Assembly of a Chromosomal Replication Machine: Two DNA Polymerases, a Clamp Loader, and Sliding Clamps in One Holoenzyme Particle

The Escherichia coli replicase, DNA polymerase III holoenzyme, derives its processivity from the β subunit sliding clamp that encircles DNA and tethers the replicase to the template. The β dimer is assembled around DNA by the γ complex clamp loader in an ATP-dependent reaction. In this report, the essential contact between the clamp loader and β is identified as mediated through the δ subunit of the γ complex. The δ subunit appears to contact the face of the β dimer ring that contains the two C termini. Surprisingly, ATP is required for the γ complex to bind β, but not for δ to bind β. This indicates that δ is buried in the γ complex and suggests a role for ATP in exposing δ for interaction with β. A protease protection assay has been developed to specifically probe the δ subunit within the γ complex. The results of the assay are consistent with an ATP-induced conformational change in the γ complex that alters the state of the δ subunit within it. The implication of these key features to the clamp loading mechanism of the γ complex is discussed.