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)

In this report, we describe the isolation, molecular genetic mapping, and phenotypic characterization of vaccinia virus mutants resistant to cytosine arabinoside (araC) and phosphonoacetic acid (PAA). At 37 degrees C, 8 microM araC was found to prevent macroscopic plaque formation by wild-type virus and to cause a 10(4)-fold reduction in viral yield. Mutants resistant to 8 microM araC were selected by serial passage of a chemically mutagenized viral stock in the presence of drug. Because recovery of mutants required that initial passages be performed under less stringent selective conditions, and because plaque-purified isolates were found to be cross-resistant to 200 micrograms of PAA per ml, it seemed likely that resistance to araC required more than one genetic lesion. This hypothesis was confirmed by genetic and physical mapping of the responsible mutations. PAAr was accorded by the acquisition of one of three G-A transitions in the DNA polymerase gene which individually alter cysteine 356 to tyrosine, glycine 372 to aspartic acid, or glycine 380 to serine. AraCr was found to require one of these substitutions plus an additional T-C transition within codon 171 of the DNA polymerase gene, a change which replaces the wild-type phenylalanine with serine. Congenic viral stocks carrying one of the three PAAr lesions, either alone or in conjunction with the upstream araCr lesion, in an otherwise wild-type background were generated. The PAAr mutations conferred nearly complete resistance to PAA, a slight degree of resistance to araC, hypersensitivity to aphidicolin, and decreased spontaneous mutation frequency. Addition of the mutation at codon 171 significantly augmented araC resistance and aphidicolin hypersensitivity but caused no further change in mutation frequency. Several lines of evidence suggest that the PAAr mutations primarily affect the deoxynucleoside triphosphate-binding site, whereas the codon 171 mutation, lying within a conserved motif associated with 3'-5' exonuclease function, is postulated to affect the proofreading exonuclease of the DNA polymerase.
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PMID:Genetic characterization of the vaccinia virus DNA polymerase: cytosine arabinoside resistance requires a variable lesion conferring phosphonoacetate resistance in conjunction with an invariant mutation localized to the 3'-5' exonuclease domain. 838 30

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 determined the DNA sequence of the mitochondrial plasmid from Neurospora intermedia strain Fiji N6-6. The plasmid contains a 1278-codon open reading frame that is 49% identical to the open reading frame of the mitochondrial plasmid from the LaBelle strain of N. intermedia, which is known to encode a DNA-dependent DNA polymerase. The results of polymerase assays and photolabeling studies, the high degree of identity with the LaBelle plasmid polymerase, and the observation that the Fiji polymerase activity in a reaction utilizing endogenous template is not affected by removal of RNA suggest that the Fiji plasmid also encodes a DNA-dependent DNA polymerase. Comparison of regions of amino acids that are highly conserved in the two plasmid polymerases to family B polymerases reveals good correlates for the three major polymerase motifs and suggests that previously identified motifs characteristic of reverse transcriptase found in the LaBelle sequence are not significant. The polymerases encoded by the Fiji and LaBelle plasmids are unusual in that the amino acid sequence Asp-Thr-Asp, which forms the core of the third motif in family B polymerases, is not present in either Fiji or LaBelle. A version of the motif containing Thr-Thr-Asp exists in both sequences.
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PMID:Two Neurospora mitochondrial plasmids encode DNA polymerases containing motifs characteristic of family B DNA polymerases but lack the sequence Asp-Thr-Asp. 848 47

The Escherichia coli dnaQ gene encodes the proofreading 3' exonuclease (epsilon subunit) of DNA polymerase III holoenzyme and is a critical determinant of chromosomal replication fidelity. We constructed by site-specific mutagenesis a mutant, dnaQ926, by changing two conserved amino acid residues (Asp-12-->Ala and Glu-14-->Ala) in the Exo I motif, which, by analogy to other proofreading exonucleases, is essential for the catalytic activity. When residing on a plasmid, dnaQ926 confers a strong, dominant mutator phenotype, suggesting that the protein, although deficient in exonuclease activity, still binds to the polymerase subunit (alpha subunit or dnaE gene product). When dnaQ926 was transferred to the chromosome, replacing the wild-type gene, the cells became inviable. However, viable dnaQ926 strains could be obtained if they contained one of the dnaE alleles previously characterized in our laboratory as antimutator alleles or if it carried a multicopy plasmid containing the E. coli mutL+ gene. These results suggest that loss of proofreading exonuclease activity in dnaQ926 is lethal due to excessive error rates (error catastrophe). Error catastrophe results from both the loss of proofreading and the subsequent saturation of DNA mismatch repair. The probability of lethality by excessive mutation is supported by calculations estimating the number of inactivating mutations in essential genes per chromosome replication.
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PMID:Mutants in the Exo I motif of Escherichia coli dnaQ: defective proofreading and inviability due to error catastrophe. 861 Jan 31

