| Abstrakt: |
The γ complex (γδδ′χψ) and τ complex (τδδ′χψ) clamp loaders require ATP hydrolysis to load β sliding clamps onto DNA. The β sliding clamp tethers the polymerase (Pol) III∗ replicase to DNA for processive synthesis. Pol III∗ contains both γ and τ, but only one each of the δ, δ′, χ, and ψ subunits. Hence, there is ambiguity with respect to which clamp loader, the γ or τ complex, exists in the Pol III∗ replicase structure. In this study, ATP-binding site mutants of γ and τ have been prepared, and these mutants, when assembled into either the γ or τ 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 γ or τ in place of wild-type γ or τ. The results show that mutation of γ inactivates Pol III∗ activity, but mutation of τ does not, indicating that the γ complex (and not the τ complex) is the clamp loader of Pol III∗. The τ subunit carries the task of dimerizing the core polymerase, and it is this association of τ with core that appears to direct the single copy subunits away from τ and onto γ. |
