stable in vivo, with an estimated half-life of greater than or equal to 15 h. In contrast, the polypeptide is extremely fragile in vitro and generates partial degradation products of p165, p140, and p125 that explain the "microheterogeneity" typically exhibited by polymerase alpha peptides in denaturing polyacrylamide gels. In addition to the catalytically active polymerase alpha polypeptide(s), the immunopurified enzyme fraction typically contains three other proteins, p77, p55, and p49, the functions of which have not yet been established. These proteins do not display polymerase alpha epitopes and have been shown by peptide mapping to be independent species that are unrelated either to the large polymerase peptides or to one another. The polypeptide p77 is also a phosphoprotein, and in both p180 and p77 the phosphorylated amino acids are exclusively serine and threonine.
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PMID:Structural and enzymological characterization of immunoaffinity-purified DNA polymerase alpha.DNA primase complex from KB cells. 242 27

The amino acid substitutions responsible for the temperature-sensitive (ts) and mutator phenotypes of the classical bacteriophage T4 DNA polymerase mutant tsL56 were determined. tsL56 DNA polymerase has two mutations in the 5' end of the DNA polymerase gene (g43) that produce two amino acid substitutions: codon 89, alanine to threonine, and codon 363, aspartate to asparagine. Both mutations are required for the strong ts and mutator phenotypes. The increased error rate of the tsL56 DNA polymerase is due to a reduction in 3'----5' exonuclease activity relative to polymerase activity (N. Muzyczka, R. L. Poland, and M. J. Bessman, J. Biol. Chem. 247:7116-7122, 1972). Thus, the locations of the tsL56 mutations suggest that the 3'----5' exonuclease domain resides in the N-terminal region. Several other ts DNA polymerase mutant strains isolated with tsL56 also have mutator or antimutator phenotypes. The nucleotide changes in these important mutant strains were also determined. This mutant collection, combined with collections of g43 amber mutants and mutants selected on the basis of a strong mutator phenotype (L. J. Reha-Krantz, J. Mol. Biol. 202:711-724, 1988), contains nearly 70 different DNA polymerase mutations. The numerous T4 DNA polymerase mutations are valuable for DNA polymerase structure-function and fidelity studies.
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PMID:Locations of amino acid substitutions in bacteriophage T4 tsL56 DNA polymerase predict an N-terminal exonuclease domain. 267 3

Treatment of HSV-infected cells with 5-10 mM beta-hydroxynorvaline (Hnv), a threonine analog, specifically affects herpesvirus DNA replication: both the rate of and total DNA synthesis are reduced, the former approximately 15-fold by Hnv (6 h post-infection) and the latter by 12-fold (between 3 and 12 h post-infection). The effect on DNA replication was due to inhibition of HSV-1 thymidine kinase (TK) and DNA polymerase (DP) activities; the former is reduced by 75% and whereas DP returns to baseline levels (when compared to untreated and/or uninfected cells). Host cell TK and DP activities are unaffected. It is suggested that beta-hydroxynorvaline is incorporated into these enzyme(s), either close to or at the active site thus perturbing viral DNA synthesis. beta-Hydroxynorvaline should have unique utility as a targeted antiviral compound, acting on both membrane-mediated phenomena (fusion, penetration and attachment) and DNA replication.
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PMID:Inhibition of herpesvirus-induced thymidine kinase and DNA polymerase by beta-hydroxynorvaline. 282 76

By site-directed mutagenesis we have changed the serine residue 232 of the phi 29 terminal protein, involved in the covalent linkage to dAMP for the initiation of replication, into a threonine residue. The mutant terminal protein has been purified to homogeneity and shown to be inactive in the formation of the initiation complex; nevertheless, the mutant protein retains its ability to interact with the phi 29 DNA polymerase and with the DNA. The results obtained indicate a high specificity in the linking site of the terminal protein.
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PMID:Site-directed mutagenesis in the DNA linking site of bacteriophage phi 29 terminal protein: isolation and characterization of a Ser232----Thr mutant. 313 31


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