Fidelity of DNA synthesis by the Thermococcus litoralis DNA polymerase--an extremely heat stable enzyme with proofreading activity.

Abstract:

We demonstrate that the DNA polymerase isolated from Thermococcus litoralis (VentTM DNA polymerase) is the first thermostable DNA polymerase reported having a 3'----5' proofreading exonuclease activity. This facilitates a highly accurate DNA synthesis in vitro by the polymerase. Mutational frequencies observed in the base substitution fidelity assays were in the range of 30 x 10(-6). These values were 5-10 times lower compared to other thermostable DNA polymerases lacking the proofreading activity. All classes of DNA polymerase errors (transitions, transversions, frameshift mutations) were assayed using the forward mutational assay (1). The mutation frequencies of Thermococcus litoralis DNA polymerase varied between 15-35 x 10(-4) being 2-4 times lower than the respective values obtained using enzymes without proofreading activity. We also noticed that the fidelity of the DNA polymerase from Thermococcus litoralis responds to changes in dNTP concentration, units of enzyme used per one reaction and the concentration of MgSO4 relative to the total concentration of dNTPs present in the reaction. The high fidelity DNA synthesis in vitro by Thermococcus litoralis DNA polymerase provides good possibilities for maintaining the genetic information of original target DNA sequences intact in the DNA amplification applications.

Polymerases:

Topics:

Fidelity, Exonuclease Activity

One line summary:

This paper is the first to report a thermostable DNA polymerase (Vent) having a 3' —5' proofreading exonuclease activity

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Vent (exo-) Mattila P1991 3-5' Exonuclease (proofreading) No
Vent (exo-) Mattila P1991 Frameshift Error Rate 0.0094 errors/bp Technique: Forward mutational (1mM dNTP)
Vent (exo-) Mattila P1991 Nucleotide Substitution Rate 0.00019 errors/bp Technique: Reversion (1mM dNTP)
Replinase Mattila P1991 3-5' Exonuclease (proofreading) No
Replinase Mattila P1991 Frameshift Error Rate 0.0064 errors/bp Technique: Forward mutational (1mM dNTP)
Replinase Mattila P1991 Nucleotide Substitution Rate 0.000103 errors/bp Technique: Reversion (1mM dNTP)
T7 Mattila P1991 3-5' Exonuclease (proofreading) Yes
T7 Mattila P1991 Frameshift Error Rate 0.0015 errors/bp Technique: Forward mutational (1mM dNTP)
T7 Mattila P1991 Nucleotide Substitution Rate 1.5E-05 errors/bp Technique: Reversion (1mM dNTP)
Vent Mattila P1991 3-5' Exonuclease (proofreading) Yes
Vent Mattila P1991 Cloned or native Native organism
Vent Mattila P1991 5-3' Exonuclease No
Vent Mattila P1991 Frameshift Error Rate 0.0034 errors/bp Technique: Forward mutational (1mM dNTP)
Vent Mattila P1991 Nucleotide Substitution Rate 5.8E-05 errors/bp Technique: Reversion (1mM dNTP)

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