scholarly journals Mutants of Escherichia coli with increased fidelity of DNA replication.

Genetics ◽  
1993 ◽  
Vol 134 (4) ◽  
pp. 1023-1030 ◽  
Author(s):  
I J Fijalkowska ◽  
R L Dunn ◽  
R M Schaaper
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Acid Resistance ◽  
Bacteriophage P1 ◽  
Epsilon Subunit ◽  
Dna Mismatch ◽  
Replication Errors ◽  
Localized Mutagenesis ◽  
Dna Replication Errors

Abstract To improve our understanding of the role of DNA replication fidelity in mutagenesis, we undertook a search for Escherichia coli antimutator strains with increased fidelity of DNA replication. The region between 4 and 5 min of the E. coli chromosome was mutagenized using localized mutagenesis mediated by bacteriophage P1. This region contains the dnaE and dnaQ genes, which encode, respectively, the DNA polymerase (alpha subunit) and 3' exonucleolytic proofreading activity (epsilon subunit) of DNA polymerase III holoenzyme, the enzyme primarily responsible for replicating the bacterial chromosome. The mutated bacteria were screened for antimutator phenotype in a strain defective in DNA mismatch repair (mutL), using a papillation assay based on the reversion of the galK2 mutation. In a mutL strain, mutations result primarily from DNA replication errors. Among 10,000 colonies, seven mutants were obtained whose level of papillation was reduced 5-30-fold. These mutants also displayed decreased mutation frequencies for rifampicin or nalidixic acid resistance as well as for other markers. Mapping by P1 transduction and complementation showed each to reside in dnaE. These observations support the idea that the mutants represent antimutators which replicate their DNA with increased fidelity. Mutation rates were reduced in both mutL and mutT backgrounds, but mutagenesis by ultraviolet light was not significantly affected, suggesting that the antimutator effect may be largely restricted to normal DNA replication.

scholarly journals Spontaneous mutation in Escherichia coli containing the dnaE911 DNA polymerase antimutator allele.

Genetics ◽  
1994 ◽  
Vol 138 (2) ◽  
pp. 263-270 ◽  
Author(s):  
A R Oller ◽  
R M Schaaper
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Mismatch Repair ◽  
Dna Polymerase ◽  
Spontaneous Mutation ◽  
Gene Encoding ◽  
Replication Errors ◽  
Lac Operator ◽  
Laci Gene ◽  
Dna Replication Errors

Abstract We have previously isolated mutants of Escherichia coli that replicate their DNA with increased fidelity. These mutants have a mutation in the dnaE gene, encoding the alpha subunit of DNA polymerase III. They were isolated in a mismatch-repair-defective mutL background, in which mutations can be considered to represent uncorrected DNA replication errors. In the present study we analyze the effect of one of these alleles, dnaE911, on spontaneous mutagenesis in a mismatch-repair-proficient background. In this background, spontaneous mutations may be the sum of uncorrected replication errors and mutations resulting from other pathways. Hence, the effect of the dnaE allele may provide insights into the contribution of uncorrected DNA replication errors to spontaneous mutation. The data show that dnaE911 decreases the level of Rifr, lacI and galK mutations in this background by 1.5-2-fold. DNA sequencing of 748 forward mutants in the lacI gene reveals that this effect has a clear specificity. Transversions are decreased by approximately 3-fold, whereas transitions, frameshifts, deletions and duplications remain essentially unchanged. Among the transversions, A.T-->T.A are affected most strongly (approximately 6-fold). In addition to this effect on transversions within the lacI gene, one previously recognized A.T-->G.C base-pair substitution hotspot in the lac operator is also reduced (approximately 5-fold). The data are discussed in the light of the role of DNA replication errors in spontaneous mutation, as well as other possible explanations for the observed antimutator effects.

scholarly journals The Bacteriophage P1 hot Gene Product Can Substitute for the Escherichia coli DNA Polymerase III θ Subunit

2005 ◽  
Vol 187 (16) ◽  
pp. 5528-5536 ◽  
Author(s):  
Anna K. Chikova ◽  
Roel M. Schaaper
Keyword(s):  
Escherichia Coli ◽  
Gene Product ◽  
Dna Polymerase ◽  
Temperature Sensitive ◽  
Bacteriophage P1 ◽  
Replication Errors ◽  
Pol Iii ◽  
Dna Replication Errors ◽  
Replication Factors

ABSTRACT The θ subunit (holE gene product) of Escherichia coli DNA polymerase (Pol) III holoenzyme is a tightly bound component of the polymerase core. Within the core (α-ε-θ), the α and ε subunits carry the DNA polymerase and 3′ proofreading functions, respectively, while the precise function of θ 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 θ 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 ε 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 θ subunit and is likely incorporated in the Pol III complex. We also show that overexpression of either θ or Hot further suppresses the dnaQ49 mutator phenotype. This suggests that the complexing of dnaQ49-ε with θ is rate limiting for its ability to proofread DNA replication errors. The possible role of hot for bacteriophage P1 is discussed.

Escherichia coli Mutator ΔpolA is Defective in Base Mismatch Correction: The Nature of in Vivo DNA Replication Errors

2005 ◽  
Vol 351 (2) ◽  
pp. 299-308 ◽  
Author(s):  
Yu-ichiro Tago ◽  
Masaru Imai ◽  
Makoto Ihara ◽  
Hironari Atofuji ◽  
Yuki Nagata ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Replication Errors ◽  
Mismatch Correction ◽  
Base Mismatch ◽  
Dna Replication Errors

DNA Replication Errors Produced by the Replicative Apparatus of Escherichia coli

1999 ◽  
Vol 289 (4) ◽  
pp. 835-850 ◽  
Author(s):  
Shingo Fujii ◽  
Masahiro Akiyama ◽  
Kazuhiro Aoki ◽  
Yutaka Sugaya ◽  
Kumiko Higuchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Replication Errors ◽  
Dna Replication Errors

scholarly journals 3′ → 5′ Exonucleases of DNA Polymerases ϵ and δ Correct Base Analog Induced DNA Replication Errors on Opposite DNA Strands in Saccharomyces cerevisiae

Genetics ◽  
1996 ◽  
Vol 142 (3) ◽  
pp. 717-726 ◽  
Author(s):  
Polina V Shcherbakova ◽  
Youri I Pavlov
Keyword(s):  
Dna Replication ◽  
Dna Polymerases ◽  
Replication Errors ◽  
Dna Strands ◽  
Base Analog ◽  
Chromosome Iii ◽  
Dna Strand ◽  
Dna Replication Errors ◽  
Yeast Mutants ◽  
Lagging Strand

Abstract The base analog 6-N-hydroxylaminopurine (HAP) induces bidirectional GC → AT and AT → GC transitions that are enhanced in DNA polymerase ϵ and δ 3′ → 5′ exonuclease-deficient yeast mutants, pol2-4 and pol3-01, respectively. We have constructed a set of isogenic strains to determine whether the DNA polymerases δ and ϵ contribute equally to proofreading of replication errors provoked by HAP during leading and lagging strand DNA synthesis. Site-specific GC → AT and AT → GC transitions in a Pol→, pol2-4 or pol3-01 genetic background were scored as reversions of ura3 missense alleles. At each site, reversion was increased in only one proofreading-deficient mutant, either pol2-4 or pol3-01, depending on the DNA strand in which HAP incorporation presumably occurred. Measurement of the HAP-induced reversion frequency of the ura3 alleles placed into chromosome III near to the defined active replication origin ARS306 in two orientations indicated that DNA polymerases ϵ and δ correct HAP-induced DNA replication errors on opposite DNA strands.

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