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

Escherichia coli DNA polymerase III holoenzyme (HE) contains a core polymerase consisting of three subunits: alpha (polymerase). epsilon (3'-5' exonuclease), and theta. Genetic experiments suggested that theta subunit stabilizes the intrinsically labile epsilon subunit and, furthermore, that theta 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 Delta holE strain (lacking theta subunit) displays reduced amounts of free epsilon. We also demonstrate the existence of a dimer of epsilon, which may be involved in the stabilization of the protein. Second, theta, when overexpressed, dissociates the epsilon dimer and significantly increases the amount of Pol III core. The stability oft epsilon also depends on cellular chaperones, including DnaK. Here, we report that: (i) temperature shift-up of Delta dnaK strains leads to rapid depletion of E, and (ii) overproduction of theta overcomes both the depletion of epsilon, and the temperature sensitivity of the strain. Overall, our data suggest that epsilon is a critical factor in the assembly of Pol III core, and that this is role is strongly influenced by the theta subunit through its prevention oft epsilon degradation.