Abstract
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.
Highlights
The I' complex (1'00' xt/l) and 'T complex ('TOO' xt/l) clamp loaders require ATP hydrolysis to load /3 sliding clamps onto DNA
The results show that mutation of I' inactivates Pol 111* activity, but mutation of r does not, indicating that the I' complex is the clamp loader of Pol 111*
The f3 clamp is assembled onto DNA by a clamp loading activity inherent in the holoenzyme structure (3-5), The first report of this series showed that a clamp loader can be assembled from the 8, 8', x, and t/J
Summary
(Received for publication, December 9, 1994, and in revised form, February 23, 1995). ATP-binding site mutants of I' and 'T have been prepared, and these mutants, when assembled into either the I' or 'T complex, are inactive in clamp loading. These mutants have been used as a tool to determine the identity of the clamp loader in Pol 111*. The f3 clamp is assembled onto DNA by a clamp loading activity inherent in the holoenzyme structure (3-5), The first report of this series showed that a clamp loader can be assembled from the 8, 8', x, and t/J subunits and from a dimer of either y or '1' to produce a y complex (Y2818'1X1 t/J1) or a '1' complex ('1'2818' 1X1 t/Jl)' respectively (6).
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