Mutational analyses of Aquifex pyrophilus DNA ligase define essential domains for self-adenylation and DNA binding activity

Jae Hwan Lim, Juhyun Choi, Wookhyun Kim, Juhyun Choi, Byung Yoon Ahn, Ye Sun Han

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

We constructed nine deletion mutants of NAD+-dependent DNA ligase from Aquifex pyrophilus to characterize the functional domains. All of DNA ligase deletion mutants were analyzed in biochemical assays for NAD+-dependent self-adenylation, DNA binding, and nick-closing activity. Although the mutant lsub1 (91-362) included the active site lysine (KxDG), self-adenylation was not shown. However, the mutants lsub6 (1-362), lsub7 (1-516), and lsub9 (1-635) showed the same adenylation activity as that of wild type. The lsub5 (91-719), which has the C-terminal domain (487-719) as to lsub4 (91-486), showed minimal adenylation activity. These results suggest that the presence of N-terminal 90 residues is essential for the formation of an enzyme-AMP complex, while C-terminal domain (487-719) appears to play a minimal role in adenylation. It was found that the presence of C-terminal domain (487-719) is indispensable for DNA binding activity of lsub5 (91-719). The mutant lsub9 (1-635) showed reduced DNA binding activity compared to that of wild type, suggesting the contribution of the domain (636-719) for the DNA binding activity. Thus, we concluded that the N-terminal 90 residues and C-terminal domain (487-719) of NAD+-dependent DNA ligase from A. pyrophilus are mutually indispensable for binding of DNA substrate.

Original languageEnglish
Pages (from-to)253-260
Number of pages8
JournalArchives of Biochemistry and Biophysics
Volume388
Issue number2
DOIs
Publication statusPublished - 2001 Apr 15

Keywords

  • Aquifex pyrophilus
  • DNA binding activity
  • Deletion mutant
  • NAD-dependent DNA ligase
  • Nick-closing activity
  • Self-adenylation

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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