Identification of Four Acidic Amino Acids that Constitute the Catalytic Center of the RuvC Holliday Junction Resolvase

• Published in: Proceedings of the National Academy of Sciences - PNAS 1995-08, Vol.92 (16), p.7470-7474
• Main Authors: Saito, Atsushi, Iwasaki, Hiroshi, Ariyoshi, Mariko, Morikawa, Kosuke, Shinagawa, Hideo
• Format: Article
• Language: English
• Subjects: Acetates; Amino acids; Aspartic Acid - chemistry; Aspartic Acid - genetics; Bacteria; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Base Sequence; Binding Sites - genetics; Biochemistry; Catalysis; Cruciform DNA; Dimers; Divalent cations; DNA; DNA, Bacterial - genetics; Endodeoxyribonucleases - chemistry; Endodeoxyribonucleases - genetics; Endodeoxyribonucleases - metabolism; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli Proteins; Gels; Genes; Genetic Complementation Test; Glutamic Acid - chemistry; Glutamic Acid - genetics; Magnesium - pharmacology; Manganese - pharmacology; Models, Molecular; Molecular Sequence Data; Molecules; Mutagenesis, Site-Directed; Mutant proteins; Mutation; Phenotype; Plasmids; Protein Conformation; