Assembly of a chromosomal replication machine: two DNA polymerases, a clamp loader, and sliding clamps in one holoenzyme particle. IV. ATP-binding site mutants identify the clamp loader.

The gamma complex (gamma delta delta' chi psi) and tau complex (tau delta delta' chi psi) clamp loaders require ATP hydrolysis to load beta sliding clamps onto DNA. The beta sliding clamp tethers the polymerase (Pol) III* replicase to DNA for processive synthesis. Pol III* contains both gamma and tau, but only one each of the delta, delta', chi, and psi subunits. Hence, there is ambiguity with respect to which clamp loader, the gamma or tau complex, exists in the Pol III* replicase structure. In this study, ATP-binding site mutants of gamma and tau have been prepared, and these mutants, when assembled into either the gamma or tau complex, are inactive in clamp loading. These mutants have been used as a tool to determine the identity of the clamp loader in Pol III*. The nine-subunit Pol III* has been assembled using either mutant gamma or tau in place of wild-type gamma or tau. The results show that mutation of gamma inactivates Pol III* activity, but mutation of tau does not, indicating that the gamma complex (and not the tau complex) is the clamp loader of Pol III*. The tau subunit carries the task of dimerizing the core polymerase, and it is this association of tau with core that appears to direct the single copy subunits away from tau and onto gamma.