Proteolysis of the proofreading subunit controls the assembly of Escherichia coli DNA polymerase III catalytic core.

The C-terminal region of the proofreading subunit (ɛ) of Escherichia coli DNA polymerase III is shown here to be labile and to contain the residues (identified between F187 and R213) responsible for association with the polymerase subunit (α). We also identify two α-helices of the polymerase subunit (comprising the residues E311–M335 and G339–D353, respectively) as the determinants of binding to ɛ. The C-terminal region of ɛ is degraded by the ClpP protease assisted by the GroL molecular chaperone, while other factors control the overall concentration in vivo of ɛ. Among these factors, the chaperone DnaK is of primary importance for preserving the integrity of ɛ. Remarkably, inactivation of DnaK confers to Escherichia coli inviable phenotype at 42 °C, and viability can be restored over-expressing ɛ. Altogether, our observations indicate that the association between ɛ and α subunits of DNA polymerase III depends on small portions of both proteins, the association of which is controlled by proteolysis of ɛ. Accordingly, the factors catalysing (ClpP, GroL) or preventing (DnaK) this proteolysis exert a crucial checkpoint of the assembly of Escherichia coli DNA polymerase III core.