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Structure-Based Mutational Study of an Archaeal DNA Ligase towards Improvement of Ligation Activity

Authors

  • Maiko Tanabe,

    1. Central Research Laboratory, Hitachi Ltd. 1-280 Higashi-koigakubo, Kokubunji, 185-8601, Tokyo (Japan)
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  • Dr. Sonoko Ishino,

    1. Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka-shi, 812-8581, Fukuoka (Japan)
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  • Prof. Masafumi Yohda,

    1. Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, 184-8588, Tokyo (Japan)
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  • Prof. Kosuke Morikawa,

    1. International Institute for Advanced Studies and Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, Yoshida Ushinomiya-cho, Sakyo-ku, 606-8501, Kyoto (Japan)
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  • Prof. Yoshizumi Ishino,

    Corresponding author
    1. Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka-shi, 812-8581, Fukuoka (Japan)
    • Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka-shi, 812-8581, Fukuoka (Japan)
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  • Dr. Hirokazu Nishida

    Corresponding author
    1. Central Research Laboratory, Hitachi Ltd. 1-280 Higashi-koigakubo, Kokubunji, 185-8601, Tokyo (Japan)
    • Central Research Laboratory, Hitachi Ltd. 1-280 Higashi-koigakubo, Kokubunji, 185-8601, Tokyo (Japan)
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Abstract

DNA ligases catalyze the joining of strand breaks in duplex DNA. The DNA ligase of Pyrococcus furiosus (PfuLig), which architecturally resembles the human DNA ligase I (hLigI), comprises an N-terminal DNA-binding domain, a middle adenylylation domain, and a C-terminal oligonucleotide-binding (OB)-fold domain. Here we addressed the C-terminal helix in the OB-fold domain of PfuLig by mutational analysis. The crystal structure of PfuLig revealed that this helix stabilizes a closed conformation of the enzyme by forming several ionic interactions with the adenylylation domain. The C-terminal helix is oriented differently in hLigI when DNA is bound; this suggested that disruption of its interaction with the adenylylation domain might facilitate the binding of DNA substrates. We indeed identified one of its residues, Asp540, as being critical for ligation efficiency. The D540R mutation improved the overall ligation activity relative to the wild-type enzyme, and at lower temperatures; this is relevant to applications such as ligation amplification reactions. Physical and biochemical analyses indicated that the improved ligation activity of the D540R variant arises from effects on the ligase adenylylation step and on substrate DNA binding in particular.

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