RRC ID 53646
Author Whatley Z, Kreuzer KN.
Title Mutations that Separate the Functions of the Proofreading Subunit of the Escherichia coli Replicase.
Journal G3 (Bethesda)
Abstract The dnaQ gene of Escherichia coli encodes the ε subunit of DNA polymerase III, which provides the 3' → 5' exonuclease proofreading activity of the replicative polymerase. Prior studies have shown that loss of ε leads to high mutation frequency, partially constitutive SOS, and poor growth. In addition, a previous study from our laboratory identified dnaQ knockout mutants in a screen for mutants specifically defective in the SOS response after quinolone (nalidixic acid) treatment. To explain these results, we propose a model whereby, in addition to proofreading, ε plays a distinct role in replisome disassembly and/or processing of stalled replication forks. To explore this model, we generated a pentapeptide insertion mutant library of the dnaQ gene, along with site-directed mutants, and screened for separation of function mutants. We report the identification of separation of function mutants from this screen, showing that proofreading function can be uncoupled from SOS phenotypes (partially constitutive SOS and the nalidixic acid SOS defect). Surprisingly, the two SOS phenotypes also appear to be separable from each other. These findings support the hypothesis that ε has additional roles aside from proofreading. Identification of these mutants, especially those with normal proofreading but SOS phenotype(s), also facilitates the study of the role of ε in SOS processes without the confounding results of high mutator activity associated with dnaQ knockout mutants.
Volume 5(6)
Pages 1301-11
Published 2015-4-15
DOI 10.1534/g3.115.017285
PII g3.115.017285
PMID 25878065
PMC PMC4478557
MeSH Amino Acid Motifs Amino Acid Sequence DNA Polymerase III / chemistry DNA Polymerase III / genetics* Escherichia coli / enzymology* Escherichia coli / genetics* Escherichia coli Proteins / chemistry Escherichia coli Proteins / genetics* Molecular Sequence Data Mutagenesis, Insertional / genetics Mutagenesis, Site-Directed Mutation / genetics* Mutation Rate Nalidixic Acid / metabolism Phenotype Protein Structure, Tertiary Protein Subunits / chemistry Protein Subunits / genetics* SOS Response, Genetics
IF 2.63
Times Cited 2
Resource
Prokaryotes E. coli