Recent advances in understanding the molecular mechanisms of inherited cancer susceptibilities have increased the interest in mutators, i.e. cell lines with higher mutation rates than wild-type cells. The bacterium Escherichia coli has been a productive model system for investigating mutators and their underlying mechanisms. Those studies have revealed mutators affected in the fidelity of DNA replication, postreplicative mismatch repair, and the repair of a wide range of DNA damages (for review, see Miller, 1996). Of central importance in mutagenesis is the fidelity of DNA replication, because it controls the production of mutations on normal and damaged DNA templates. Replication of the E. coli chromosome is performed by DNA polymerase III holoenzyme (HE), a large dimeric complex composed of two polymerase III core assemblies and a number of accessory factors. The accessory subunits are responsible for high processivity and for co-ordinating simultaneous synthesis of leading- and lagging-strands (for reviews, see Kelman and O’Donnell, 1995 or McHenry, 2003). The core polymerase contains three tightly bound subunits: α, the polymerase encoded by the dnaE gene; ɛ, the 3′→5′ proofreading subunit encoded by the dnaQ gene; and θ, a subunit encoded by the holE gene, which may function to stabilize the ɛ subunit (Taft-Benz and Schaaper, 2004). The accessory proteins include the β subunit, which forms a ring-shaped structure (sliding clamp), tethering the core to the DNA (Kong et al., 1992) to ensure high processivity, and the seven-subunit DnaX complex (τ2γδδ′χψ) (Dallmann and McHenry, 1995; Onrust et al., 1995; McHenry, 2003). Within the DnaX complex, the products of the dnaX gene (τ and γ subunits) play important roles. The complex functions as a clamp loader for the β-sliding clamp (Naktinis et al., 1996). The τ subunit (τ2) serves to dimerize the two Pol III cores (McHenry, 1982; Stukenberg and O’Donnell, 1995; Gao and McHenry, 2001) enabling a physical coupling of the leading- and lagging-strand polymerase (Kim et al., 1996; McInerney and O’Donnell, 2004).
With regard to fidelity, two of the HE subunits have received most of the attention. The α (polymerase) subunit is responsible for discrimination against incorrect nucleotides during the insertion step (base selection), while the ɛ subunit is responsible for proofreading misinserted nucleotides using its 3′→5′ exonuclease. θ subunit many play an indirect role by stabilizing the ɛ subunit (Studwell-Vaughan and O’Donnell, 1993; Taft-Benz and Schaaper, 2004). Together, base selection and proofreading are thought to maintain the accuracy of polymerization at about one error per 107 bases replicated (Schaaper, 1993a). Although genetic and biochemical studies have revealed insights into the organization and functions of the accessory proteins, little is known about their role in replication fidelity. To gain insight into this matter, we have initiated studies of E. coli mutants affected in genes encoding the HE subunits. Here, we show that a classical temperature-sensitive allele, dnaX36, encoding a mutant τ subunit, increases significantly the frequency of frameshift and base pair substitution replication errors. Among the base substitutions, transversions are enhanced preferentially. A search for new transversion mutators by localized mutagenesis targeting the dnaX gene yielded six new, τ-specific dnaX mutator alleles, which, like dnaX36, specifically enhance transversions and (−1) frameshifts. Thus, we have discovered a new class of E. coli mutators. The possible mechanism(s) by which the τ subunit contributes to high replication fidelity are discussed. Specifically, we develop a model in which τ may act as a sensor of certain Pol III HE misinsertion errors.