Stabilization of the Escherichia coli DNA polymerase III ε subunit by the θ subunit favors in vivo assembly of the Pol III catalytic core

Abstract

Escherichia coli DNA polymerase III holoenzyme (HE) contains a core polymerase consisting of three subunits: α(polymerase), ε(3′-5′ exonuclease), and θ. Genetic experiments suggested that θ subunit stabilizes the intrinsically labile ε subunit and, furthermore, that θ might affect the cellular amounts of Pol III core and HE. Here, we provide biochemical evidence supporting this model by analyzing the amounts of the relevant proteins. First, we show that a ΔholE strain (lacking θ subunit) displays reduced amounts of free ε. We also demonstrate the existence of a dimer of ε, which may be involved in the stabilization of the protein. Second, θ, when overexpressed, dissociates the ε dimer and significantly increases the amount of Pol III core. The stability of ε also depends on cellular chaperones, including DnaK. Here, we report that: (i) temperature shift-up of ΔdnaK strains leads to rapid depletion of ε, and (ii) overproduction of θ overcomes both the depletion of ε and the temperature sensitivity of the strain. Overall, our data suggest that ε is a critical factor in the assembly of Pol III core, and that this is role is strongly influenced by the θ subunit through its prevention of ε degradation.

Single‐molecule mechanochemical characterization of E. coli pol III core catalytic activity

“…Pol III is composed of 10 subunits that coordinate leading and lagging strand synthesis. The core of the polymerase (pol III core) contains the polymerase subunit α, the proofreading exonuclease ε, and a subunit of unknown function θ 13, 14. The exact role of the θ subunit is still to be determined, but its presence increases the accuracy of pol III and it has been suggested to stabilize the interaction between α and ε 15, 16, 17.…”

“…The core of the polymerase (pol III core) contains the polymerase subunit a, the proofreading exonuclease e, and a subunit of unknown function u. 13,14 The exact role of the u subunit is still to be determined, but its presence increases the accuracy of pol III and it has been suggested to stabilize the interaction between a and e. [15][16][17] Although the dimer is asymmetric 7,18 to allow simultaneous polymerization of both template strands, the core of each branch consists of the same a e u complex. 13 The polymerase subunit a is a C family DNA polymerase, a family that is found only in prokaryotes.…”

The DnaE polymerase from Deinococcus radiodurans features RecA-dependent DNA polymerase activity

“…Elution of proteins from acrylamide gels, trypsin digestion and separation of peptides were performed as previously described [17]. The resulting peptides were analysed by LC-MS/MS using an Orbitrap XL instrument (Thermo Fisher Scientific) equipped with a nano-ESI source coupled with a nano-Acquity capillary UPLC (Waters).…”

“…E. coli DNA Pol III (αEc) is composed of a catalytic core, a DnaX complex and a β-clamp. The catalytic core does contain the α, ε and θ subunits [13] respectively featuring 5′–3′ polymerase [14], 3′–5′ exonuclease (proofreading) [15] and ε-stabilizing activity [16,17]. Notably, DNA Pol III α subunit, which is coded by the dnaE gene, is the only essential replicase in E. coli .…”

Polymerase manager protein UmuD directly regulates Escherichia coli DNA polymerase III binding to ssDNA

“…It is composed of three subassemblies: the polymerase core, the β processivity clamp and the clamp loader complex. Polymerase and proofreading activities are conducted by the core sub-assembly, which consists of the polymerase subunit α, the proofreading subunit ε and the θ subunit, which has a role in stabilizing the core (3,4). The β processivity clamp encircles the DNA and provides a platform for the polymerase core to bind, providing α with access to the primer-template and facilitating processive replication.…”