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)

Several of the vaccinia virus core proteins are synthesized as large precursor proteins which are subsequently processed to smaller products during the course of viral maturation. Amino acid alignment of these proteins reveals a conserved Ala-Gly-X motif (AG*X) at their confirmed cleavage sites. To better understand the regulation of cleavage site selection, the sequence of the entire vaccinia virus genome was searched for the occurrence of this AG*X motif in predicted open reading frames. Of the 82 sites found, 19 resembled cleavage sites which have previously been shown to be actively processed, namely AG*A of P25K and P4b, and AG*S and AG*T of P4a. To test the universality of the AG*X motif utilization, immunological methods in concert with N-terminal microsequencing procedures have been used to determine which of the subset of predicted proteins containing AG*A sites are utilized in vivo. Of the seven AG*A-containing substrates, four were cleaved and three were not. Considering all the known AG*X processing events, it appears that only those proteins expressed at late times during infection and associated with the assembling virion are candidate substrates for proteolytic cleavage. Such proteins include P4a, P4b, P25K, and the newly identified P21K and P17K (derived from genes A17L and A12L, respectively). Although proteins such as DNA polymerase, P37K, and a host range protein contain a consensus cleavage site, they are excluded from processing. This proteolytic exclusion presumably occurs because these proteins do not meet both of the above criteria, which suggests that temporal expression or compartmentalization (substrate presentation) in the assembling virion may play a regulatory role in proteolysis.
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PMID:Differential utilization of a conserved motif for the proteolytic maturation of vaccinia virus proteins. 812 19

The vaccinia virus genome encodes a DNA polymerase that is similar to other DNA polymerases. A mutation in the polymerase gene at a site that is adjacent to conserved residues allows viral replication in the presence of aphidicolin. Since wild-type virus is converted to aphidicolin-resistance by site-directed mutagenesis, it was feasible that active virus with substituted conserved residues could be detected by linking alterations to the aphidicolin-resistance mutation. Altered DNA, from a PCR, was introduced into virus by a marker transfer procedure. DNA from plaques of drug-resistant virus was amplified, and the product was sequenced to check for the conserved residue alteration. An alteration that introduced a Bg1I site was designed to facilitate the selection of drug-resistant virus containing substituted residues. One positive result was the replacement of two amino acids, tyrosine and alanine, by tryptophan and threonine. The failure to substitute aspartic acid for tyrosine indicates that drastic changes of the conserved sequence are not tolerated. Although the limitations associated with negative results apply, the method provides an in vivo assay for selecting a polymerase with conserved residue changes.
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PMID:A biological method for examining the effect of codon changes in a conserved region of DNA polymerase. 813 25

Conserved site-directed mutations were introduced into the second most conserved amino acid region, region II, of the human DNA polymerase alpha catalytic subunit. These mutants were expressed in the baculovirus system and purified to near homogeneity. The mutants had polymerase activity ranging from 4 to 60% compared with the wild type polymerase alpha. Steady-state kinetic analysis of mutants G841A, D860A, D860S, D860N, Y865S, and Y865F demonstrated no significant difference in their Km values for primer-template compared with that of the wild type enzyme. In contrast, mutants D860A, Y865S, and Y865F showed a 5-10-fold increase in the Km for deoxynucleotide triphosphate (dNTP) compared with the wild type enzyme. DNA synthetic fidelity studies of these mutants showed that mutant Y865S but not Y865F had a greater than 10-fold higher misinsertion efficiency than the wild type enzyme in Mg(2+)-catalyzed reactions. However, with Mn2+ as the metal activator, Y865S and Y865F demonstrated a 2- and 9-fold higher misinsertion efficiency, respectively. These results indicate that Asp860 and Tyr865 in region II of human DNA polymerase alpha are involved in incoming dNTP substrate binding. Using three deoxynucleotide structural analogs as probes, we show that the nucleotide base is the structural requirement for dNTP binding with Tyr865. Furthermore, abolishing the hydrophobic phenyl ring side chain of Tyr865 by replacing tyrosine with serine rendered the enzyme resistant to aphidicolin. Results of these studies strongly suggest that the phenyl ring of Tyr865 directly interacts with the nucleotide base moiety of the dNTP and plays a critical role in the misinsertion fidelity of DNA synthesis. Although mutation of Gly841 to Ala did not affect the binding of primer-template, it had a significant decrease in kcat, an increase in Km for dNTP, a striking decrease of processivity, and also resistance to aphidicolin. Thus, mutation of this residue, Gly841, which is highly conserved among the alpha-like DNA polymerases, appears to affect both catalysis and substrate deoxynucleotide binding. This suggests that Gly841 is essential for the maintenance of the overall structure of the polymerase alpha catalytic site.
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PMID:Mutational studies of human DNA polymerase alpha. Identification of residues critical for deoxynucleotide binding and misinsertion fidelity of DNA synthesis. 822 63

