Characterization of a DNA polymerase from the hyperthermophile archaea Thermococcus litoralis. Vent DNA polymerase, steady state kinetics, thermal stability, processivity, strand displacement, and exonuclease activities.

Abstract:

We have isolated, cloned, and characterized a DNA polymerase from the hyperthermophile archaea Thermococcus litoralis, the Tli DNA polymerase (also referred to as Vent DNA polymerase). The enzyme is extremely thermostable, having a half-life of 8 h at 95 degrees C and about 2 h at 100 degrees C. Pseudo-first-order kinetics at 70 degrees C reveal an extremely low Km for a primed M13mp18 substrate (0.1 nM), coupled with a relatively high Km for dNTPs (50 microM). Accompanying extension rates are on the order of 1000 nucleotides/min. Synthesis by the polymerase is largely distributive, adding an average of 7 nucleotides/initiation event. This distributive synthesis can generate products of at least 10,000 bases. Tli DNA polymerase contains a 3'-->5' exonuclease activity that enhances the fidelity of replication by the enzyme (Mattila, P., Korpela, J., Tenkanen, T. and Pitkanen, K. (1991) Nucleic Acids Res. 19, 4967-4973). A 2-amino acid substitution within the conserved exonuclease domain abolishes both double and single strand-dependent exonuclease activity, without altering kinetic parameters for polymerization on a primed single-stranded template. Strand displacement activity by the mutated and unmutated forms increases with increasing temperature and is enhanced in the exonuclease-deficient form of the enzyme.

Polymerases:

Topics:

Kinetic Parameters, Nucleotide Incorporation, Exonuclease Activity, Source / Purification

One line summary:

This paper characterizes native and recombinant Vent polymerases and compares the parameters to other polymerases

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
T4 Kong H1993 Processivity 12bp
T4 Kong H1993 KM 2uM Reaction: Nucleotide incorporation; Substrate: dNTPs; Technique: Filter binding ; Experimental conditions: Temp (72°C)
Klenow fragment Kong H1993 Processivity 12bp
Klenow fragment Kong H1993 KM 2.3uM Reaction: Nucleotide incorporation; Substrate: dNTPs; Technique: Filter binding ; Experimental conditions: Temp (72°C)
T7 Kong H1993 Processivity 1kb
T7 Kong H1993 KM 18uM Reaction: Nucleotide incorporation; Substrate: dNTPs; Technique: Filter binding ; Experimental conditions: Temp (72°C)
Vent Kong H1993 3-5' Exonuclease (proofreading) Yes
Vent Kong H1993 Cloned or native Native organism
Vent Kong H1993 5-3' Exonuclease No
Vent Kong H1993 Full length or truncated Full length
Vent Kong H1993 Processivity 7bp
Vent Kong H1993 KM 41uM Reaction: Nucleotide incorporation; Technique: Filter binding ; Experimental conditions: Temp (72°C)
Vent Kong H1993 KM 0.07uM Reaction: Polymerase-DNA interaction; Substrate: DNA template; Technique: Filter binding ; Experimental conditions: Temp (72°C)
Vent Kong H1993 Vmax 1000 /minute Reaction: Nucleotide incorporation; Substrate: dNTPs
Eco Pol I Kong H1993 Full length or truncated Full length
Eco Pol I Kong H1993 Processivity 97bp
Eco Pol I Kong H1993 KM 2uM Reaction: Nucleotide incorporation; Substrate: dNTPs; Technique: Filter binding ; Experimental conditions: Temp (72°C)
Taq pol I Kong H1993 Full length or truncated Full length
Taq pol I Kong H1993 Processivity 42bp
Taq pol I Kong H1993 KM 16uM Reaction: Nucleotide incorporation; Substrate: dNTPs; Technique: Filter binding ; Experimental conditions: Temp (72°C)

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