The C-terminal domain of DnaQ contains the polymerase binding site

Abstract

The Escherichia coli dnaQ gene encodes the 3'→5' exonucleolytic proofreading (ε) subunit of DNA polymerase III (Pol III). Genetic analysis of dnaQ mutants has suggested that ε might consist of two domains, an N- terminal domain containing the exonuclease and a C-terminal domain essential for binding the polymerase (α) subunit. We have created truncated forms of dnaQ resulting in ε subunits that contain either the N-terminal or the C- terminal domain. Using the yeast two-hybrid system, we analyzed the interactions of the single-domain ε subunits with the α and θ subunits of the Pol III core. The DnaQ991 protein, consisting of the N-terminal 186 amino acids, was defective in binding to the α subunit while retaining normal binding to the θ subunit. In contrast, the NΔ186 protein, consisting of the C-terminal 57 amino acids, exhibited normal binding to the α subunit but was defective in binding to the θ subunit. A strain carrying the dnaQ991 allele exhibited a strong, recessive mutator phenotype, as expected from a defective α binding mutant. The data are consistent with the existence of two functional domains in ε, with the C-terminal domain responsible for polymerase binding.