Clamp loader ATPases and the evolution of DNA replication machinery

Clamp loaders are pentameric ATPases of the AAA+ family that operate to ensure processive DNA replication. They do so by loading onto DNA the ring-shaped sliding clamps that tether the polymerase to the DNA. Structural and biochemical analysis of clamp loaders has shown how, despite differences in composition across different branches of life, all clamp loaders undergo the same concerted conformational transformations, which generate a binding surface for the open clamp and an internal spiral chamber into which the DNA at the replication fork can slide, triggering ATP hydrolysis, release of the clamp loader, and closure of the clamp round the DNA. We review here the current understanding of the clamp loader mechanism and discuss the implications of the differences between clamp loaders from the different branches of life

A direct proofreader-clamp interaction stabilizes the Pol III replicase in the polymerization mode

<p>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.</p>