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...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.