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


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.


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


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)


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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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