title: 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.
authors.affiliation: Department of Industrial Chemistry, University of Bologna, Viale Risorgimento 4, 40136 Bologna, Italy.
authors.forename: Emanuele
authors.initials: E
authors.lastname: Conte
authors.forename: Gabriele
authors.initials: G
authors.lastname: Vincelli
authors.forename: Roel M
authors.initials: RM
authors.lastname: Schaaper
authors.forename: Daniela
authors.initials: D
authors.lastname: Bressanin
authors.forename: Alessandra
authors.initials: A
authors.lastname: Stefan
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Dal Piaz
authors.forename: Alejandro
authors.initials: A
authors.lastname: Hochkoeppler
mesh_terms: D020134:Catalytic Domain; D004258:DNA Polymerase III / Q000737:chemistry* / Q000172:deficiency / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary
chemical_list: D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; C505238:holE protein, E coli; D004258:DNA Polymerase III; C063842:dnaK protein, E coli