We describe catalytically active mutants of HIV RT (human immunodeficiency virus reverse transcriptase) generated by random sequence mutagenesis and selected in Escherichia coli for ability to complement the temperature-sensitive phenotype of a DNA polymerase I (Pol Its) mutant. We targeted amino acids Asp-67 through Arg-78 in HIV RT, which form part of the beta3-beta4 flexible loop and harbor many of the currently known mutations that confer resistance to nucleoside analogs. DNA sequencing of 109 selected mutants that complement the Pol Its phenotype revealed substitutions at all 12 residues targeted, indicating that none of the wild-type amino acids is essential. However, single mutations were not observed at Trp-71, Arg-72, and Arg-78, consistent with evolutionary conservation of these residues among viral RTs and lack of variation at these positions among isolates from patients. The mutations we recovered included most of those associated with drug resistance as well as previously unidentified mutations. Purification and assay of 14 mutant proteins revealed correlation between their DNA-dependent DNA polymerize activity in vitro and ability to complement the Pol Its phenotype. Activity of several mutants was resistant to 3'-azidothymidine triphosphate. We conclude that random sequence mutagenesis coupled with positive genetic selection in E. coli yields large numbers of functional HIV RT mutants. Among these are less active variants which are unlikely to be isolated from HIV-infected individuals and which will be informative of the roles of individual amino acids in the catalytic functions of the enzyme.
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PMID:Human immunodeficiency virus reverse transcriptase. Functional mutants obtained by random mutagenesis coupled with genetic selection in Escherichia coli. 861 58

We have previously described the mutator alleles mutA and mutC, which map at 95 minutes and 42 minutes, respectively, on the Escherichia coli genetic map and which stimulate transversions; the A.T-->T.A and G.C-->T.A substitutions are the most prominent. In this study we show that both mutA and mutC result from changes in the anticodon in one of four copies of the same glycine tRNA, at either the glyV or the glyW locus. This change results in a tRNA that inserts glycine at aspartic acid codons. In view of previous studies of missense suppressor tRNAs, the mistranslation of aspartic acid codons is assumed to occur at approximately 1-2%. We postulate that the mutator tRNA effect is exerted by generating a mutator polymerase and suggest that the epsilon subunit of DNA polymerase, which provides a proofreading function, is the most likely target. The implications of these findings for the contribution of mistranslation to observed spontaneous mutation rates in wild-type strains, as well as other cellular phenomena such as aging, are discussed.
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PMID:Mutator tRNAs are encoded by the Escherichia coli mutator genes mutA and mutC: a novel pathway for mutagenesis. 863 75

Upon infection of Escherichia coli, bacteriophage T7 annexes a host protein, thioredoxin, to serve as a processivity factor for its DNA polymerase, T7 gene 5 protein. In a previous communication (Himawan, J., and Richardson, C. C. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9774-9778), we reported that an E. coli strain encoding a Gly-74 to Asp-74 (G74D) thioredoxin mutation could not support wild-type T7 growth and that in vivo, six mutations in T7 gene 5 could individually suppress this G74D thioredoxin defect. In the present study, we report the purification and biochemical characterization of the G74D thioredoxin mutant and two suppressor gene 5 proteins, a Glu-319 to Lys-319 (E319K) mutant of gene 5 protein and an Ala-45 to Thr-45 (A45T) mutant. The suppressor E319K mutation, positioned within the DNA polymerization domain of gene 5 protein, appears to suppress the parental thioredoxin mutation by compensating for the binding defect that was caused by the G74D alteration. We suggest that the Glu-319 residue of T7 gene 5 protein and the Gly-74 residue of E. coli thioredoxin define a contact point or site of interaction between the two proteins. In contrast, the A45T mutation in gene 5 protein, located within the 3' to 5' exonuclease domain, does not suppress the G74D thioredoxin mutation by simple restoration of binding affinity. Based upon our understanding of the mechanisms of suppression, we propose a model for the T7 gene 5 protein-E. coli thioredoxin interaction.
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PMID:Amino acid residues critical for the interaction between bacteriophage T7 DNA polymerase and Escherichia coli thioredoxin. 870 17

