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

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, chi and psi, which are not present in the crystal structures, were determined by measuring the activities of three forms of the clamp loader, gamma(3)delta delta', gamma(3)delta delta'psi, and gamma(3)delta delta'psi chi. The psi subunit is important for stabilizing an ATP-induced conformational. state with high affinity for DNA, whereas the X subunit does not contribute directly to clamp loading in our assays lacking single-stranded DNA-binding protein. The psi subunit also increases the affinity of the clamp loader for the clamp in assays in which ATP gamma S is substituted for ATP. Interestingly, the affinity of the gamma(3)delta delta' complex for beta is no greater in the presence than in the absence of ATP gamma S. A role for psi in stabilizing or promoting ATP- and ATP gamma S-induced conformational changes may explain why large conformational differences were not seen in gamma(3)delta delta' structures with and without bound ATP gamma S. The beta clamp partially compensates for the activity of psi 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 psi subunit may introduce a temporal order to the clamp loading reaction in which clamp binding precedes DNA binding.