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

Maiko Tanabe, Sonoko Ishino, Masafumi Yohda, Kosuke Morikawa, Yoshizumi Ishino, Hirokazu Nishida

研究成果: ジャーナルへの寄稿学術誌査読

13 被引用数 (Scopus)

抄録

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.

本文言語英語
ページ(範囲)2575-2582
ページ数8
ジャーナルChemBioChem
13
17
DOI
出版ステータス出版済み - 11月 2012

!!!All Science Journal Classification (ASJC) codes

  • 生化学
  • 分子医療
  • 分子生物学
  • 有機化学

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