DNA: Replication, Repair, and Recombination
A Function for the ψ Subunit in Loading the Escherichia coli DNA Polymerase Sliding Clamp*

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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′, γ3δδ′ψ, and γ3δδ′ψχ. The ψ subunit is important for stabilizing an ATP-induced conformational state with high affinity for DNA, whereas the χ 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 γ3δδ′ 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.

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This work was supported by National Institutes of Health Grants GM055596 (to L. B. B.) and GM38839 (to M. O. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S4.