DNA Polymerase III Holoenzyme from Thermus thermophilus Identification, Expression, Purification of Components, and Use to Reconstitute a Processive Replicase

Abstract

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...

The Rate and Character of Spontaneous Mutation in Thermus thermophilus

“…However, they have dramatically different DNA base compositions (69 vs. 38 mol% G 1 C) and represent different domains of cellular life (bacteria vs. archaea), resulting in different molecular contexts of genome replication. Thus, T. thermophilus employs a family-A DNA polymerase for genome replication (Bullard et al 2002), whereas Sulfolobus spp. and other archaea possess only family-B polymerases capable of genome replication (Kawarabayasi et al 2001;She et al 2001).…”

Involvement of DnaE, the Second Replicative DNA Polymerase from Bacillus subtilis, in DNA Mutagenesis

“…Protein concentrations were quantified using the Coomassie Protein Assay Kit (Pierce) using BSA as a standard. A gel filtration chromatography was performed using a Superose 12 column (Amersham Biosciences) and buffer TEN 50 supplemented with 10% glycerol, to determine the oligomerization state of DnaE. DnaE eluted as a dimer on the gel filtration column.…”

Conservation of Eubacterial Replicases

“…2, Schaeffer et al (1)). A. aeolicus and P. aeruginosa a subunits complex with an e subunit (5, 8), whereas in T. thermophilus, the a subunit is devoid of both e and y subunits (6). The y subunit is not widespread in bacteria, but the e subunit (or similar proteins) is common.…”

“…While E. coli expresses g and t subunits, S. pyogenes, S. aureus, P. aeruginosa, A. aeolicus and B. subtilis express only the complete DnaX product, the t subunit (4, 5, 8 -10). On the other hand, the g subunit is seen in T. thermophilus and a number of Enterobacteriaceae, while Aeromonas and Vibrio may express an intermediate form (6,11). The use of combinations of t and g subunits in the clamp loaders may impact on the clamp loaders' interactions with the replicative polymerases within the replisome complex.…”