Assembly of a chromosomal replication machine: Two DNA polymerases, a clamp loader, and sliding clamps in one holoenzyme particle. I. Organization of the clamp loader

Abstract

The γ complex of DNA polymerase III holoenzyme, the replicase of Escherichia coli, couples ATP hydrolysis to the loading of β sliding clamps onto primed DNA. The β sliding clamp tethers the holoenzyme replicase to DNA for rapid and processive synthesis. In this report, the γ complex has been constituted from its five different subunits. Size measurements and subunit stoichiometry studies show a composition of γ2δ1δ1χ1ψ1. Strong intersubunit contacts have been identified by gel filtration, and weaker contacts were identified by surface plasmon resonance measurements. An analogous τ complex has also been constituted and characterized; it is nearly as active as the γ complex in clamp loading activity, but as shown in the fourth report of this series, it is at a disadvantage in binding the δ, δ', χ, and ψ subunits when core is present (Xiao, H., Naktinis, V., and O'Donnell, M. (1995) J. Biol. Chem. 270, 13378-13383). The single copy subunits within the γ complex provide the basis for the structural asymmetry inherent within DNA polymerase III holoenzyme.