Temperature-dependent mutational specificity of an Escherichia coli mutator, dnaQ49, defective in 3'---&gt; 5' exonuclease (proofreading) activity

Isbell, Roberta J.

doi:10.31979/etd.qfwc-5gnu

Cited by 4 publications

(12 citation statements)

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“…However, as they are unpaired much of the time, their MIs are much higher (2.3 and 2.5) than that of the wild‐type G (0.3). The relative MI values calculated for the three mutable bases in these mutants compare favourably with their relative reversion frequencies (Isbell and Fowler, 1989; Bhamre et al ., 2001; see Fig. 3, table inset).…”

Section: Resultsmentioning

confidence: 92%

“…In the trpA gene (Yanofsky et al ., 1981; Isbell and Fowler, 1989; Bhamre et al ., 2001), the locations of seven mutable bases in four codons are known, and the sequences containing these bases are shown (Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

“…If mutable bases are located in unpaired bases of SLSs predicted by the Zuker DNA folding program, MI values may predict relative mutation frequencies. Sequences and reversion frequencies have been determined for bases in the same codons of seven trpA mutant alleles (Isbell and Fowler, 1989; Bhamre et al ., 2001). In Fig.…”

Section: Resultsmentioning

confidence: 99%

“… b . Taken from Table 4 in Isbell and Fowler (1989 ). Only class I full revertant frequencies were used.…”

Section: Resultsmentioning

confidence: 99%

“… Linear regression analyses for the data in Tables 1 and 3 . Mutation frequencies or number of mutations are plotted against MIs for seven mutable alleles in the derepressed trpA gene ( Isbell and Fowler, 1989 ; Bhamre et al ., 2001 ), and for 35 background mutable alleles in lacI ( Schaaper and Dunn, 1991 ). …”

Section: Resultsmentioning

confidence: 99%

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Predicting mutation frequencies in stem–loop structures of derepressed genes: implications for evolution

Wright¹,

Reschke

Schmidt

et al. 2003

Molecular Microbiology

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SummaryThis work provides evidence that, during transcription, the mutability (propensity to mutate) of a base in a DNA secondary structure depends both on the stability of the structure and on the extent to which the base is unpaired. Zuker ' s DNA folding computer program reveals the most probable stem-loop structures (SLSs) and negative energies of folding (-∆ ∆ ∆ ∆ G) for any given nucleotide sequence. We developed an interfac-

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Section: Resultsmentioning

confidence: 92%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

“… b . Taken from Table 4 in Isbell and Fowler (1989 ). Only class I full revertant frequencies were used.…”

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Predicting mutation frequencies in stem–loop structures of derepressed genes: implications for evolution

Wright¹,

Reschke

Schmidt

et al. 2003

Molecular Microbiology

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Dysfunctional proofreading in the Escherichia coli DNA polymerase III core

Lehtinen

Perrino

2004

Biochemical Journal

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The epsilon-subunit contains the catalytic site for the 3'-->5' proofreading exonuclease that functions in the DNA pol III (DNA polymerase III) core to edit nucleotides misinserted by the alpha-subunit DNA pol. A novel mutagenesis strategy was used to identify 23 dnaQ alleles that exhibit a mutator phenotype in vivo. Fourteen of the epsilon mutants were purified, and these proteins exhibited 3'-->5' exonuclease activities that ranged from 32% to 155% of the activity exhibited by the wild-type epsilon protein, in contrast with the 2% activity exhibited by purified MutD5 protein. DNA pol III core enzymes constituted with 11 of the 14 epsilon mutants exhibited an increased error rate during in vitro DNA synthesis using a forward mutation assay. Interactions of the purified epsilon mutants with the alpha- and theta;-subunits were examined by gel filtration chromatography and exonuclease stimulation assays, and by measuring polymerase/exonuclease ratios to identify the catalytically active epsilon511 (I170T/V215A) mutant with dysfunctional proofreading in the DNA pol III core. The epsilon511 mutant associated tightly with the alpha-subunit, but the exonuclease activity of epsilon511 was not stimulated in the alpha-epsilon511 complex. Addition of the theta;-subunit to generate the alpha-epsilon511-theta; DNA pol III core partially restored stimulation of the epsilon511 exonuclease, indicating a role for the theta;-subunit in co-ordinating the alpha-epsilon polymerase-exonuclease interaction. The alpha-epsilon511-theta; DNA pol III core exhibited a 3.5-fold higher polymerase/exonuclease ratio relative to the wild-type DNA pol III core, further indicating dysfunctional proofreading in the alpha-epsilon511-theta; complex. Thus the epsilon511 mutant has wild-type 3'-->5' exonuclease activity and associates physically with the alpha- and theta;-subunits to generate a proofreading-defective DNA pol III enzyme.

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Recovery of active beta-lactamases from Proteus vulgaris and RTEM-1 hybrid by random mutagenesis by using a dnaQ strain of Escherichia coli

Hosseini-Mazinani

Nakajima

Ihara

et al. 1996

Antimicrob Agents Chemother

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Proteus vulgaris and RTEM-1 beta-lactamases that belong to molecular class A with 37% amino acid similarity were examined to find the relationship between amino acid residues and activity of enzymes. MICs of ampicillin were > 2,000 micrograms/ml for Escherichia coli cells producing these enzymes. We have made 18 hybrid genes by substituting the coding region of the P. vulgaris beta-lactamase gene with the equivalent portions from the RTEM-1 gene. Most of these hybrids produced inactive proteins, but a few hybrid enzymes had partial or trace activity. From one of the hybrid genes (MIC of ampicillin, 100 micrograms/ml), we recovered three kinds of active mutants which provided ampicillin MICs of 1,000 micrograms/ml by the selection of spontaneous mutations in a dnaQ strain of E. coli. In these mutants, Leu-148, Met-182, and Tyr-274 were replaced with Val, Thr, and His, respectively. These amino acids have not been identified as residues with functional roles in substrate hydrolysis. Furthermore, from these hybrid mutants, we obtained a second set of mutants which conferred ampicillin MICs of 1,500 micrograms/ml. Interestingly, the second mutations were limited to these three amino acid substitutions. These amino acid residues which do not directly interact with substrates have an effect on enzyme activity. These mutant enzymes exhibited lower K(m) values for cephaloridine than both parental enzymes.

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Temperature-dependent mutational specificity of an Escherichia coli mutator, dnaQ49, defective in 3'---> 5' exonuclease (proofreading) activity

Cited by 4 publications

References 0 publications

Predicting mutation frequencies in stem–loop structures of derepressed genes: implications for evolution

Predicting mutation frequencies in stem–loop structures of derepressed genes: implications for evolution

Dysfunctional proofreading in the Escherichia coli DNA polymerase III core

Recovery of active beta-lactamases from Proteus vulgaris and RTEM-1 hybrid by random mutagenesis by using a dnaQ strain of Escherichia coli

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