Abstract
Processive DNA synthesis by the alpha epsilon theta core of the Escherichia coli Pol III replicase requires it to be bound to the beta(2) clamp via a site in the a polymerase subunit. How the epsilon proofreading exonuclease subunit influences DNA synthesis by alpha was not previously understood. In this work, bulk assays of DNA replication were used to uncover a non-proofreading activity of epsilon. Combination of mutagenesis with biophysical studies and single-molecule leading-strand replication assays traced this activity to a novel beta-binding site in e that, in conjunction with the site in a, maintains a closed state of the alpha epsilon theta-beta(2) replicase in the polymerization mode of DNA synthesis. The epsilon-beta interaction, selected during evolution to be weak and thus suited for transient disruption to enable access of alternate polymerases and other clamp binding proteins, therefore makes an important contribution to the network of protein-protein interactions that finely tune stability of the replicase on the DNA template in its various conformational states.