DNA replication in bacteria is performed by a specialized multicomponent replicase, the DNA polymerase III holoenzyme, that consist of three essential components: a polymerase, the ␤ sliding clamp processivity factor, and the DnaX complex clamp-loader. We report here the assembly of the minimal functional holoenzyme from Thermus thermophilus (Tth), an extreme thermophile. The minimal holoenzyme consists of ␣ (pol III catalytic subunit), ␤ (sliding clamp processivity factor), and the essential DnaX ( /␥), ␦ and ␦ components of the DnaX complex. We show with purified recombinant proteins that these five components are required for rapid and processive DNA synthesis on long single-stranded DNA templates. Subunit interactions known to occur in DNA polymerase III holoenzyme from mesophilic bacteria including ␦-␦ interaction, ␦␦ -/␥ complex formation, and ␣-interaction, also occur within the Tth enzyme. As in mesophilic holoenzymes, in the presence of a primed DNA template, these subunits assemble into a stable initiation complex in an ATP-dependent manner. However, in contrast to replicative polymerases from mesophilic bacteria, Tth holoenzyme is efficient only at temperatures above 50°C, both with regard to initiation complex formation and processive DNA synthesis. The minimal Tth DNA polymerase III holoenzyme displays an elongation rate of 350 bp/s at 72°C and a processivity of greater than 8.6 kilobases, the length of the template that is fully replicated after a single association event.DNA replication in all biological systems is performed by specialized multiprotein replicases (1, 2). Cellular replicases consist of three major subassemblies: a sliding clamp processivity factor, a clamp loader, and a specialized polymerase. Replicases, especially bacterial replicases, are rapid and processive consistent with the requirement for them to synthesize a several megabase genome from a single origin in less than one hour.In the prototypic Escherichia coli replication system, a key determinant of processive DNA synthesis is the interaction between the ␤ processivity factor and pol III 1 (3, 4). The dimeric ␤ subunit is a bracelet-shaped molecule that clamps around DNA permitting it to rapidly slide along duplex DNA without dissociating (5). ␤ binds to the pol III ␣ subunit through protein-protein contacts preventing the polymerase from dissociating from the template, ensuring high processivity. Efficient loading of the ␤ subunit onto DNA requires ATP-dependent opening and closing of the clamp by the DnaX complex. The DnaX complex contains the essential DnaX, ␦ and ␦Ј subunits plus two ancillary proteins, and (6 -9). The dnaX gene encodes two proteins, ␥ and , by programmed ribosomal frameshifting (10 -15). Both and the shorter ␥ product share ATPbinding domain I, domain II, and domain III that is responsible for DnaX oligomerization, -binding, and binding of ␦-␦Ј (16 -19). contains two unique domains. domain IV forms a link with the DnaB helicase and domain V binds pol III (17,20,21). Pol III consists of ␣, the catalyti...