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Characterization of a dITPase from the hyperthermophilic archaeon Thermococcus onnurineus NA1 and its application in PCR amplification


In this study, we found that deoxyinosine triphosphate (dITP) could inhibit polymerase chain reaction (PCR) amplification of various family B-type DNA polymerases, and 0.93% dITP was spontaneously generated from deoxyadenosine triphosphate during PCR amplification. Thus, it was hypothesized that the generated dITP might have negative effect on PCR amplification of family B-type DNA polymerases. To overcome the inhibitory effect of dITP during PCR amplification, a dITP pyrophosphatase (dITPase) from Thermococcus onnurineus NA1 was applied to PCR amplification. Genomic analysis of the hyperthermophilic archaeon T. onnurineus NA1 revealed the presence of a 555-bp open reading frame with 48% similarity to HAM1-like dITPase from Methanocaldococcus jannaschii DSM2661 (NP_247195). The dITPase-encoding gene was cloned and expressed in Escherichia coli. The purified protein hydrolyzed dITP, not deoxyuridine triphosphate. Addition of the purified protein to PCR reactions using DNA polymerases from T. onnurineus NA1 and Pyrococcus furiosus significantly increased product yield, overcoming the inhibitory effect of dITP. This study shows the first representation that removing dITP using a dITPase enhances the PCR amplification yield of family B-type DNA polymerase.

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  1. Bae SS, Kim YJ, Yang SH, Lim JK, Jeon JH, Lee HS, Kang SG, Kim S-J, Lee J-H (2006) Thermococcus onnurineus sp. nov., a hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent area at the PACMANUS field. J Microbiol Biotechnol 16:1826–1831

  2. Barnes WM (1994) PCR amplification of up to 35-kb DNA with high fidelity and high yield from l bacteriophage templates. Proc Natl Acad Sci U S A 91:2216–2220

  3. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254

  4. 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 9:450–458

  5. Chung JH, Back JH, Park YI, Han YS (2001) Biochemical characterization of a novel hypoxanthine/xanthine dNTP pyrophosphatase from Methanocaldococcus jannaschii. Nucleic Acids Res 29:3099–3107

  6. Dabrowski S, Ahring BK (2003) Cloning, expression, and purification of the His6-tagged hyper-thermostable dUTPase from Pyrococcus woesei in Escherichia coli: application in PCR. Protein Expr Purif 31:72–78

  7. Fogg MJ, Pearl LH, Connolly BA (2002) Structural basis for uracil recognition by archaeal family B DNA polymerases. Nature Struct Biol 9:922–927

  8. Friedberg EC, Walker GC, Siede W (1995) DNA repair and mutagenesis. ASM, Washington, DC

  9. Gill S, O’Neill R, Lewis RJ, Connolly BA (2007) Interaction of the family-B DNA polymerase from the archaeon Pyrococcus furiosus with deaminated bases. J Mol Biol 372:855–863

  10. Greagg MA, Fogg MJ, Panayootu G, Evans SJ, Connolly BA, Pearl LH (1999) A read-ahead function in archaeal DNA polymerases detects pro-mutagenic template-strand uracil. Proc Natl Acad Sci U S A 96:9045–9050

  11. Grogan DW (1998) Hyperthermophiles and the problems of DNA instability. Mol Microbiol 28:1043–1049

  12. Grogan DW (2000) The question of DNA repair in hyperthermophilic archaea. Trends Microbiol 8:180–185

  13. Gruz P, Shimizu M, Pisani FM, De Felice M, Kanke Y, Nohmi T (2003) Processing of DNA lesions by archaeal DNA polymerases from Sulfolobus solfataricus. Nucleic Acids Res 31:4024–4030

  14. Hill-Perkins M, Jones MD, Karran P (1986) Site-specific mutagenesis in vivo by single methylated or deaminated purine bases. Mutat Res 162:153–163

  15. Hogrefe HH, Hansen CJ, Scott BR, Nelson KB (2002) Archaeal dUTPase enhances PCR amplifications with archaeal DNA polymerases by preventing dUTP incorporation. Proc Natl Acad Sci U S A 99:596–601

