Advertisement

Biotechnology Letters

, Volume 36, Issue 5, pp 985–992 | Cite as

Enhancing the processivity of a family B-type DNA polymerase of Thermococcus onnurineus and application to long PCR

  • Yun Jae Kim
  • Hyun Sook Lee
  • Suk-Tae Kwon
  • Jung-Hyun Lee
  • Sung Gyun KangEmail author
Original Research Paper

Abstract

Mechanisms that allow replicative DNA polymerases to attain high processivity are often specific to a given polymerase and cannot be generalised to others. Amplification efficiency is lower in family B-type DNA polymerases than in family A-type (Taq) polymerases because of their strong 3′–5′ exonuclease-activity. Here, we have red the exonuclease domain of the Thermococcus onnurineus NA1 (TNA1) DNA polymerase, especially Asn210 to Asp215 residues in Exo II motif (NXXXFD), to improve the processivity. N213D mutant protein had higher processivity and extension rate than the wild-type TNA1 DNA polymerase, retaining a lower mutation frequency than recombinant Taq DNA polymerase. Consequently, the N213D mutant could amplify target DNA up to 13.5 kb in length from human genomic DNA and 16.2 kb in length from human mitochondrial DNA while wild-type TNA1 amplified target DNA of 2.7 kb in length from human genomic DNA.

Keywords

DNA polymerase Error rate 3′–5′ Exonuclease domain Processivity Thermococcus DNA polymerase TNA1 DNA polymerase 

Notes

Acknowledgments

This work was supported by the KIOST in-house Program (PE98983, PE98993), the Marine and Extreme Genome Research Center Program and the Development of Biohydrogen Production Technology using Hyperthermophilic Archaea Program of the Ministry of Ocean and Fisheries, Republic of Korea.

Supplementary material

10529_2013_1441_MOESM1_ESM.docx (22 kb)
Supplementary material 1 (DOCX 21 kb)

