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 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

The human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) is a protein of 66 kDa, p66, which contains two domains, an amino-terminal DNA polymerase and an RNase H at the carboxy terminus of the molecule. In order to characterize the mode of action of the RNase H, two previously described mutant enzymes were used, with substitutions in the highly conserved histidine 539, which was mutated to the neutral amino acid asparagine and to the negatively charged aspartate. The purified wild-type (wt) and mutant (mt) enzyme activities are analyzed here using RNA-DNA hybrids consisting of in vitro transcribed RNA that harbors the polypurine tract (PPT) from HIV-1 and DNA oligonucleotides complementary to the PPT or to other regions of the RNA. Analysis of the radioactively labeled RNA of these model hybrids after RNase H treatment indicates that both, wt and mt enzymes, are capable of cleaving the RNA in an endonucleolytic manner. The mt enzymes exhibit a severely reduced exonuclease activity. They are more sensitive towards salt and competition with excess of unlabeled hybrid, suggesting a reduced substrate binding affinity. DNA elongation by the RT is coupled with RNA hydrolysis by the 3'-5' exonuclease of the wt RNase H. The RNase Hmt of the mt enzymes, however, does not exhibit such processive 3'-5' exonuclease activity during DNA synthesis but gives rise to sporadic endonucleolytic cuts, whereas the RT is not affected. The endonuclease activities of the RNase H mt enzymes exhibit cleavage preferences in the absence or presence of DNA synthesis different from those of the wt enzyme. They cannot recognize specific sequences required to generate a PPT-primer and therefore cannot initiate plus-strand DNA synthesis in vitro at the 3' end of the PPT, which is essential for viral replication.
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PMID:Mutations of a conserved residue within HIV-1 ribonuclease H affect its exo- and endonuclease activities. 171 5

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

A mutation (asparagine 815 to serine 815) was introduced into the herpes simplex virus type 1 (HSV-1) DNA polymerase (pol). The HSV-1 pol enzyme in lysates of Saccharomyces cerevisiae cells expressing the mutant protein showed increased resistance to acyclovir triphosphate and increased sensitivity to phosphonoacetate but was not substantially altered with respect to sensitivity to phosphonoformate or aphidicolin. These results directly demonstrate that both resistance to acyclovir triphosphate and sensitivity to phosphonoacetate can be conferred by this mutation in the absence of other viral factors and that the yeast expression system can be used for structure-function studies on HSV-1 pol.
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PMID:In vitro mutagenesis of the herpes simplex virus type 1 DNA polymerase gene results in altered drug sensitivity of the enzyme. 255 70

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

Protein splicing is a self-catalyzed, posttranslational process which converts a precursor polypeptide into two new proteins by the excision of an internal polypeptide segment and the ligation of the flanking polypeptides. Evidence has been presented that protein splicing involves a branched intermediate, which is resolved into the two protein products by the cyclization of an asparagine residue to aminosuccinimide [Xu, M. Q., Comb, D. G., Paulus, H., Noren, C. J., Shao, Y., & Perler, F. (1994) EMBO J. 13, 5517-5522]. This report describes the chemical synthesis of a peptide with a C-terminal aminosuccinimide residue, corresponding to the putative C-terminus of the excised intervening sequence (intein) derived from the thermostable DNA polymerase of Pyrococcus species GB-D. The synthetic aminosuccinimide peptide was compared with the C-terminal cyanogen bromide peptide of the excised intein and found to be indistinguishable in terms of its chromatographic properties, high-resolution mass spectrum, and colorimetric assay involving reaction with hydroxylamine. This establishes definitively that protein splicing is accompanied by the cyclization of asparagine to yield an aminosuccinimide residue at the C-terminus of the excised intein and that this unusual residue is therefore a natural constituent of spliced proteins. The effects of pH and temperature on the stability of the synthetic aminosuccinimide peptide are described.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Protein splicing: characterization of the aminosuccinimide residue at the carboxyl terminus of the excised intervening sequence. 766 64

All known family B DNA polymerases contain a conserved region of amino acids, KX6-7YG, which appears to be correspond to the 'finger' alpha helix O of the Klenow fragment of E. coli DNA polymerase I, a family A DNA polymerase. Toward the goal of establishing the evolutionary relationship between the family A and B DNA polymerases, we have employed site-directed mutagenesis to access the functional role of the invariant amino acid lysine-340 of the PRD1 DNA polymerase. We have replaced the lysine-340 with three amino acids: histidine, asparagine and glutamic acid, respectively. Mutant DNA polymerases were overexpressed and purified to near homogeneity. Our results showed that the modification of the lysine-340 of the PRD1 DNA polymerase abolishes the polymerase activity without affecting the 3' to 5' exonuclease activity. These results support the proposal that the KX6-7YG motif of the family B DNA polymerases may be analogous to the KX7YG motif of the family A DNA polymerases, suggesting that two family DNA polymerases share a common ancestor.
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PMID:Mutagenesis of a highly conserved lysine 340 of the PRD1 DNA polymerase. 791 20

The gene encoding DNA polymerase alpha from the human malaria parasite Plasmodium falciparum has been sequenced and characterised. The deduced amino acid sequence possesses the seven sequence motifs which characterise eukaryotic replicative DNA polymerases (I-VII) and four of five motifs (A-E) identified in alpha DNA polymerases. The predicted protein also contains sequences which are reminiscent of Plasmodium proteins but absent from other DNA polymerases. These include four blocks of additional amino acids interspersed with the conserved motifs of the DNA polymerases, four asparagine rich sequences and a novel carboxy-terminal extension. Repetitive sequences similar to those found in other malarial proteins are also present. cDNA-directed PCR was used to establish the presence of these features in the approximately 7kb mRNA. The coding sequence contains a single intron. The gene for DNAPol alpha is located on chromosome 4 and is transcribed in both asexual and sexual erythrocytic stages of the parasite.
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PMID:The gene encoding DNA polymerase alpha from Plasmodium falciparum. 836 80

We have isolated two high copy, allele-specific suppressors of the temperature sensitivity of mutations in POL1, the gene that encodes the catalytic subunit of DNA polymerase alpha in the yeast Saccharomyces cerevisiae. Both genes, PSP1 and PSP2, also partially suppressed a mutation in POL3 which encodes DNA polymerase delta, and both also affected a mutation in CDC6, which acts in initiation of DNA replication. Suppression was not general, since ts mutations in several genes unrelated to replication were not affected, PSP1 was partially effective on low-copy-number vectors, while PSP2 required high copy numbers. The presence of suppressing plasmids did not alter the steady-state level of Pol1 protein, so suppression does not appear to be due to an increase in production or stability of Pol1p. Deletion of either PSP gene or both in combination resulted in apparently normal viable cells. While neither gene is homologous to genes with known functions, PSP1 and PSP2 both have unusual amino acid compositions: PSP1 is rich in asparagine and glutamine, while PSP2 is rich in asparagine and contains "RGG" motifs that have been associated with RNA-binding proteins. We also describe a transposon-mediated strategy that should be generally effective for rapid characterization of multicopy suppressors.
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PMID:Suppressors of the temperature sensitivity of DNA polymerase alpha mutations in Saccharomyces cerevisiae. 952 27


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