Mutational Analyses of Aquifex pyrophilus DNA Ligase Define Essential Domains for Self-Adenylation and DNA Binding Activity

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.