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Applied Microbiology and Biotechnology

, Volume 103, Issue 9, pp 3795–3806 | Cite as

Biochemical characterization of a thermostable DNA ligase from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5

  • Haoqiang Shi
  • Yanchao Huang
  • Qi Gan
  • Mianwen Rui
  • Hongxun Chen
  • Chuandeng Tu
  • Zhihui YangEmail author
  • Philippe OgerEmail author
  • Likui ZhangEmail author
Biotechnologically relevant enzymes and proteins
  • 82 Downloads

Abstract

DNA ligases are essential enzymes for DNA replication, repair, and recombination processes by catalyzing a nick-joining reaction in double-stranded DNA. The genome of the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 encodes a putative ATP-dependent DNA ligase (Tba ligase). Herein, we characterized the biochemical properties of the recombinant Tba ligase. The enzyme displays an optimal nick-joining activity at 65–70 °C and retains its DNA ligation activity even after heated at 100 °C for 2 h, suggesting the enzyme is a thermostable DNA ligase. The enzyme joins DNA over a wide pH spectrum ranging from 5.0–10.0, and its optimal pH is 6.0–9.0. Tba ligase activity is dependent on a divalent metal ion: Mn2+, Mg2+, or Ca2+ is an optimal ion for the enzyme activity. The enzyme activity is inhibited by NaCl with high concentrations. Tba ligase is ATP-dependent and can also use UTP as a weak cofactor; however, the enzyme with high concentrations could function without an additional nucleotide cofactor. Mass spectrometric result shows that the residue K250 of Tba ligase is AMPylated, suggesting that the enzyme is bound to AMP. The substitution of K250 of Tba ligase with Ala abolishes the enzyme activity. In addition, the mismatches at the first position 3′ to the nick suppress Tba ligase activity more than those at the first position 5′ to the nick. The enzyme also discriminates more effectively mismatches at 3′ to the nick than those at 5′ to the nick in a ligation cycling reaction, suggesting that the enzyme might have potential application in single nucleotide polymorphism.

Keywords

DNA ligase Thermococcus barophilus Thermo-tolerance Nucleotide cofactor Ligation cycling reaction 

Notes

Author contributions

LZ, ZY, and PO designed experiments; HS, YL, QG, YH, MR, HC, and CT performed experiments; LZ, ZY, HS, and YH analyzed data; LZ, ZY, and PO wrote and revised the paper.

Funding information

This work was supported by the National Natural Science Foundation of China Grant (No. 41306131) to L.Z., the Academic Leader of Middle and Young People of Yangzhou University Grant to L.Z.; the practice innovation training program for college students in Yangzhou University to H.S. (No. XKYCX18_072); the National Natural Science Foundation of Jiangsu Province (No. BK20180937) to C.T.

Compliance with ethical standards

Conflict of interest

All authors declare that they have no conflict of interest.

Ethical statement

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2019_9736_MOESM1_ESM.rar (132 kb)
ESM 1 (RAR 131 kb)
253_2019_9736_MOESM2_ESM.pdf (147 kb)
ESM 2 (PDF 146 kb)

