The C-Terminal Domain of DnaQ Contains the Polymerase Binding Site

The Escherichia coli dnaQ gene encodes the 3*35* exonucleolytic proofreading («) subunit of DNA polymerase III (Pol III). Genetic analysis of dnaQ mutants has suggested that « might consist of two domains, an N-terminal domain containing the exonuclease and a C-terminal domain essential for binding the polymerase (a) subunit. We have created truncated forms of dnaQ resulting in « 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 « subunits with the a and u subunits of the Pol III core. The DnaQ991 protein, consisting of the N-terminal 186 amino acids, was defective in binding to the a subunit while retaining normal binding to the u subunit. In contrast, the ND186 protein, consisting of the C-terminal 57 amino acids, exhibited normal binding to the a subunit but was defective in binding to the u subunit. A strain carrying the dnaQ991 allele exhibited a strong, recessive mutator phenotype, as expected from a defective a binding mutant. The data are consistent with the existence of two functional domains in «, with the C-terminal domain responsible for polymerase binding. The Escherichia coli DNA polymerase III (Pol III) holoenzyme (HE) is responsible for the efficient and accurate replication of the bacterial chromosome (for a review, see references 9, 10, and 16). This multisubunit enzyme contains a core polymerase with both polymerizing and exonucleolytic activities. Unlike those of most other proofreading polymerases, these two activities of Pol III are contained in two separate polypeptides. The polymerase activity is contained in the a subunit (dnaE gene product), while the 39359 exonuclease (Exo) activity, which serves as a proofreader for replication errors, is contained in the e subunit (dnaQ gene product). The Pol III core also contains the u subunit (holE gene product), but the function of this subunit is unclear (4, 20, 21). The subunits are arranged in a linear fashion, a-e-u, with e binding both a and u (21). The precise interactions, both functionally and structurally, between the core subunits and of the core subunits with the additional accessory factors of the HE are the subject of active current interest (2, 7, 14). Previously, in an effort to better understand the roles of the e subunit, we undertook a genetic analysis of a large collection of dnaQ mutator mutants (23). These studies confirmed and extended the importance of three conserved exonuclease motifs in the catalytic activity of the DnaQ enzyme (see Fig. 1) and also suggested that the C terminus of e might be required for interaction with the a subunit. dnaQ932, a mutant containing a stop codon 3 amino acids from the C-terminal end of e, was recessive in the presence of a single copy of dnaQ 1 , suggesting that the DnaQ932 protein does not compete efficiently with the wild-type protein for binding to a. Furthermore, the region between residues 190 and 212 has been identified as a so-called Q-linker (24). In certain proteins, Q-linkers act as a hinge to link domains that have different functions (22, 24). In e, this linker may tether the N-terminal exonuclease domain to the