Mutational analysis of the Pyrococcus furiosus holliday junction resolvase hjc revealed functionally important residues for dimer formation, junction DNA binding, and cleavage activities.
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.
Komori
K
K
Departments of Molecular Biology, Bioinformatics, and Structural Biology, Biomolecular Engineering Research Institute, Suita, Osaka, Japan.
Sakae
S
S
Daiyasu
H
H
Toh
H
H
Morikawa
K
K
Shinagawa
H
H
Ishino
Y
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D045565:Holliday Junction Resolvases; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D020027:Pyrococcus furiosus / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D004269:DNA, Bacterial; D004706:Endodeoxyribonucleases; D045565:Holliday Junction Resolvases