Mutational analyses of the thermostable NAD+-dependent DNA ligase from Thermus filiformis

被引:21
作者
Jeon, HJ
Shin, HJ
Choi, JJ
Hoe, HS
Kim, HK
Suh, SW
Kwon, ST [1 ]
机构
[1] Sungkyunkwan Univ, Dept Genet Engn, Suwon 440746, South Korea
[2] Seoul Natl Univ, Dept Chem, Seoul 151742, South Korea
[3] Super Bio Co Ltd, Genet Resource R&D Inst, Suwon 440746, South Korea
关键词
Thermus filiformis (Tfi) DNA ligase; self-adenylation; DNA binding activity; nick-closing activity;
D O I
10.1016/j.femsle.2004.06.018
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
The crystal structure of NAD(+)-dependent DNA ligase from Thermus filiformis (Tfi) revealed that the protein comprised four structural domains. In order to investigate the biochemical activities of these domains, seven deletion mutants were constructed from the Tfi DNA ligase. The mutants Tfi-M1 (residues 1-581), Tfi-M2 (residues 1-448), Tfi-M3 (residues 1-403) and Tfi-M4 (residues 1-314) showed the same adenylation activity as that of wild-type. This result indicates that only the adenylation domain (domain 1) is essential for the formation of enzyme-AMP complex. It was found that the zinc finger and helix-hairpin-helix (HhH) motif domain (domain 3) and the oligomer binding (OB)-fold domain (domain 2) are important for the formation of enzyme-DNA complex. The mutant Tfi-M1 alone showed the activities for in vitro nick-closing and in vivo complementation in Escherichia coli as those of wildtype. These results indicate that the BRCT domain (domain 4) of Tfi DNA ligase is not essential for the enzyme activity. The enzymatic properties of Tfi-M1 mutant (deleted the BRCT domain) were slightly different from those of wild-type and the nick-closing activity of Tfi-M1 mutant was approximately 50% compared with that of wild-type. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
引用
收藏
页码:111 / 118
页数:8
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