References

  1. Barany F (1991) Genetic disease detection and DNA amplification using cloned thermostable ligase. Proc Natl Acad Sci U S A 88:189–193CrossRefGoogle Scholar
  2. Chambers CR, Patrick WM (2015) Archaeal nucleic acid ligases and their potential in biotechnology. Archaea: 170571Google Scholar
  3. Chen J, Tomkinson AE, Ramos W, Mackey ZB, Danehower S, Walter CA, Schultz RA, Besterman JM, Husain I (1995) Mammalian DNA ligase III: molecular cloning, chromosomal localization, and expression in spermatocytes undergoing meiotic recombination. Mol Cell Biol 15:5412–5422CrossRefGoogle Scholar
  4. Doherty AJ, Suh SW (2000) Structural and mechanistic conservation in DNA ligases. Nucleic Acids Res 28:4051–4058CrossRefGoogle Scholar
  5. Edgell DR, Doolittle WF (1997) Archaea and the origin(s) of DNA replication proteins. Cell 89:995–998CrossRefGoogle Scholar
  6. Ellenberger T, Tomkinson AE (2008) Eukaryotic DNA ligases: structural and functional insights. Annu Rev Biochem 77:313–338CrossRefGoogle Scholar
  7. Grogan DW, Carver GT, Drake JW (2001) Genetic fidelity under harsh conditions: analysis of spontaneous mutation in the thermoacidophilic archaeon Sulfolobus acidocaldarius. Proc Natl Acad Sci U S A 98:7928–7933CrossRefGoogle Scholar
  8. Jackson BR, Noble C, Lavesa-Curto M, Bond PL, Bowater RP (2007) Characterization of an ATP-dependent DNA ligase from the acidophilic archaeon “Ferroplasma acidarmanus” Fer1. Extremophiles 11:315–327CrossRefGoogle Scholar
  9. Jacobs KL, Grogan DW (1997) Rates of spontaneous mutation in an archaeon from geothermal environments. J Bacteriol 179:3298–3303CrossRefGoogle Scholar
  10. Jeon SJ, Ishikawa K (2003) A novel ADP-dependent DNA ligase from Aeropyrum pernix K1. FEBS Lett 550:69–73CrossRefGoogle Scholar
  11. Keppetipola N, Shuman S (2005) Characterization of a thermophilic ATP-dependent DNA ligase from the euryarchaeon Pyrococcus horikoshii. J Bacteriol 187:6902–6908CrossRefGoogle Scholar
  12. Kim YJ, Lee HS, Bae SS, Jeon JH, Yang SH, Lim JK, Kang SG, Kwon ST, Lee JH (2006) Cloning, expression, and characterization of a DNA ligase from a hyperthermophilic archaeon Thermococcus sp. Biotechnol Lett 28:401–407CrossRefGoogle Scholar
  13. Kim YJ, Lee HS, Kim ES, Bae SS, Lim JK, Matsumi R, Lebedinsky AV, Sokolova TG, Kozhevnikova DA, Cha SS, Kim SJ, Kwon KK, Imanaka T, Atomi H, Bonch-Osmolovskaya EA, Lee JH, Kang SG (2010) Formate-driven growth coupled with H(2) production. Nature 467:352–355CrossRefGoogle Scholar
  14. Kim JH, Lee KK, Sun Y, Seo GJ, Cho SS, Kwon SH, Kwon ST (2013) Broad nucleotide cofactor specificity of DNA ligase from the hyperthermophilic crenarchaeon Hyperthermus butylicus and its evolutionary significance. Extremophiles 17:515–522CrossRefGoogle Scholar
  15. Lai X, Shao H, Hao F, Huang L (2002) Biochemical characterization of an ATP-dependent DNA ligase from the hyperthermophilic crenarchaeon Sulfolobus shibatae. Extremophiles 6:469–477CrossRefGoogle Scholar
  16. Lehman IR (1974) DNA ligase: structure, mechanism, and function. Science 186:790–797CrossRefGoogle Scholar
  17. Li J, Chu X, Liu Y, Jiang JH, He Z, Zhang Z, Shen G, Yu RQ (2005) A colorimetric method for point mutation detection using high-fidelity DNA ligase. Nucleic Acids Res 33:e168CrossRefGoogle Scholar
  18. Li Y, Wark AW, Lee HJ, Corn RM (2006) Single-nucleotide polymorphism genotyping by nanoparticle-enhanced surface plasmon resonance imaging measurements of surface ligation reactions. Anal Chem 78:3158–3164CrossRefGoogle Scholar
  19. Lindahl T, Barnes DE (1992) Mammalian DNA ligases. Annu Rev Biochem 61:251–281CrossRefGoogle Scholar
  20. Lindahl T, Nyberg B (1974) Heat-induced deamination of cytosine residues in deoxyribonucleic acid. Biochemistry 13:3405–3410CrossRefGoogle Scholar
  21. Marteinsson VT, Birrien JL, Reysenbach AL, Vernet M, Marie D, Gambacorta A, Messner P, Sleytr UB, Prieur D (1999) Thermococcus barophilus sp. nov., a new barophilic and hyperthermophilic archaeon isolated under high hydrostatic pressure from a deep-sea hydrothermal vent. Int J Syst Bacteriol 49:351–359CrossRefGoogle Scholar
  22. Nakatani M, Ezaki S, Atomi H, Imanaka T (2000) A DNA ligase from a hyperthermophilic archaeon with unique cofactor specificity. J Bacteriol 182:6424–6433CrossRefGoogle Scholar
  23. Nishida H, Kiyonari S, Ishino Y, Morikawa K (2006) The closed structure of an archaeal DNA ligase from Pyrococcus furiosus. J Mol Biol 360:956–967CrossRefGoogle Scholar
