Mutational analysis of the Pyrococcus furiosus Holliday junction resolvase Hjc revealed functionally important residues for dimer formation, junction DNA binding, and cleavage activities

被引:29
作者
Komori, K
Sakae, S
Daiyasu, H
Toh, H
Morikawa, K
Shinagawa, H
Ishino, Y
机构
[1] Biomol Engn Res Inst, Dept Mol Biol, Suita, Osaka 5650874, Japan
[2] Biomol Engn Res Inst, Dept Bioinformat, Suita, Osaka 5650874, Japan
[3] Biomol Engn Res Inst, Dept Biol Struct, Suita, Osaka 5650874, Japan
[4] Osaka Univ, Microbial Dis Res Inst, Dept Mol Microbiol, Osaka 5650874, Japan
关键词
D O I
10.1074/jbc.M006294200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The Holliday junction cleavage protein, Hjc resolvase of Pyrococcus furiosus, is the first Holliday junction resolvase to be discovered in Archaea. Although the archaeal resolvase shares certain biochemical properties with other non-archaeal junction resolvases, no amino acid sequence similarity has been identified. To investigate the structure-function relationship of this new Holliday junction resolvase, we constructed a series of mutant hjc genes using site-directed mutagenesis targeted at the residues conserved among the archaeal orthologs. The products of these mutant genes were purified to homogeneity. With analysis of the activity of the mutant proteins to bind and cleave synthetic Holliday junctions, one acidic residue, Glu-9, and two basic residues, Arg-10 and Arg-25, were found to play critical roles in enzyme action. This is in addition to the three conserved residues, Asp-33, Glu-46, and Lys-48, which are also conserved in the motif found in the type II restriction endonuclease family proteins. Two aromatic residues, Phe-68 and Phe-72, are important for the formation of the homodimer probably through hydrophobic interactions. The results of these studies have provided insights into the structure-function relationships of the archaeal Holliday junction resolvase as well as the universality and diversity of the Holliday junction cleavage reaction.
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页码:40385 / 40391
页数:7
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