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 beta 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 alpha (pol III catalytic subunit), beta (sliding clamp processivity factor), and the essential DnaX (tau/gamma), delta and delta' 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 delta-delta' interaction, deltadelta'-tau/gamma complex formation, and alpha-tau 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 degrees 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 degrees C and a processivity of greater than 8.6 kilobases, the length of the template that is fully replicated after a single association event.
Highlights
DNA replication in all biological systems is performed by specialized multiprotein replicases [1, 2]
The purified novel polymerase cross-reacted with a subset of monoclonal antibodies directed against E. coli Pol III and was found associated with two proteins that exhibited strong sequence similarity with the ␥ and subunits of E. coli complex composed of an oligomer of DnaX ( and/or ␥), ␦ and ␦Ј; RFII, replicative form II; Tth, Thermus thermophilus; ATP␥S, adenosine 5Ј-O-(thiotriphosphate)
The identification of the gene encoding ␣ from Tth was aided by partial NH2-terminal and internal peptide fragment sequencing of the pol III ␣ subunit isolated previously from Tth extracts [40]
Summary
Pol III, DNA polymerase III (␣⑀); Pol III holoenzyme, DNA polymerase III holoenzyme; DnaX complex, a. Pol III (␣⑀) forms a stable, isolable complex held together by ␣-⑀ and ⑀- interactions (22, 28 –30). Within the DnaX complex, three DnaX subunits form a pentameric core with structurally related proteins, ␦ and ␦Ј [19, 27, 33]. The core of a replicase-like polymerase from a thermophile was isolated by pursuing a minor activity that could be resolved chromatographically from Tth DNA polymerase I [40]. The purified novel polymerase cross-reacted with a subset of monoclonal antibodies directed against E. coli Pol III and was found associated with two proteins that exhibited strong sequence similarity with the ␥ and subunits of E. coli complex composed of an oligomer of DnaX ( and/or ␥), ␦ and ␦Ј; RFII, replicative form II; Tth, Thermus thermophilus; ATP␥S, adenosine 5Ј-O-(thiotriphosphate). We describe the expression and purification of the ␣, DnaX (/␥), ␦, ␦Ј, and  subunits of Tth holoenzyme and their use to reconstitute a processive thermophilic replicase
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