References

  1. Bae H, Kim KP, Song JM, Kim JH, Yang JS, Kwon ST (2009) Characterization of intein homing endonuclease encoded in the DNA polymerase gene of Thermococcus marinus. FEMS Microbiol Lett 297:180–188PubMedCrossRefGoogle Scholar
  2. Barnes WM (1994) PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates. Proc Natl Acad Sci USA 91:2216–2220PubMedCentralPubMedCrossRefGoogle Scholar
  3. Beese LS, Steitz TA (1991) Structural basis for the 3′–5′ exonuclease activity of Escherichia coli DNA polymerase I: a two metal ion mechanism. EMBO J 10:25–33PubMedCentralPubMedGoogle Scholar
  4. Bernad A, Blanco L, Lazaro JM, Martin G, Salas M (1989) A conserved 3′–5′ exonuclease active site in prokaryotic and eukaryotic DNA polymerases. Cell 59:219–228PubMedCrossRefGoogle Scholar
  5. Cho Y, Lee HS, Kim YJ, Kang SG, Kim SJ, Lee JH (2007) Characterization of a dUTPase from the hyperthermophilic archaeon Thermococcus onnurineus NA1 and its application in polymerase chain reaction amplification. Mar Biotechnol 9:450–458PubMedCrossRefGoogle Scholar
  6. Cho SS, Kim KP, Lee KK, Youn MH, Kwon ST (2012) Characterization and PCR application of a new high-fidelity DNA polymerase from Thermococcus waiotapuensis. Enzyme Microbiol Technol 51:334–341CrossRefGoogle Scholar
  7. Chung JH, Back JH, Park YI, Han YS (2001) Biochemical characterization of a novel hypoxanthine/xanthine dNTP pyrophosphatase from Methanococcus jannaschii. Nucleic Acid Res 29:3099–3107PubMedCentralPubMedCrossRefGoogle Scholar
  8. Davidson JF, Fox R, Harris DD, Lyons-Abbott S, Loeb LA (2003) Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase. Nucleic Acid Res 31:4702–4709PubMedCentralPubMedCrossRefGoogle Scholar
  9. Gueguen Y, Rolland JL, Lecompte O, Azam P, Le Romancer G, Flament D, Raffin JP, Dietrich J (2001) Characterization of two DNA polymerases from the hyperthermophilic euryarchaeon Pyrococcus abyssi. Eur J Biochem 268:5961–5969PubMedCrossRefGoogle Scholar
  10. Hogrefe HH, Hansen CJ, Scott BR, Nielson KB (2002) Archaeal dUTPase enhances PCR amplifications with archaeal DNA polymerases by preventing dUTP incorporation. Proc Natl Acad Sci USA 99:596–601PubMedCentralPubMedCrossRefGoogle Scholar
  11. Kim YJ, Lee HS, Bae SS, Jeon JH, Lim JK, Cho Y, Nam KH, Kang SG, Kim SJ, Kwon ST, Lee JH (2007) Cloning, purification, and characterization of a new DNA polymerase from a hyperthermophilic archaeon, Thermococcus sp. NA1. J Microbiol Biotechnol 17:1090–1097PubMedGoogle Scholar
  12. Kim KP, Bae H, Kim IH, Kwon ST (2011a) Cloning, expression, and PCR application of DNA polymerase from the hyperthermophilic archaeon, Thermococcus celer. Biotechnol Lett 33:339–346PubMedCrossRefGoogle Scholar
  13. Kim KP, Cho SS, Lee KK, Youn MH, Kwon ST (2011b) Improved thermostability and PCR efficiency of Thermococcus celericrescens DNA polymerase via site-directed mutagenesis. J Biotechnol 155:156–163PubMedCrossRefGoogle Scholar
  14. Kong H, Kucera RB, Jack WE (1993) 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. J Biol Chem 268:1965–1975PubMedGoogle Scholar
  15. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  16. Lee JI, Kim YJ, Bae H, Cho SS, Lee JH, Kwon ST (2010) Biochemical properties and PCR performance of a family B DNA polymerase from hyperthermophilic Euryarchaeon Thermococcus peptonophilus. Appl Biochem Biotechnol 160:1585–1599PubMedCrossRefGoogle Scholar
  17. Lundberg KS, Shoemaker DD, Adams MW, Short JM, Sorge JA, Mathur EJ (1991) High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus. Gene 108:1–6PubMedCrossRefGoogle Scholar
  18. Marsic D, Flaman JM, Ng JD (2008) New DNA polymerase from the hyperthermophilic marine archaeon Thermococcus thioreducens. Extremophiles 12:775–788PubMedCrossRefGoogle Scholar
  19. Mattila P, Korpela J, Tenkanen T, Pitkanen K (1991) Fidelity of DNA synthesis by the Thermococcus litoralis DNA polymerase-an extremely heat stable enzyme with proofreading activity. Nucleic Acid Res 19:4967–4973PubMedCentralPubMedCrossRefGoogle Scholar
  20. Morrison A, Bell JB, Kunkel TA, Sugino A (1991) Eukaryotic DNA polymerase amino acid sequence required for 3′–5′ exonuclease activity. Proc Natl Acad Sci USA 88:9473–9477PubMedCentralPubMedCrossRefGoogle Scholar
  21. Motz M, Kober I, Girardot C, Loeser E, Bauer U, Albers M, Moeckel G, Minch E, Voss H, Kilger C, Koegl M (2002) Elucidation of an archaeal replication protein network to generate enhanced PCR enzymes. J Biol Chem 277:16179–16188PubMedCrossRefGoogle Scholar
  22. Nishioka M, Mizuguchi H, Fujiwara S, Komatsubara S, Kitabayashi M, Uemura H, Takagi M, Imanaka T (2001) Long and accurate PCR with a mixture of KOD DNA polymerase and its exonuclease deficient mutant enzyme. J Biotechnol 88:141–149PubMedCrossRefGoogle Scholar
  23. Pavlov AR, Belova GI, Kozyavkin SA, Slesarev AI (2002) Helix-hairpin-helix motifs confer salt resistance and processivity on chimeric DNA polymerases. Proc Natl Acad Sci USA 99:13510–13515PubMedCentralPubMedCrossRefGoogle Scholar
  24. Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA (1998) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487–491CrossRefGoogle Scholar
  25. Southworth MW, Kong H, Kucera RB, Ware J, Jannasch HW, Perler FB (1996) Cloning of thermostable DNA polymerases from hyperthermophilic marine Archaea with emphasis on Thermococcus sp. 9 degrees N-7 and mutations affecting 3′–5′ exonuclease activity. Proc Natl Acad Sci USA 93:5281–5285PubMedCentralPubMedCrossRefGoogle Scholar
  26. Takagi M, Nishioka M, Kakihara H, Kitabayashi M, Inoue H, Kawakami B, Oka M, Imanaka T (1997) Characterization of DNA polymerase from Pyrococcus sp. strain KOD1 and its application to PCR. Appl Environ Microbiol 63:4504–4510PubMedCentralPubMedGoogle Scholar
  27. Wang Y, Prosen DE, Mei L, Sullivan JC, Finney M, Vander Horn PB (2004) A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro. Nucleic Acid Res 32:1197–1207PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Yun Jae Kim
    • 1
    • 2
  • Hyun Sook Lee
    • 1
    • 2
  • Suk-Tae Kwon
    • 3
  • Jung-Hyun Lee
    • 1
    • 2
  • Sung Gyun Kang
    • 1
    • 2
    Email author
  1. 1.Korea Institute of Ocean Science and TechnologyAnsanKorea
  2. 2.Department of Marine BiotechnologyUniversity of Science and TechnologyTaejonKorea
  3. 3.Department of Genetic EngineeringSungkyunkwan UniversitySuwonKorea

Personalised recommendations