The second most conserved region of alpha-like DNA polymerases, region II, spans a block of 40 amino acid residues centered at the core sequence -DFNSLYPSII-. In the previous paper, we described mutational studies of 3 amino acid residues in region II which includes 2 amino acid residues in the core sequence. We showed that residues Asp860 and Tyr865 in the core sequence are involved in substrate deoxynucleotide triphosphate (dNTP) binding. We further showed that the phenyl moiety of the Tyr865 side chain interacts with the incoming dNTP and is responsible for the misinsertion fidelity of the enzyme. In this report, we investigated the function of 2 serine residues, Ser863 and Ser867, in this core sequence. Mutation of these 2 Ser residues to either Ala or Thr yielded mutant enzymes with similar Km for dNTPs, kcat, processivity, and misinsertion fidelity of DNA synthesis as the wild type enzyme. However, mutation of Ser867 to Ala demonstrated a 30-fold increase in Km for primer-template and a 5-fold higher KD for binding primer-template. DNA footprinting experiments of primer with the dideoxynucleotide terminus indicated that the structural feature of the primer recognized by Ser867 is the 3'-OH terminus. Single-stranded DNA inhibition data suggest that removal of the hydroxyl side chain of Ser867 affects the polymerase's interaction with primer and not with template. Mutation of Ser867 to Ala also decreases the mutant enzyme's Km for dNTP to extend a mispaired primer and thus enhances its capacity to extend a mispaired primer terminus. These data support the conclusion that the hydroxyl side chain of Ser867 of human DNA polymerase alpha is involved in primer interaction during DNA synthesis and plays an essential role in mispair extension fidelity of DNA synthesis.
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PMID:Mutational studies of human DNA polymerase alpha. Serine 867 in the second most conserved region among alpha-like DNA polymerases is involved in primer binding and mispair primer extension. 822 64

DR6 is a complex allele family composed of at least 16 different alleles. Although 25% of Koreans express DR6 alleles, this allele family has not been well studied in the population. DNA samples obtained from 252 unrelated individuals were screened by PCR using Taq DNA polymerase and a DRB1 group-specific PCR primer set that amplifies the polymorphic second exon of DR3, DR11, and DR6 DRB1 alleles. To identify the DR6 allelic frequencies in this population, PCR-positive samples were further analyzed by dot-blot hybridization using digoxigenin-labeled SSOPs. In this process, a new DRB1 allele was identified by its unique hybridization pattern and was further characterized by direct sequencing after PCR. The new DRB1 sequence is similar to DRB1*1101, differing at codon 47 (TAC[Tyr]/TTC[Phe]) and at codon 58 (GCC[Ala]/GAG[Glu]). Based on sequence comparisons as well as its DRB3 and DQ associations, the new allele may have arisen by a gene conversion event from DRB1*1101. The resultant DR molecule bears DR6 serologic determinants as determined by serologic typing and, based on sequence, is probably a DR13 and not a DR14 allele. These data suggest that the DR11 allele has frequently acted as a recipient gene in the gene conversion events that created the subfamily of DR13 alleles, DRB1*1303, *1304, *1305, and the new allele described here.
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PMID:DR6 in Koreans. DR11 frequently acts as a recipient gene to create DR13 alleles. 830 Apr 8

Adenovirus DNA polymerase (AdPol) exists as a complex with the preterminal protein (pTP) and is essential for both initiation and elongation stages of viral DNA replication. Recent evidence from our laboratory indicates that AdPol is a phosphoprotein and that the major in vivo phosphorylation site, serine 67, occurs within the consensus substrate recognition sequence for cdc2 kinases. In this study, we found that a protein kinase which also exhibits histone H1 phosphorylation activity is stably associated with AdPol. AdPol forms a multimeric complex with this histone H1 kinase and pTP in HeLa cells infected with adenovirus or coinfected with recombinant vaccinia viruses encoding AdPol and pTP. The associated protein kinase and the p34cdc2 kinase phosphorylate AdPol at the same sites which are utilized in vivo, suggesting that the p34cdc2 kinase or a related kinase may be involved in the in vivo phosphorylation of AdPol. Serine 67 is also one of the major in vitro phosphorylation sites, and the substitution of alanine for serine at this position abolishes DNA replication initiation activity of AdPol.
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PMID:Adenovirus DNA polymerase is phosphorylated by a stably associated histone H1 kinase. 834 26

Ganciclovir-resistant mutant 759rD100 derived from human cytomegalovirus strain AD169 contains two resistance mutations, one of which is in the UL97 gene and results in decreased ganciclovir phosphorylation in infected cells [V. Sullivan, C. L. Talarico, S. C. Stanat, M. Davis, D. M. Coen, and K. K. Biron, Nature (London) 358:162-164, 1992]. In the present study, we mapped the second mutation to a 4.1-kb DNA fragment containing the DNA polymerase gene and showed that it confers ganciclovir resistance without impairing phosphorylation. Sequence analysis of the 4.1-kb region revealed a single nucleotide change that resulted in a glycine-to-alanine substitution at position 987 within conserved region V of the DNA polymerase. Recombinant viruses constructed to contain the DNA polymerase mutation but not the phosphorylation defect displayed intermediate resistance (4- to 6-fold) to ganciclovir relative to the original mutant 759rD100 (22-fold); the recombinant viruses also displayed resistance to ganciclovir cyclic phosphate (7-fold), 1-(dihydroxy-2-propoxymethyl)-cytosine (12-fold), and the phosphonylmethoxyalkyl derivatives (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)adenine and (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (8- to 10-fold). However, the recombinant viruses remained susceptible to certain related compounds. These results imply that the human cytomegalovirus DNA polymerase is a selective target for the antiviral activities of ganciclovir, certain of its derivatives and phosphonomethoxyalkyl derivatives; support a role for region V in substrate recognition; and suggest the possibility of clinical resistance of human cytomegalovirus to these compounds because of polymerase mutations.
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PMID:A point mutation in the human cytomegalovirus DNA polymerase gene confers resistance to ganciclovir and phosphonylmethoxyalkyl derivatives. 838 37

