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)

Gene 5 protein of bacteriophage T7 is a nonprocessive DNA polymerase. During infection of Escherichia coli, T7 annexes the host protein thioredoxin for use as a processivity factor for T7 DNA polymerase. We describe here a genetic method to investigate the interaction between T7 gene 5 protein and E. coli thioredoxin. The strategy is to use thioredoxin mutants that are unable to support the growth of wild-type T7 phage to select for T7 revertant phage that suppress the defect in thioredoxin. A thioredoxin mutation that replaces glycine at position 74 with aspartic acid fails to support the growth of wild-type T7. This mutation is suppressed by six different mutations within T7 gene 5, each of which results in a single amino acid substitution within gene 5 protein. Three of the suppressor mutations are located within the putative polymerization domain of gene 5 protein, and three are located within the putative 3'-to-5' exonucleolytic domain. Each suppressor mutation alone is necessary and sufficient to confer the revertant phenotype.
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PMID:Genetic analysis of the interaction between bacteriophage T7 DNA polymerase and Escherichia coli thioredoxin. 140 97

The inherent infidelity of Taq DNA polymerase in the polymerase chain reaction was exploited to produce random mutations in the trp A gene. Screening of the resulting clones allowed selection of non-interactive mutant alpha subunits retaining their intrinsic catalytic activity. Two single changes responsible for this phenotype were identified by DNA sequencing as: alpha 126 valine (GTG)----glutamic acid (GAG) and alpha 128 valine (GTT)----aspartic acid (GAT). Three single changes giving a non-interactive phenotype with an impaired intrinsic catalytic activity were identified by DNA sequencing as alpha 66 asparagine (AAC)----aspartic acid (GAC); alpha 109 lysine (AAA)----arginine (AGA); alpha 118 cysteine (TGC)----arginine (CGC). Where possible, we individually assessed the importance of these residues in alpha beta interaction in light of structural information from X-ray crystallography and by intergeneric protein sequence comparison.
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PMID:Selection and analysis of non-interactive mutants in the Escherichia coli tryptophan synthase alpha subunit. 160 55

We have determined the nucleotide sequence of the polC gene of Bacillus subtilis which codes for DNA polymerase III. Our recent analysis has revealed that the gene comprises 4311 nucleotides, from the start to the stop codon, 306 nucleotides more than we reported earlier. The plasmid reported by us and by N.C. Brown's laboratory contained a sequence at the end of the gene which is not related to the polC region of B. subtilis. We have isolated the rest of the gene, the sequence of which is presented in this paper. The new stop codon is followed by a hyphenated palindromic sequence of 13 nucleotides. The C-terminus of the coding region contains the novel mutation, dnaF, which results in a defect in the initiation of replication due to a change in the codon TCC to TTC (serine to phenylalanine). The hypermutator mutation mut-1 is due to two point mutations in the 3' to 5' exonuclease domain, the proof reading function. The codon changes are GGA to GAA (glycine to glutamic acid) and AGC to AAC (serine to asparagine). The elongation defective mutation, polC26, affecting the catalytic site that adds nucleotides to the growing chain, is due to a change in the codon GTC to GAC (valine to aspartic acid). It is separated from the mutation reported earlier, azp-12, by 306 nucleotides. Knowing the locations of the mutational sites allowed us to deduce the domains of the gene and the enzyme it encodes, and permitted us to present a precise map of the gene at the molecular level.
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PMID:Genetic structure and domains of DNA polymerase III of Bacillus subtilis. 184 Jun 38

We have applied the polymerase chain reaction (PCR) technique to analyse mutations in the thymidine kinase (TK) gene of varicella-zoster virus (VZV) associated with resistance to the 5-bromovinyl (BVaraU) and 5-propynyl (PYaraU) analogues of arabinofuranosyl deoxyuridine. The results from this study allow three clear conclusions to be drawn. Firstly, the technique clearly shows that populations of VZV derived from plaque purification were truly clonal only when the plaques were initiated from cell-free virus (representing a tiny fraction of infectious virus) and plaques initiated by infected cells contained a mixture of variants. Secondly, despite the background mutations caused by errors of the Taq DNA polymerase, mutations relevant to drug resistance can easily be distinguished. The BVaraU-resistant mutant, 7-1, contained an aspartic acid to asparagine mutation at residue 18 and a single base deletion (position 65298 of the VZV DNA sequence), resulting in a frameshift and premature termination of the polypeptide chain, was found in the BVaraU-resistant mutant YSR. PYaraU-resistant virus populations contained viruses with one or more of three independent mutations, i.e. single base substitutions resulting in mutations from leucine to proline at residue 92, histidine to arginine at residue 97 and a deletion of 20bp (residues 65,135 to 65,154). Finally, the technique has uncovered novel sites in the virus TK associated with drug resistance. We conclude that in vitro amplification using the PCR combined with cloning and sequencing is a relatively rapid method for identifying mutations in small virus populations even when they are not homogeneous.
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PMID:Analysis of mutations in the thymidine kinase genes of drug-resistant varicella-zoster virus populations using the polymerase chain reaction. 184 97

