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Updated: May 12, 2026

Using Modified Synthetic Oligonucleotides to Assay Nucleic Acid-Metabolizing Enzymes
Published on: July 5, 2024
Broad nucleotide cofactor specificity of DNA ligase from the hyperthermophilic crenarchaeon Hyperthermus butylicus
Jun-Hwan Kim1, Kang-Keun Lee, Younguk Sun
1Department of Genetic Engineering, Sungkyunkwan University, 300 Cheoncheon-dong, Jangan-gu, Suwon 440-746, Korea.
Abstract:
The nucleotide cofactor specificity of the DNA ligase from the hyperthermophilic crenarchaeon Hyperthermus butylicus (Hbu) was studied to investigate the evolutionary relationship of DNA ligases. The Hbu DNA ligase gene was expressed under control of the T7lac promoter of pTARG in Escherichia coli BL21-CodonPlus(DE3)-RIL. The expressed enzyme was purified using the IMPACT™-CN system (intein-mediated purification with an affinity chitin-binding tag) and cation-ion (Arg-tag) chromatography. The optimal temperature for Hbu DNA ligase activity was 75 °C, and the optimal pH was 8.0 in Tris-HCl. The activity was highly dependent on MgCl2 or MnCl2 with maximal activity above 5 mM MgCl2 and 2 mM MnCl2. Notably, Hbu DNA ligase can use ADP and GTP in addition to ATP. The broad nucleotide cofactor specificity of Hbu DNA ligase might exemplify an undifferentiated ancestral stage in the evolution of DNA ligases. This study provides new evidence for possible evolutionary relationships among DNA ligases.
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