The 3'-5' proofreading exonuclease of bacteriophage T4 DNA polymerase is stimulated by other T4 DNA replication proteins.

Abstract:

The bacteriophage T4 DNA polymerase has an intrinsic 3'-5' proofreading exonuclease activity that plays a central role in determining the fidelity of T4 DNA replication. In order to monitor this activity, we have measured the rate at which the polymerase decreases the size of a double-stranded DNA substrate in the absence of deoxyribonucleoside triphosphates. With this assay, we find that the addition of the polymerase accessory proteins, 45 protein and 44/62 protein, increases the rate at which the polymerase-associated exonuclease digests the DNA substrate 3- to 4-fold. This stimulation requires the continuous hydrolysis of ATP catalyzed by the accessory protein complex. When added alone, the T4 helix-destabilizing protein, 32 protein, inhibits the exonuclease rate at high concentrations (greater than 100 micrograms/ml), while stimulating about 3-fold at low concentrations. The 32 protein and the accessory proteins together increase the exonuclease rate 8- to 10-fold above that found for the polymerase alone. The bacteriophage T7 DNA polymerase displays a similar 3'-5' exonuclease activity, but this exonuclease is not stimulated by any of the T4 replication proteins. It therefore appears that specific protein-protein interactions are involved.

Polymerases:

T4,T7

Topics:

Kinetic Parameters, Fidelity, Accessory Proteins/Complexes, Exonuclease Activity, Enzyme Substrate Interactions

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
T4 Bedinger P1983 Exonuclease accessory protein(s) gp 45, gp 44/62, gp 32

Entry validated by:

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