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

We constructed nine deletion mutants of NAD(+)-dependent DNA ligase from Aq uifex pyrophilus to characterize the functional domains. All of DNA ligase deletion mutants were analyzed in biochemical assays for NAD(+)-dependent s elf-adenylation, DNA binding, and nick-closing activity. Although the mutan t lsub1 (91-362) included the active site lysine (KxDG), self-adenylation w as 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 lsub 5 (91-719), which has the C-terminal domain (487-719) as to lsub4 (91-486), showed minimal adenylation activity. These results suggest that the presen ce of N-terminal 90 residues is essential for the formation of an enzyme-AM P 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 (487-7 19) 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 bind ing activity. Thus, we concluded that the N-terminal 90 residues and C-term inal domain (487-719) of NAD(+)-dependent DNA ligase from A. pyrophilus are mutually indispensable for binding of DNA substrate. (C) 2001 Academic Pre ss.