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.

H Kong,R.B Kucera,W.E Jack

doi:10.1016/s0021-9258(18)53949-1

H Kong, R.B Kucera + Show 1 more

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https://doi.org/10.1016/s0021-9258(18)53949-1

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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.

Highlights

We have isolated, cloned, and characterized a DNA ardson, 1987)
The enzyme is extremely thermostable, having a half-life of 8 h at 95 “C and about 2 h at 100 “C. Pseudo-first-order kinetics at 70 “C reveal an extremelylow K, for a primed M13mp18 substrate (0.1 nM), coupled with a relatively high K, for dNTPs (50 MM)A. ccompanying extension rates are on the order of 1000 nucleotides/ min
A 197-kDa protein was encoded by the polymerase open reading frame,over twice as large as the active peptiodfe93 kDa observed by SDS-PAGE.’

Summary

Hydroxylapatite

Incubation of units of Fraction VI1 at 37 or 75 "C for 4.5 h with 1 Fg of 0x174 RFI DNA in Tli DNA polymerase assay buffer containing 0.4 mM dNTPs resulted in no detectable linearization or nicking of the substrate. The resulting Uninhibited reactions contained Tli DNA polymerase buffer and substrate had a specific activity of 9 X lo4cpm 3H and 8 X lo4cpm 0.2 mM dATP, dGTP, dCTP0, .08 mM [3H]TTP (0.25 Ci/mmol), 0.15. Polymerase reaction buffer containing 0.12 p M M13mp singlestranded template and 1.2 p M M13 24-mer sequencing primer(1224, Table I) was heated at 93 "C for 3 min and annealed at25 "C for 20 in these experiments was [3H]TTP, and incorporation into the inhibitor was not monitored.

RESULTS

DISCUSSION

Methods