The θ Subunit of Escherichia coli DNA Polymerase III: a Role in Stabilizing the ε Proofreading Subunit

The DNA polymerase III (Pol III) holoenzyme (HE) is the major chromosomal replication enzyme in Escherichia coli (19, 22, 30, 31). The enzyme is composed of 17 subunits, 10 of which are distinct (19, 31). HE contains two polymerase core molecules, each consisting of an α, ɛ, and θ subunit arranged in the linear order α-ɛ-θ. The two cores are connected through a dimer of the τ subunit, establishing the basic arrangement of a dimeric polymerase that simultaneously replicates the leading and lagging strands (14). In addition, the HE contains, for each core, a β-clamp (β2) tethering the polymerase to the DNA to ensure high processivity and the five-subunit γ-complex (γδδ′χψ) responsible for loading and unloading the β-clamp. The precise functioning of the HE complex in chromosomal replication is under active investigation (5, 24, 26, 31). Within the Pol III core, α (the dnaE gene product) is the DNA polymerase, while ɛ (the dnaQ gene product) is the 3′→5′ proofreading exonuclease. The function of θ (the holE gene product), which is tightly bound to ɛ, is unclear. Genetic analysis of the Pol III core constituents has provided insight into the role of its constituents. For example, dnaE(Ts) mutants, encoding temperature-sensitive polymerase subunits, are conditionally lethal, as expected in view of the essential nature of the Pol III replication function. Several dnaE mutants display mutator or antimutator effects (9, 28), indicating the important fidelity role of this enzyme. Many dnaQ mutants exhibit strong mutator phenotypes, indicating the importance of the 3′-exonuclease activity for replication fidelity (45). Deletion mutants of dnaQ (23, 27) or mutants lacking the domain necessary for interaction with the polymerase (46) have been generated, but these mutants proved essentially inviable unless accompanied by a suppressing mutation in dnaE. Based on these studies, ɛ is assigned at least two functions: a fidelity function through its 3′-exonuclease activity and a structural function based on its tight, and presumably stabilizing, interaction with the polymerase (23, 27, 45, 46). In contrast, the role of the θ subunit of the Pol III core is unknown. Loss of θ (ΔholE) results in healthy cells with no morphology changes and little or no change in mutant frequencies (42). Based on these studies, it was suggested that θ is not necessary or important for proper functioning of the Pol III core. θ does not affect DNA synthesis by α or α-ɛ (43); however, gel filtration (44), coexpression (1), and yeast two-hybrid experiments (18) have demonstrated a tight interaction between θ and ɛ, but none between θ and α. Interestingly, θ was shown to moderately affect the 3′→5′ proofreading exonuclease activity, as addition of θ to an exonuclease assay measuring the removal of a G · T mispair increased ɛ-mediated excision of the terminal T residue by about 2.5-fold (44). The above findings suggest that θ, while not essential, could play a role in DNA replication and its fidelity, presumably indirectly through its interaction with the ɛ subunit. The precise nature of this interaction is being pursued structurally by both nuclear magnetic resonance (NMR) and crystallography studies. Structures of both ɛ and θ have been reported (6, 15, 20), as well as the ɛ-θ interaction surface on ɛ (7). Here, we report on a series of genetic experiments on the ɛ-θ interaction. Specifically, we have studied (i) in greater detail, the possible mutator effect resulting from a holE deletion, (ii) the effect of the holE deletion on dnaQ mutator mutants, and (iii) the effect of θ on the α-ɛ interaction as measured by yeast two- and three-hybrid assays. The results suggest that θ may be a stabilizing factor for the ɛ subunit, which has been shown to be intrinsically unstable (11).