Phosphorylation of simian virus 40 (SV40) T antigen on threonine 124 activates viral DNA replication in vivo and in vitro. We have manipulated the modification of T-antigen residue 124 both genetically and biochemically and have investigated individual replication functions of T antigen under conditions suitable for in vitro DNA replication. We find that the hexamer assembly, helicase, DNA polymerase alpha-binding, and transcriptional-autoregulation functions are independent of phosphorylation of threonine 124. In contrast, neither T antigen with an alanine mutation of threonine 124 made in human cells nor unphosphorylated T antigen made in Escherichia coli binds the SV40 replication origin as stably as phosphorylated wild-type T antigen does. Furthermore, modification of threonine 124 is essential for complete unwinding of the SV40 replication origin. We conclude that phosphorylation of threonine 124 enhances specific interactions of T antigen with SV40 origin DNA. Our findings do not exclude the possibility that phosphorylation of threonine 124 may affect additional undefined steps in DNA replication. We also show that DNase footprinting and KMnO4 modification assays are not as stringent as immunoprecipitation and origin-dependent strand displacement assays for detecting defects in the origin-binding and -unwinding functions of T antigen. Differences in the assays may explain discrepancies in previous reports on the role of T-antigen phosphorylation in DNA binding.
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PMID:cdc2 phosphorylation of threonine 124 activates the origin-unwinding functions of simian virus 40 T antigen. 839 45

Bacteriophage T4 DNA polymerase has a proofreading 3'-->5' exonuclease that plays an important role in maintaining the accuracy of DNA replication. We have constructed a T4 DNA polymerase deficient in this exonuclease by converting Asp-219 to Ala. The exonuclease activity of the mutant T4 DNA polymerase has been reduced by a factor of at least 10(7), but it retains a polymerase activity whose kinetic parameters, kcat, Kd DNA, and Kd dATP, are very close to those of the wild-type enzyme. Bacteriophage T4 with the mutant polymerase gene has a markedly increased mutation frequency. Asp-219 in T4 DNA polymerase is within a sequence similar to those surrounding Asp residues previously shown to be essential for the exonuclease activities of the Klenow fragment of Escherichia coli DNA polymerase I (Asp-424), bacteriophage phi 29 DNA polymerase (Asp-66), and Saccharomyces cerevisiae DNA polymerase delta (Asp-405). Thus, these studies support the proposal that there are similar sequences in the active sites for the proofreading exonucleases of these and related DNA polymerases.
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PMID:Construction and characterization of a bacteriophage T4 DNA polymerase deficient in 3'-->5' exonuclease activity. 846 64

We have reported that a domain containing Arg682 in the Klenow fragment of Escherichia coli DNA polymerase I (pol I) is important for the template-dependent dNTP-binding function [Pandey, V.N., Kaushik, N. A., Pradhan, D. S. & Modak, M. J. (1990) J. Biol. Chem. 265, 3679-3884]. In order to further define the role of Arg682 in the catalytic process, we have performed site-directed mutagenesis of this residue. For this purpose the Klenow-coding region of the DNA-pol-I gene was selectively amplified from the genomic DNA of E. coli and was cloned in an expression vector, pET-3a. This clone under appropriate conditions overproduces the Klenow fragment in E. coli. Using this clone (pET-3a-K) as the template, two mutant polymerase clones were constructed in which arginine has been replaced with either alanine, [R682A] pol I, or lysine [R682K] pol I. Both mutant enzymes showed significantly lower specific activity as compared to the wild-type enzyme. The kinetic analyses of the mutant enzymes indicated a 3-4-fold increase in the Km for the substrate dNTP, a 20-25-fold decrease in the Vmax and an overall decrease in the processive nature of DNA synthesis in both the mutant enzymes. The reverse mutation of Ala682 to the wild-type form Arg682 fully restored the processive nature and the polymerase activity of the enzyme. These observations suggest that the positively charged guanidino group in the side chain of Arg682 is catalytically important but not absolutely essential for synthesis of DNA. Furthermore it appears to maintain high processivity of the DNA synthesis catalyzed by the enzyme.
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PMID:Site directed mutagenesis of DNA polymerase I (Klenow) from Escherichia coli. The significance of Arg682 in catalysis. 850 7


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