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 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.

Polymerases:

Topics:

Nucleotide Incorporation, Kinetic Parameters, Historical Protein Properties (MW, pI, ...), Exonuclease Activity, Source / Purification

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Tth Pol III Bullard JM2002 Molecular Weight 1.35E+05 Dalton Technique: SDS-PAGE
Tth Pol III Bullard JM2002 3-5' Exonuclease (proofreading) Yes
Tth Pol III Bullard JM2002 Cloned or native Cloned in E. coli
Tth Pol III Bullard JM2002 Tagged Yes
Tth Pol III Bullard JM2002 Tag Name Histidine
Tth Pol III Bullard JM2002 Full length or truncated Full length
Tth Pol III Bullard JM2002 Processivity 8.6kb
Tth Pol III Bullard JM2002 Vmax 350 /second Reaction: Nucleotide incorporation; Substrate: dNTPs; Experimental conditions: Temp (72°C)

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