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 language | English |
|---|---|
| Pages (from-to) | 253-260 |
| Number of pages | 8 |
| Journal | Archives of Biochemistry and Biophysics |
| Volume | 388 |
| Issue number | 2 |
| DOIs | |
| Publication status | Published - 2001 Apr 15 |
Fingerprint
Keywords
- Aquifex pyrophilus
- Deletion mutant
- DNA binding activity
- NAD-dependent DNA ligase
- Nick-closing activity
- Self-adenylation
ASJC Scopus subject areas
- Biochemistry
- Biophysics
- Molecular Biology
Cite this
Mutational analyses of Aquifex pyrophilus DNA ligase define essential domains for self-adenylation and DNA binding activity. / Lim, J. H.; Choi, J.; Kim, W.; Ahn, B. Y.; Ahn, Byung-Yoon.
In: Archives of Biochemistry and Biophysics, Vol. 388, No. 2, 15.04.2001, p. 253-260.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Mutational analyses of Aquifex pyrophilus DNA ligase define essential domains for self-adenylation and DNA binding activity
AU - Lim, J. H.
AU - Choi, J.
AU - Kim, W.
AU - Ahn, B. Y.
AU - Ahn, Byung-Yoon
PY - 2001/4/15
Y1 - 2001/4/15
N2 - 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.
AB - 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.
KW - Aquifex pyrophilus
KW - Deletion mutant
KW - DNA binding activity
KW - NAD-dependent DNA ligase
KW - Nick-closing activity
KW - Self-adenylation
UR - http://www.scopus.com/inward/record.url?scp=0035870144&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0035870144&partnerID=8YFLogxK
U2 - 10.1006/abbi.2001.2291
DO - 10.1006/abbi.2001.2291
M3 - Article
C2 - 11368162
AN - SCOPUS:0035870144
VL - 388
SP - 253
EP - 260
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
SN - 0003-9861
IS - 2
ER -