The high error rates characteristic of human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT) are a presumptive source of the viral hypermutability that impedes prevention and therapy of acquired immunodeficiency syndrome (AIDS). We have analyzed two mutants of HIV-1 RT by conducting a comparative study of the accuracy of DNA synthesis. Each mutant bears a single amino acid substitution adjacent to the two aspartic acid residues at positions 185 and 186 in the highly conserved DNA polymerase active site. The first mutant, Met 184-->Leu (M184L), displays a marked reduction in both misinsertion and mispair extension, suggesting a fidelity of DNA synthesis significantly higher than that of the wild-type HIV-1 RT. The second mutant, Tyr 183-->Phe (Y183F), shows a decrease in mispair extension with no significant change in misincorporation. Thus, the overall pattern of error-proneness of DNA synthesis is: wild-type HIV-1 RT > Y183F > M184L. Taken together, it is possible that residues 183 and 184 contribute to the low fidelity of DNA synthesis characteristic of the reverse transcriptases of HIV-1, HIV-2 and possibly, of other lentiviruses. Our observations may bear on the nature of potential mutations responsible for resistance to the nucleoside analogs used in chemotherapy of AIDS.
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PMID:Mutational studies of human immunodeficiency virus type 1 reverse transcriptase: the involvement of residues 183 and 184 in the fidelity of DNA synthesis. 876 85

We studied the 5' --> 3' exonuclease activity of Bacillus caldotenax DNA polymerase by site-directed mutagenesis. Among seven mutants constructed, two mutant DNA polymerases with an amino acid substitution of Gly184 --> Asp or Gly192 --> Asp were confirmed to be deficient in this exonuclease. The two positions corresponded to those of the Escherichia coli DNA polymerase I mutants defective in 5' --> 3' exonuclease, polA480ex and polA214. These results provide experimental support for the proposed amino acid sequence essential for the 5' --> 3' exonuclease activity associated with eubacterial polymerase I-like DNA polymerases (family A), including E.coli and Thermus aquaticus.
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PMID:The amino acid sequence required for 5' --> 3' exonuclease activity of Bacillus caldotenax DNA polymerase. 881 83

When crystals of human DNA polymerase beta (pol beta) complexed with DNA [Pelletier, H., Sawaya, M. R., Wolfle, W., Wilson, S. H., & Kraut, J. (1996) Biochemistry 35, 12742-12761] are soaked in the presence of dATP and Mn2+, X-ray structural analysis shows that nucleotidyl transfer to the primer 3'-OH takes place directly in the crystals, even though the DNA is blunt-ended at the active site. Under similar crystal-soaking conditions, there is no evidence for a reaction when Mn2+ is replaced by Mg2+, which is thought to be the divalent metal ion utilized by most polymerases in vivo. These results suggest that one way Mn2+ may manifest its mutagenic effect on polymerases is by promoting greater reactivity than Mg2+ at the catalytic site, thereby allowing the nucleotidyl transfer reaction to take place with little or no regard to instructions from a template. Non-template-directed nucleotidyl transfer is also observed when pol beta-DNA cocrystals are soaked in the presence of dATP and Zn2+, but the reaction products differ in that the sugar moiety of the incorporated nucleotide appears distorted or otherwise cleaved, in agreement with reports that Zn2+ may act as a polymerase inhibitor rather than as a mutagen [Sirover, M. A., & Loeb, L. A. (1976) Science 194, 1434-1436]. Although no reaction is observed when crystals are soaked in the presence of dATP and other metal ions such as Ca2+, Co2+, Cr3+, or Ni2+, X-ray structural analyses show that these metal ions coordinate the triphosphate moiety of the nucleotide in a manner that differs from that observed with Mg2+. In addition, all metal ions tested, with the exception of Mg2+, promote a change in the side-chain position of aspartic acid 192, which is one of three highly conserved active-site carboxylate residues. Soaking experiments with nucleotides other than dATP (namely, dCTP, dGTP, dTTP, ATP, ddATP, ddCTP, AZT-TP, and dATP alpha S) reveal a non-base-specific binding site on pol beta for the triphosphate and sugar moieties of a nucleotide, suggesting a possible mechanism for nucleotide selectivity whereby triphosphate-sugar binding precedes a check for correct base pairing with the template.
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PMID:A structural basis for metal ion mutagenicity and nucleotide selectivity in human DNA polymerase beta. 884 Nov 19


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