title: Mutations that Separate the Functions of the Proofreading Subunit of the Escherichia coli Replicase.
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.
authors: University Program in Genetics and Genomics, Duke University, Durham, North Carolina 27705.
authors: Zakiya
authors: Z
authors: Whatley
authors: University Program in Genetics and Genomics, Duke University, Durham, North Carolina 27705 Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710 kenneth.kreuzer@duke.edu.
authors: Kenneth N
authors: KN
authors: Kreuzer
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D004258:DNA Polymerase III / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D059645:Mutation Rate; D009268:Nalidixic Acid / Q000378:metabolism; D010641:Phenotype; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics*; D013014:SOS Response, Genetics
chemical_list: D029968:Escherichia coli Proteins; D021122:Protein Subunits; D009268:Nalidixic Acid; D004258:DNA Polymerase III; C468316:dnaQ protein, E coli
keywords: DNA polymerase; SOS response; epsilon subunit; linker-scanning mutagenesis; mutation rate