The high mutator activity of the dnaQ49 allele of Escherichia coli is medium-dependent and results from both defective 3′→5′ proofreading and methyl-directed mismatch repair

Krishnaswamy, Shalini; Rogers, Jacquelyne A.; Isbell, Roberta J.; Fowler, Robert G.

doi:10.1016/0027-5107(93)90099-2

Cited by 10 publications

(5 citation statements)

References 27 publications

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“…Mutagenesis studies were also performed with another allele defective in proofreading, dnaQ49 (12,21), which has a temperature-dependent mutator activity. Transductions of the dnaE911 through dnaE917 alleles into the dnaQ49 strain were performed at 29ЊC, and then mutation frequencies were measured at 35ЊC, at which temperature the mutagenesis of dnaQ49 is maximal (12,16). All seven alleles produced a significantly reduced level of mutagenesis (data not shown).…”

Section: Resultsmentioning

confidence: 96%

Effects of Escherichia coli dnaE antimutator alleles in a proofreading-deficient mutD5 strain

Fijałkowska

Schaaper

1995

J Bacteriol

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We have previously isolated seven mutants of Escherichia coli which replicate their DNA with increased fidelity. These mutants were isolated as suppressors of the elevated mutability of a mismatch-repair-defective mutL strain. Each mutant was shown to contain a single amino acid substitution in the dnaE gene product, the ␣ (i.e., polymerase) subunit of DNA polymerase III holoenzyme responsible for replicating the E. coli chromosome. The mechanism(s) by which these antimutators exert their effect is of interest. Here, we have examined the effects of the antimutator alleles in a mutD5 mutator strain. This strain carries a mutation in the dnaQ gene, which results in defective exonucleolytic proofreading. Our results show that dnaE mutations also confer a strong antimutator phenotype in this background, the effects being generally much greater than those observed previously in the mutL background. The results suggest that the dnaE antimutator alleles can exert their effect independently of exonucleolytic proofreading activity. The large magnitude of the antimutator effects in the mutD5 background can be ascribed, at least in part, to the (additional) restoration of DNA mismatch repair, which is generally impaired in mutD5 strains because of error saturation. The high mutability of mutD5 strains was exploited to isolate a strong new dnaE antimutator allele on the basis of its ability to suppress the high reversion rate of an A ⅐ T3T ⅐ A transversion in this background. A model suggesting how the dnaE antimutator alleles might exert their effects in proofreading-proficient and -deficient backgrounds is presented.Mutations in DNA may arise during DNA replication as a result of mistakes made by the DNA polymerase in copying a DNA template strand. It is important to understand the factors which contribute to the accuracy of this process. In general, three discrete steps to ensure fidelity have been identified (7,11). In the first, the base selection step, the polymerase strongly favors the insertion of correct nucleotides over incorrect ones. In the second step, fidelity is improved by a polymerase-associated 3Ј35Ј exonuclease, whose joint action with the DNA polymerase allows for the preferential removal of incorrectly inserted nucleotides (proofreading). Third, following replication, mismatches resulting from replication errors are subject to correction by postreplicative mismatch repair (for review, see reference 25), further enhancing overall replication fidelity.In the bacterium Escherichia coli, DNA polymerase III holoenzyme is responsible for most of the chromosomal replication and is the major determinant of fidelity (15,22,40). The polymerase III holoenzyme is a large asymmetric dimer containing some 10 distinct subunits (18 total). From these, the ␣, ε, and subunits bind together tightly to form the polymerase III core. The ␣ subunit encoded by the dnaE gene contains the 5Ј 3 3Ј polymerase activity. The ε subunit encoded by the dnaQ gene contains the 3Ј35Ј exonuclease (proofreading) activity. The role of is as ...

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Section: Resultsmentioning

confidence: 96%

Effects of Escherichia coli dnaE antimutator alleles in a proofreading-deficient mutD5 strain

Fijałkowska

Schaaper

1995

J Bacteriol

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“…Addition of α to ε stimulated the ε exonuclease activity by 8.5-fold ( Figure 5, compare lanes 2 and 3), whereas addition of θ to ε had little measurable effect on ε exonuclease Exonuclease reactions containing partial duplex DNA were prepared as described in the Experimental section. Wild-type ε (lanes 2-5) or mutant ε511 (lanes 6-9), ε513 (lanes [10][11][12][13] or ε504 (lanes [14][15][16][17] protein was mixed with the α-subunit (lanes 3, 7, 11 and 15), the θ-subunit (lanes 4, 8, 12 and 16), or the α-and θ-subunits (lanes 5, 9, 13 and 17) at 10× the final concentration (0.5 nM) and incubated for 10 min at 24 • C. Samples (1 µl) of the mixtures were added in reactions. The reaction in lane 1 (NE) contained no enzyme.…”

