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 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.
Lim
J H
JH
Structural Biology Research Center, Korea Institute of Science and Technology, Seoul.
Choi
J
J
Kim
W
W
Ahn
B Y
BY
Han
Y S
YS
D000249:Adenosine Monophosphate / Q000096:biosynthesis / Q000737:chemistry; D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D011088:DNA Ligases / Q000235:genetics* / Q000378:metabolism; D004252:DNA Mutational Analysis; D017931:DNA Primers; D017353:Gene Deletion; D008969:Molecular Sequence Data; D009243:NAD / Q000737:chemistry; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D009243:NAD; D000249:Adenosine Monophosphate; D004247:DNA; D011088:DNA Ligases