DNA polymerase proofreading: active site switching catalyzed by the bacteriophage T4 DNA polymerase.

Abstract:

DNA polymerases achieve high-fidelity DNA replication in part by checking the accuracy of each nucleotide that is incorporated and, if a mistake is made, the incorrect nucleotide is removed before further primer extension takes place. In order to proofread, the primer-end must be separated from the template strand and transferred from the polymerase to the exonuclease active center where the excision reaction takes place; then the trimmed primer-end is returned to the polymerase active center. Thus, proofreading requires polymerase-to-exonuclease and exonuclease-to-polymerase active site switching. We have used a fluorescence assay that uses differences in the fluorescence intensity of 2-aminopurine (2AP) to measure the rates of active site switching for the bacteriophage T4 DNA polymerase. There are three findings: (i) the rate of return of the trimmed primer-end from the exonuclease to the polymerase active center is rapid, >500 s(-1); (ii) T4 DNA polymerase can remove two incorrect nucleotides under single turnover conditions, which includes presumed exonuclease-to-polymerase and polymerase-to-exonuclease active site switching steps and (iii) proofreading reactions that initiate in the polymerase active center are not intrinsically processive.

Polymerases:

Topics:

Biotech Applications, Kinetic Parameters, Structure and Structure/Function, Fidelity, Nucleotide Incorporation, Exonuclease Activity, Methods

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
T4 W213S Fidalgo da Silva E2007 Methods Featured Fluorescence Spectroscopy
T4 W213S Fidalgo da Silva E2007 3-5' Exonuclease (proofreading) No
T4 W213S Fidalgo da Silva E2007 kcat 1.5 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover ; Experimental conditions: Mg++ (8mM), Temp (37°C)
T4 D112AE114A Fidalgo da Silva E2007 3-5' Exonuclease (proofreading) No
T4 Fidalgo da Silva E2007 kcat 145 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Single Turnover (with heparin); Experimental conditions: Temp (37°C), Mg++ (8mM)
T4 Fidalgo da Silva E2007 kcat 106 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Multiple Turnover (without heparin); Experimental conditions: Mg++ (8mM), Temp (37°C)

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