Section: Stimulation Of ε Exonuclease Activity By the θ -Subunit In Tmentioning

confidence: 99%

“…Addition of the α-subunit Reactions containing partial duplex DNA were prepared as described in the Experimental section. Wild-type ε (lanes 3-6) or mutant ε511 (lanes 7-10), ε513 (lanes [11][12][13][14] or ε504 (lanes [15][16][17][18] protein was mixed with the α-subunit (lanes 4, 8, 12 and 16), the θ-subunit (lanes 5, 9, 13 and 17) or the α-and θ-subunits (lanes 6, 10, 14 and 18) at 10× the final concentration (1 nM) and incubated for 10 min at 24 • C. Samples (2 µl) of the mixtures were added to reactions and incubated at 37 • C for 5 min prior to initiation by addition of MgCl 2 . The reaction in lane 1 (NE) contained no enzyme.…”

Section: Polymerase/exonuclease Ratios In the Constituted Dna Pol IIImentioning

confidence: 99%

“…A functional dnaQ allele encoding the ε subunit is required to achieve high-fidelity replication in E. coli. Mutations in dnaQ increase the spontaneous mutation rates in cells, resulting in a mutator phenotype [13][14][15][16][17][18]. Targeted mutagenesis of the dnaQ gene and subsequent detection of mutators has been used to identify dnaQ alleles encoding defective ε proteins [19][20][21][22][23].…”

Section: Introductionmentioning

confidence: 99%

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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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“…The dnaQ49 mutant (containing the V96G mutation) carries a defective polymerase III ε subunit whose stability is greatly dependent on the subunit (50). The dnaQ49 mutant is only a modest mutator at low temperatures (25°C), but it becomes a very strong mutator (up to 1,000-fold enhanced) at 37°C due to collapse of its proofreading ability (13,17,36,40). In contrast, the dnaQ49 ⌬holE strain is an exceptionally strong mutator even at the lowest temperature (50).…”

Section: Vol 187 2005mentioning

confidence: 99%

The Bacteriophage P1hotGene Product Can Substitute for theEscherichia coliDNA Polymerase III θ Subunit

Chikova

Schaaper

2005

J Bacteriol

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The subunit (holE gene product) of Escherichia coli DNA polymerase (Pol) III holoenzyme is a tightly bound component of the polymerase core. Within the core (␣--), the ␣ and subunits carry the DNA polymerase and 3 proofreading functions, respectively, while the precise function of is unclear. holE homologs are present in genomes of other enterobacteriae, suggestive of a conserved function. Putative homologs have also been found in the genomes of bacteriophage P1 and of certain conjugative plasmids. The presence of these homologs is of interest, because these genomes are fully dependent on the host replication machinery and contribute few, if any, replication factors themselves. To study the role of these homologs, we have constructed an E. coli strain in which holE is replaced by the P1 homolog, hot. We show that hot is capable of substituting for holE when it is assayed for its antimutagenic action on the proofreading-impaired dnaQ49 mutator, which carries a temperature-sensitive subunit. The ability of hot to substitute for holE was also observed with other, although not all, dnaQ mutator alleles tested. The data suggest that the P1 hot gene product can substitute for the subunit and is likely incorporated in the Pol III complex. We also show that overexpression of either or Hot further suppresses the dnaQ49 mutator phenotype. This suggests that the complexing of dnaQ49-with is rate limiting for its ability to proofread DNA replication errors. The possible role of hot for bacteriophage P1 is discussed.Escherichia coli DNA polymerase III holoenzyme (HE) is the enzyme responsible for the faithful duplication of the bacterial chromosome. HE is a multisubunit, dimeric complex that replicates simultaneously the leading and lagging strands at the replication fork (for a review, see reference 31). Each half of the dimeric complex contains a polymerase (Pol) III core unit consisting of three subunits, ␣, ε, and , which are tightly bound in the linear order ␣-ε-. Of these, ␣ (135 kDa) is the DNA polymerase (dnaE gene product), ε (28 kDa) is the exonucleotidic proofreader responsible for editing polymerase insertion errors (dnaQ gene product), and (8 kDa) is a small subunit whose function is not well defined (holE gene product). The HE (17 subunits total, 10 distinct) further consists of the two ␤-clamps (one for each core) that serve to tether the polymerase to the DNA and the DnaX complex ( 2 ␥␦␦Ј), which is responsible for connecting the two polymerases and for loading and unloading the ␤-clamp in the discontinuously synthesized lagging strand. The precise mechanisms by which HE is capable of replicating the bacterial chromosome with high speed and high accuracy are the subject of active investigation (21, 31, 33, 52).Our laboratory has been interested in the roles and mechanisms of the ε and subunits within the Pol III core, particularly with respect to their contributions to the fidelity of the replication process (37,(48)(49)(50). ε is a critical subunit with at least dual functions. It is a fidelity subunit whose...

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The high mutator activity of the dnaQ49 allele of Escherichia coli is medium-dependent and results from both defective 3′→5′ proofreading and methyl-directed mismatch repair

Cited by 10 publications

References 27 publications

Effects of Escherichia coli dnaE antimutator alleles in a proofreading-deficient mutD5 strain

Effects of Escherichia coli dnaE antimutator alleles in a proofreading-deficient mutD5 strain

Dysfunctional proofreading in the Escherichia coli DNA polymerase III core

The Bacteriophage P1hotGene Product Can Substitute for theEscherichia coliDNA Polymerase III θ Subunit

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