  16. Holden JF, Takai K, Summit M, Bolton S, Zyskowski J, Baross JA (2001) Diversity among three novel groups of hyperthermophilic deep-sea Thermococcus species from three sites in the northeastern Pacific Ocean. FEMS Microbiol Ecol 36:51–60

  17. Ito J, Braithwaite DK (1991) Compilation and alignment of DNA polymerases. Nucleic Acids Res 19:4045–4057

  18. Karran P, Lindahl T (1980) Hypoxanthine in deoxyribonucleic acid: generation by heat-induced hydrolysis of adenine residues and release in free form by a deoxyribonucleic acid glycosylase from calf thymus. Biochemistry 19:6005–6011

  19. 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 17:1090–1097

  20. 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 268:1965–1975

  21. Kow YW (2002) Repair of deaminated bases in DNA. Free Radic Biol Med 33:886–893

  22. Lasken RS, Schuster DM, Rashtchian A (1996) Archaebacterial DNA polymerases tightly bind uracil-containing DNA. J Biol Chem 271:17692–17696

  23. Lindahl T, Nyberg B (1974) Heat-induced deamination of cytosine residues in deoxyribonucleic acid. Biochemistry 13:3405–3410

  24. Lindahl T (1979) DNA glycosylases, endonucleases for apurinic/apyrimidinic sites and base excision-repair. Prog Nucleic Acid Res Mol Biol 22:135–192

  25. Lindahl T (1993) Instability and decay of the primary structure of DNA. Nature 362:709–715

  26. 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 108:1–6

  27. 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 Acids Res 19:4967–4973

  28. Perler FB, Kumar S, Kong H (1996) Thermostable DNA polymerases. Adv Protein Chem 48:377–435

  29. Robb FT, Place AR, Sowers KR, Schreier HJ, DasSarma S, Fleischmann EM (1995) Archaea: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, pp 3–29

  30. Saiki RK, Gelfand DH, Stoffel S, Higuchi R, Horn G, Mullis KB, Erlich HA (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487–491

  31. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor

  32. Sandigursky M, Franklin WA (2000) Uracil-DNA glycosylase in the extreme thermophile Archaeoglobus fulgidus. J Biol Chem 275:19146–19149

  33. Sartori AA, Schar P, Fitz-Gibbon S, Miller JE, Jiriciny J (2001) Biochemical characterization of uracil processing activities in the hyperthermophilic archaeon Pyrobaculum aerophilum. J Biol Chem 276:29979–29986

  34. Shapiro R, Pohl SH (1968) The reaction of ribonucleotides with nitrous acid. Side products and kinetics. Biochemistry 7:448–455

  35. Shuttleworth G, Fogg MJ, Kurpiewski MR, Jen-Jacobson L, Connolly BA (2004) Recognition of the pro-mutagenic base uracil by family B DNA polymerases from archaea. J Mol Biol 337:621–634

  36. 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 U S A 93:5281–5285

  37. 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 63:4504–4510

  38. Yang H, Fitz-Gibbon S, Marcotte EM, Tai JH, Hyman EC, Miller JH (1999) Characterization of a thermostable DNA glycosylase specific for U/G and T/G mismatches from the hyperthermophilic archaeon Pyrobaculum aerophilum. J Bacteriol 182:1272–1279

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This work was supported by KORDI in-house program (PE98210) and the Marine and Extreme Genome Research Center program of Ministry of Maritime Affairs and Fisheries, Republic of Korea.

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Correspondence to Sung Gyun Kang.

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Yun-Jae Kim and Yong-Gu Ryu contributed equally to this work.

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Kim, Y., Ryu, Y., Lee, H.S. et al. Characterization of a dITPase from the hyperthermophilic archaeon Thermococcus onnurineus NA1 and its application in PCR amplification. Appl Microbiol Biotechnol 79, 571 (2008). https://doi.org/10.1007/s00253-008-1467-5

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  • dITPase
  • dITP generation
  • Family B-type DNA polymerase
  • Hypoxanthine
  • Thermococcus