The nine-subunit DNA polymerase (Pol) III* coupled to its beta sliding clamp is a rapid and highly processive replicating machine. The multiple subunits are needed for the complicated task of duplicating the Escherichia coli chromosome. In this report, Pol III* was constituted from individual pure proteins, and its structure was studied. Constitution of the Pol III* particle requires an ordered addition of the subunits, and the final structure contains 14 polypeptides in the ratio alpha 2 epsilon 2 theta 2 tau 2 gamma 2 delta 1 delta' 1 chi 1 psi 1. The structure can be summarized as being composed of two core polymerases (alpha epsilon theta) held together by a dimer of tau and one gamma complex clamp loader (gamma 2 delta 1 delta' 1 chi 1 psi 1) for loading beta onto DNA. At the center of the structure, the related tau and gamma subunits form a heterotetramer upon which the two core polymerases and clamp loader proteins assemble. The single copy nature of the delta, delta', chi, and psi subunits confers a structural asymmetry with respect to the two polymerases, presumably for the different functions of replicating the leading and lagging strands.

The gamma complex of DNA polymerase III holoenzyme, the replicase of Escherichia coli, couples ATP hydrolysis to the loading of beta sliding clamps onto primed DNA. The beta sliding clamp tethers the holoenzyme replicase to DNA for rapid and processive synthesis. In this report, the gamma complex has been constituted from its five different subunits. Size measurements and subunit stoichiometry studies show a composition of gamma 2 delta 1 delta' 1 1 chi 1 psi 1. Strong intersubunit contacts have been identified by gel filtration, and weaker contacts were identified by surface plasmon resonance measurements. An analogous tau complex has also been constituted and characterized; it is nearly as active as the gamma complex in clamp loading activity, but as shown in the fourth report of this series, it is at a disadvantage in binding the delta, delta', chi, and psi 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 gamma complex provide the basis for the structural asymmetry inherent within DNA polymerase III holoenzyme.

Replication machines use ring-shaped clamps that encircle DNA to tether the polymerase to the chromosome. The clamp is assembled on DNA by a clamp loader. This report shows that the polymerase and clamp loader coordinate their actions with the clamp by competing for it through overlapping binding sites. The competition is modulated by DNA. In the absence of DNA, the clamp associates with the clamp loader. But after the clamp is placed on DNA, the polymerase develops a tight grip on the clamp and out-competes the clamp loader. After replication of the template, the polymerase looses affinity for the clamp. Now the clamp loader regains access to the clamp and removes it from DNA thus recycling it for future use.