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 amino acid sequence, arginine-glycine-aspartic acid (RGD), found in some cell adhesive proteins, is a recognition signal for the receptor protein. It is interesting that we have found the RGD sequence in terminal protein (TP) of bacteriophages phi 29 and M2 near an amino acid, the serine residue at 232, covalently linked to the terminal nucleotide of their DNAs. At the initiation of protein-primed DNA replication, TP is essential for the recognition of replication machinery containing DNA polymerase and primer protein (PP; PP becomes TP upon linking the first nucleotide, and hence the primary structure of TP is the same as that of PP). Synthetic peptide RGD specifically inhibited transfection of phi 29 and M2. The target of the RGD peptide is shown to be TP by marker rescue experiments, suggesting that a receptor for the RGD sequence exists in TP. Furthermore, the peptide inhibited the in vitro protein-priming reaction of DNA replication. We propose that the RGD sequence of PP and a putative receptor on TP is utilized for the molecular recognition initiating DNA replication.
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PMID:An inhibitory effect of RGD peptide on protein-priming reaction of bacteriophages phi 29 and M2. 260 28

A 5400-base-pair segment of the vaccinia virus genome was sequenced and an open reading frame of 938 codons was found precisely where the DNA polymerase had been mapped by transfer of a phosphonoacetate-resistance marker. A single nucleotide substitution changing glycine at position 347 to aspartic acid accounts for the drug resistance of the mutant vaccinia virus. The 5' end of the DNA polymerase mRNA was located 80 base pairs before the methionine codon initiating the open reading frame. Correspondence between the predicted Mr 108,577 polypeptide and the 110,000 purified enzyme indicates that little or no proteolytic processing occurs. Extensive homology, extending over 435 amino acids, was found upon comparing the DNA polymerase of vaccinia virus and DNA polymerase of Epstein-Barr virus. A highly conserved sequence of 14 amino acids in the carboxyl-terminal regions of the above DNA polymerases is also present at a similar location in adenovirus DNA polymerase. This structure, which is predicted to form a turn flanked by beta-pleated sheets, may form part of an essential binding or catalytic site that accounts for its presence in DNA polymerases of poxviruses, herpesviruses, and adenoviruses.
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PMID:Homology between DNA polymerases of poxviruses, herpesviruses, and adenoviruses: nucleotide sequence of the vaccinia virus DNA polymerase gene. 301 24

Thioredoxin was purified to homogeneity from the Escherichia coli mutant tsnC 7007 that is defective in phage T7 DNA replication and previously shown to contain a missense thioredoxin. Tryptic peptide maps of reduced and carboxymethylated 7007 thioredoxin combined with amino acid sequence analysis revealed one amino acid substitution; Gly-92 in thioredoxin is exchanged to an aspartic acid residue in the 7007 protein. The missense thioredoxin gave no activity with the gene 5 protein of phage T7 in the complementation to active T7 DNA polymerase. It competitively inhibited the complementation of wild type thioredoxin and gene 5 protein and formed a complex with the gene 5 protein that was retained by antithioredoxin Sepharose. The 7007 thioredoxin has reduced catalytic activity with thioredoxin reductase, ribonucleotide reductase, or as a protein disulfide reductase. The apparent Km value of 7007 thioredoxin as a substrate for thioredoxin reductase was increased 3-fold relative to normal thioredoxin, and the Vmax value was decreased 7-fold. The position of GLy-92 in the known three-dimensional structure of thioredoxin-S2 is correlated with the changed functional properties of the substituted mutant protein.
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PMID:A mutant thioredoxin from Escherichia coli tsnC 7007 that is nonfunctional as subunit of phage T7 DNA polymerase. 700 7

Previously constructed Swiss mouse 3T3 fibroblasts producing polyomavirus large T antigen after addition of dexamethasone were used to study the transcriptional activation by the viral protein of five genes coding for enzymes involved in DNA synthesis and precursor production, namely, dihydrofolate reductase, thymidine kinase, thymidylate synthase, DNA polymerase alpha, and proliferating-cell nuclear antigen. It was found that all these genes, whose expression is stimulated at the G1/S boundary of the cell cycle after growth stimulation by serum addition, are coordinately trans activated when T antigen is induced in cells previously growth arrested by serum withdrawal. Cell lines carrying the information for a mutant form of large T antigen, in which a glutamic acid residue in the binding site for the retinoblastoma protein was changed into aspartic acid, were constructed to test the involvement of an interaction of T antigen with the retinoblastoma protein in this reaction. It was found that the mutated T protein is incapable of stimulating transcription of any one of the genes. The promoter of three of the genes (dihydrofolate reductase, thymidine kinase, and DNA polymerase alpha) unequivocally carries binding sites for transcription factor E2F, suggesting that complexes forming with this growth- and cell cycle-regulating transcription factor are the targets for T antigen. Although there is so far no evidence that thymidylate synthase and proliferating cell nuclear antigen are regulated via E2F, our data indicate that the retinoblastoma protein still is involved in the control of these genes. mRNA for E2F itself increases in amount at the G1/S border in serum-stimulated cells but not during polyomavirus T antigen-induced transcriptional activation of DNA synthesis enzymes in arrested cells.
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PMID:Coordinated trans activation of DNA synthesis- and precursor-producing enzymes by polyomavirus large T antigen through interaction with the retinoblastoma protein. 790 59

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


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