  24. Oger P, Sokolova TG, Kozhevnikova DA, Taranov EA, Vannier P, Lee HS, Kwon KK, Kang SG, Lee JH, Bonch-Osmolovskaya EA, Lebedinsky AV (2016) Complete genome sequence of the hyperthermophilic and piezophilic archaeon Thermococcus barophilus Ch5, capable of growth at the expense of hydrogenogenesis from carbon monoxide and formate. Genome Announc 4:e01534–e01515CrossRefGoogle Scholar
  25. Olsen GJ, Woese CR (1997) Archaeal genomics: an overview. Cell 89:991–994CrossRefGoogle Scholar
  26. Pack SP, Doi A, Choi YS, Kim HB, Makino K (2010) Accurate guanine:cytosine discrimination in T4 DNA ligase-based single nucleotide polymorphism analysis using an oxanine-containing ligation fragment. Anal Biochem 398:257–259CrossRefGoogle Scholar
  27. Poidevin L, MacNeill SA (2006) Biochemical characterisation of LigN, an NAD+-dependent DNA ligase from the halophilic euryarchaeon Haloferax volcanii that displays maximal in vitro activity at high salt concentrations. BMC Mol Biol 7:44CrossRefGoogle Scholar
  28. Qi X, Bakht S, Devos KM, Gale MD, Osbourn A (2001) L-RCA (ligation-rolling circle amplification): a general method for genotyping of single nucleotide polymorphisms (SNPs). Nucleic Acids Res 29:e116CrossRefGoogle Scholar
  29. Rolland JL, Gueguen Y, Persillon C, Masson JM, Dietrich J (2004) Characterization of a thermophilic DNA ligase from the archaeon Thermococcus fumicolans. FEMS Microbiol Lett 236:267–273CrossRefGoogle Scholar
  30. Seo MS, Kim YJ, Choi JJ, Lee MS, Kim JH, Lee JH, Kwon ST (2007) Cloning and expression of a DNA ligase from the hyperthermophilic archaeon Staphylothermus marinus and properties of the enzyme. J Biotechnol 128:519–530CrossRefGoogle Scholar
  31. Shuman S (1995) Vaccinia virus DNA ligase: specificity, fidelity, and inhibition. Biochemistry 34:16138–16147CrossRefGoogle Scholar
  32. Shuman S (2009) DNA ligases: progress and prospects. J Biol Chem 284:17365–17369CrossRefGoogle Scholar
  33. Sriskanda V, Kelman Z, Hurwitz J, Shuman S (2000) Characterization of an ATP-dependent DNA ligase from the thermophilic archaeon Methanobacterium thermoautotrophicum. Nucleic Acids Res 28:2221–2228CrossRefGoogle Scholar
  34. Sun Y, Seo MS, Kim JH, Kim YJ, Kim GA, Lee JI, Lee JH, Kwon ST (2008) Novel DNA ligase with broad nucleotide cofactor specificity from the hyperthermophilic crenarchaeon Sulfophobococcus zilligii: influence of ancestral DNA ligase on cofactor utilization. Environ Microbiol 10:3212–3224CrossRefGoogle Scholar
  35. Tanabe M, Ishino Y, Nishida H (2015) From structure-function analyses to protein engineering for practical applications of DNA ligase. Archaea: 267570Google Scholar
  36. Tomkinson AE, Vijayakumar S, Pascal JM, Ellenberger T (2006) DNA ligases: structure, reaction mechanism and function. Chem Rev 106:687–699CrossRefGoogle Scholar
  37. van Wolferen M, Ajon M, Driessen AJM, Albers SV (2013) How hyperthermophiles adapt to change their lives: DNA exchange in extreme conditions. Extremophiles 17:545–563CrossRefGoogle Scholar
  38. Wang Y, Xie J, Han Z, Liu J, Liu X (2013) Expression, purification and biochemical characterization of Methanocaldococcus jannaschii DNA ligase. Protein Expr Purif 87:79–86CrossRefGoogle Scholar
  39. Wilkinson A, Day J, Bowater R (2001) Bacterial DNA ligases. Mol Microbiol 40:1241–1248CrossRefGoogle Scholar
  40. Zakabunin AI, Kamynina TP, Khodyreva SN, Pyshnaya IA, Pyshnyi DV, Khrapov EA, Filipenko ML (2011) Gene cloning, purification, and characterization of recombinant DNA ligases of the thermophilic archaea Pyrococcus abyssi and Methanobacterium thermoautotrophicum. Mol Biol 45:229–236CrossRefGoogle Scholar
  41. Zhang L, Huang Y, Xu D, Yang L, Qian K, Chang G, Gong Y, Zhou X, Ma K (2016) Biochemical characterization of a thermostable HNH endonuclease from deep-sea thermophilic bacteriophage GVE2. Appl Microbiol Biotechnol 100:8003–8012CrossRefGoogle Scholar
  42. Zhao A, Gray FC, MacNeill SA (2006) ATP- and NAD+-dependent DNA ligases share an essential function in the halophilic archaeon Haloferax volcanii. Mol Microbiol 59:743–752CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Environmental Science and Engineering, Marine Science & Technology InstituteYangzhou UniversityYangzhouChina
  2. 2.College of Plant ProtectionAgricultural University of HebeiBaoding CityChina
  3. 3.INSA de Lyon, CNRS UMR 5240Université de LyonLyonFrance

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