A function for the ψ subunit in loading the Escherichia coli DNA polymerase sliding clamp

Abstract

Crystal structures of an Escherichia coli clamp loader have provided insight into the mechanism by which this molecular machine assembles ring-shaped sliding clamps onto DNA. The contributions made to the clamp loading reaction by two subunits, χ and ψ, which are not present in the crystal structures, were determined by measuring the activities of three forms of the clamp loader, γ3δδ′, γ 3δδ′ψ, and γ 3δδ′ψχ. The ψ subunit is important for stabilizing an ATP-induced conformational state with high affinity for DNA, whereas the ψ subunit does not contribute directly to clamp loading in our assays lacking single-stranded DNA-binding protein. The ψ subunit also increases the affinity of the clamp loader for the clamp in assays in which ATPγS is substituted for ATP. Interestingly, the affinity of the γ3δδ′ complex for β is no greater in the presence than in the absence of ATPγS. A role for ψ in stabilizing or promoting ATP- and ATPγS-induced conformational changes may explain why large conformational differences were not seen in γ 3δδ′ structures with and without bound ATPγS. The β clamp partially compensates for the activity of ψ when this subunit is not present and possibly serves as a scaffold on which the clamp loader adopts the appropriate conformation for DNA binding and clamp loading. Results from our work and others suggest that the ψ subunit may introduce a temporal order to the clamp loading reaction in which clamp binding precedes DNA binding. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.