DNA Polymerase III Holoenzyme From Thermus Thermophilus Identification Expression Purification of Components and use to Reconstitute a Processive Replicase
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 β 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 α (pol III catalytic subunit), β (sliding clamp processivity factor), and the essential DnaX (τ/γ), δ and δ′ 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 δ-δ′ interaction, δδ′-τ/γ complex formation, and α-τ 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 minimalTth 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.
Bullard, J. M., Williams, J. C., Acker, W. K., Jacobi, C., Janjic, N., & McHenry, C. S. (2002). DNA Polymerase III Holoenzyme from Thermus thermophilus Identification, Expression, Purification of Components, and Use to Reconstitute a Processive Replicase. Journal of Biological Chemistry, 277(16), 13401–13408. https://doi.org/10.1074/jbc.M110833200
Journal of Biological Chemistry
© 2002 American Society for Biochemistry and Molecular Biology Inc. Original published version available at http://doi.org/10.1074/jbc.M110833200