Interplay of Clamp Loader Subunits in Opening the β Sliding Clamp of Escherichia coli DNA Polymerase III Holoenzyme

Abstract

The Escherichia coli β dimer is a ring-shaped protein that encircles DNA and acts as a sliding clamp to tether the replicase, DNA polymerase III holoenzyme, to DNA. The γ complex (γδδ′χψ) clamp loader couples ATP to the opening and closing of β in assembly of the ring onto DNA. These proteins are functionally and structurally conserved in all cells. The eukaryotic equivalents are the replication factor C (RFC) clamp loader and the proliferating cell nuclear antigen (PCNA) clamp. The δ subunit of the E. coli γ complex clamp loader is known to bind β and open it by parting one of the dimer interfaces. This study demonstrates that other subunits of γ complex also bind β, although weaker than δ. The γ subunit like δ, affects the opening of β, but with a lower efficiency than δ. The δ′ subunit regulates both γ and δ ring opening activities in a fashion that is modulated by ATP interaction with γ. The implications of these actions for the workings of the E. coli clamp loading machinery and for eukaryotic RFC and PCNA are discussed.