The mutational specificity of two Escherichia coli dnaE antimutator alleles as determined from lacI mutation spectra.

Schaaper, Roel M.

doi:10.1093/genetics/134.4.1031

Cited by 39 publications

(4 citation statements)

References 43 publications

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“…It is an interesting question whether other types of mutations, such as G. C + A e T transitions or the various transversions or frameshifts are also subject to the antimutator effect. Results described in detail in a companion paper (SCHAAPER 1993) suggest that for at least two of the antimutators, in the mutL background, both A . T + G.C and G-C -+ A .…”

Section: Discussionmentioning

confidence: 93%

Mutants of Escherichia coli with increased fidelity of DNA replication.

Fijałkowska¹,

Dunn²,

Schaaper³

1993

Genetics

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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.

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Section: Discussionmentioning

confidence: 93%

Mutants of Escherichia coli with increased fidelity of DNA replication.

Fijałkowska¹,

Dunn²,

Schaaper³

1993

Genetics

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“…An important approach that significantly improved our understanding of the molecular mechanisms ensuring accurate replication of the bacterial chromosome is based on genetic selection and screening strains for altered fidelity (reviewed in [Fijalkowska et al., 2012 ]). Using this approach, several attempts were made to isolate mutations in the dnaE gene (Fijalkowska et al., 1993 ; Fijalkowska & Schaaper, 1993 ; Hiratsuka & Reha‐Krantz, 2000 ; Maki et al., 1991 ; Makiela‐Dzbenska et al., 2019 ; Oller et al., 1993 ; Oller & Schaaper, 1994 ; Sevastopoulos & Glaser, 1977 ; Sugaya et al., 2002 ; Vandewiele et al., 2002 ; Yanagihara et al., 2007 ), as well as genes encoding other pol III HE subunits (Fijalkowska & Schaaper, 1996 ; Gawel et al., 2008 , 2011 ; Oller et al., 1993 ; Pham et al., 2006 ; Schaaper, 1993 , 1996 , 1998 ; Taft‐Benz & Schaaper, 1998 , 2004 ) that conferred either antimutator or mutator phenotypes.…”

Section: Introductionmentioning

confidence: 99%

“…The isolation of a series of dnaE mutants that result in an altered replication fidelity uncovered an important role of wild‐type pol III in contributing to the normally low replication error rates in E . coli (Fijalkowska et al., 1993 ; Fijalkowska & Schaaper, 1993 ; Oller & Schaaper, 1994 ; Schaaper, 1993 , 1996 , 1998 ). Mapping of the various mutations, which are spread throughout the entire dnaE gene, suggested that although some of them are potentially located close to the active site of the enzyme (Kim et al., 1997 ; Lamers et al., 2006 ; Parasuram et al., 2018 ; Pritchard & McHenry, 1999 ), the effect on replication fidelity was more often indirect.…”

Section: Introductionmentioning

confidence: 99%

Novel Escherichia coli active site dnaE alleles with altered base and sugar selectivity

Vaisman

Łazowski

Reijns

et al. 2021

Molecular Microbiology

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The Escherichia coli dnaE gene encodes the α‐catalytic subunit (pol IIIα) of DNA polymerase III, the cell’s main replicase. Like all high‐fidelity DNA polymerases, pol III possesses stringent base and sugar discrimination. The latter is mediated by a so‐called “steric gate” residue in the active site of the polymerase that physically clashes with the 2′‐OH of an incoming ribonucleotide. Our structural modeling data suggest that H760 is the steric gate residue in E.coli pol IIIα. To understand how H760 and the adjacent S759 residue help maintain genome stability, we generated DNA fragments in which the codons for H760 or S759 were systematically changed to the other nineteen naturally occurring amino acids and attempted to clone them into a plasmid expressing pol III core (α‐θ‐ε subunits). Of the possible 38 mutants, only nine were successfully sub‐cloned: three with substitutions at H760 and 6 with substitutions at S759. Three of the plasmid‐encoded alleles, S759C, S759N, and S759T, exhibited mild to moderate mutator activity and were moved onto the chromosome for further characterization. These studies revealed altered phenotypes regarding deoxyribonucleotide base selectivity and ribonucleotide discrimination. We believe that these are the first dnaE mutants with such phenotypes to be reported in the literature.

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“…Experimentally, this appears not to be the case. Both in vitro and in vivo, misalignment mutagenesis specificity and frequency is strongly influenced by the DNA polymerase (RIPLEY and SHOEMAKER 1983;RIPLEY et al 1983;DE BOER and RIPLEY 1984;KUNKEL 1985a,b;FIX et al 1987;RIPLEY 1989, 1991;BEBEBNEK et al 1990;MASKER and CRISSEY 1993;SCHAAPER 1993).…”

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confidence: 99%

DNA sequence effects on single base deletions arising during DNA polymerization in vitro by Escherichia coli Klenow fragment polymerase.

Wang

Ripley

1994

Genetics

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Most single base deletions detected after DNA polymerization in vitro directed by either Escherichia coli DNA polymerase I or its Klenow fragment are opposite Pu in the template. The most frequent mutations were previously found to be associated with the consensus template context 5'-PyTPu-3'. In this study, the predictive power of the consensus sequence on single base deletion frequencies was directly tested by parallel comparison of mutations arising in four related DNAs differing by a single base. G, a deletion hotspot within the template context 5'-TTGA-3', was substituted by each of the 3 other bases. Previous studies had shown that deletions opposite the G were frequent but that deletions opposite its neighboring A were never detected. Based on the predictions of the consensus, the substitution of T for G should produce frequent deletions opposite the neighboring A due to its new 5'-TTTA-3' template context. This prediction was fulfilled; no deletions of this A were detected in the other templates. The consensus further predicted that deletions opposite template C would be lower than those opposite either A or G at the same site and this prediction was also fulfilled. The C substitution also produced a new hotspot for 1 bp deletions 14 bp away. The new hotspot depends on quasi-palindromic misalignment of the newly synthesized DNA strand during polymerization; accurate, but ectopically templated synthesis is responsible for this mutagenesis. Mutations templated by quasi-palindromic misalignments have previously been recognized when they produced complex sequence changes; here we show that this mechanism can produce frequent single base deletions. The unique stimulation of misalignment mutagenesis by the C substitution in the template is consistent with the singular ability of C at that site to contribute to extended complementary pairing during the DNA misalignment that precedes mutagenesis.

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The mutational specificity of two Escherichia coli dnaE antimutator alleles as determined from lacI mutation spectra.

Cited by 39 publications

References 43 publications

Mutants of Escherichia coli with increased fidelity of DNA replication.

Mutants of Escherichia coli with increased fidelity of DNA replication.

Novel Escherichia coli active site dnaE alleles with altered base and sugar selectivity

DNA sequence effects on single base deletions arising during DNA polymerization in vitro by Escherichia coli Klenow fragment polymerase.

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