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Nucleic Acids Res. 1991 September 25; 19(18): 4967–4973.
PMCID: PMC328798

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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Kunkel TA. The mutational specificity of DNA polymerase-beta during in vitro DNA synthesis. Production of frameshift, base substitution, and deletion mutations. J Biol Chem. 1985 May 10;260(9):5787–5796. [PubMed]
  • Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985 Dec 20;230(4732):1350–1354. [PubMed]
  • Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. [PubMed]
  • Tindall KR, Kunkel TA. Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase. Biochemistry. 1988 Aug 9;27(16):6008–6013. [PubMed]
  • Eckert KA, Kunkel TA. High fidelity DNA synthesis by the Thermus aquaticus DNA polymerase. Nucleic Acids Res. 1990 Jul 11;18(13):3739–3744. [PMC free article] [PubMed]
  • Kunkel TA, Soni A. Exonucleolytic proofreading enhances the fidelity of DNA synthesis by chick embryo DNA polymerase-gamma. J Biol Chem. 1988 Mar 25;263(9):4450–4459. [PubMed]
  • Thomas DC, Kunkel TA, Casna NJ, Ford JP, Sancar A. Activities and incision patterns of ABC excinuclease on modified DNA containing single-base mismatches and extrahelical bases. J Biol Chem. 1986 Nov 5;261(31):14496–14505. [PubMed]
  • Vo-Quang T, Malpiece Y, Buffard D, Kaminski PA, Vidal D, Strosberg AD. Rapid large-scale purification of plasmid DNA by medium or low pressure gel filtration. Application: construction of thermoamplifiable expression vectors. Biosci Rep. 1985 Feb;5(2):101–111. [PubMed]
  • Kunkel TA. Mutational specificity of depurination. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1494–1498. [PubMed]
  • Kunkel TA, Sabatino RD, Bambara RA. Exonucleolytic proofreading by calf thymus DNA polymerase delta. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4865–4869. [PubMed]
  • Kunkel TA. Frameshift mutagenesis by eucaryotic DNA polymerases in vitro. J Biol Chem. 1986 Oct 15;261(29):13581–13587. [PubMed]
  • Kunkel TA. The mutational specificity of DNA polymerases-alpha and -gamma during in vitro DNA synthesis. J Biol Chem. 1985 Oct 15;260(23):12866–12874. [PubMed]
  • Kunkel TA, Gopinathan KP, Dube DK, Snow ET, Loeb LA. Rearrangements of DNA mediated by terminal transferase. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1867–1871. [PubMed]
  • Kunkel TA, Alexander PS. The base substitution fidelity of eucaryotic DNA polymerases. Mispairing frequencies, site preferences, insertion preferences, and base substitution by dislocation. J Biol Chem. 1986 Jan 5;261(1):160–166. [PubMed]
  • Engler MJ, Lechner RL, Richardson CC. Two forms of the DNA polymerase of bacteriophage T7. J Biol Chem. 1983 Sep 25;258(18):11165–11173. [PubMed]
  • Hori K, Mark DF, Richardson CC. Deoxyribonucleic acid polymerase of bacteriophage T7. Characterization of the exonuclease activities of the gene 5 protein and the reconstituted polymerase. J Biol Chem. 1979 Nov 25;254(22):11598–11604. [PubMed]
  • Nordström B, Randahl H, Slaby I, Holmgren A. Characterization of bacteriophage T7 DNA polymerase purified to homogeneity by antithioredoxin immunoadsorbent chromatography. J Biol Chem. 1981 Mar 25;256(6):3112–3117. [PubMed]
  • Keohavong P, Thilly WG. Fidelity of DNA polymerases in DNA amplification. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9253–9257. [PubMed]
  • Goodenow M, Huet T, Saurin W, Kwok S, Sninsky J, Wain-Hobson S. HIV-1 isolates are rapidly evolving quasispecies: evidence for viral mixtures and preferred nucleotide substitutions. J Acquir Immune Defic Syndr. 1989;2(4):344–352. [PubMed]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press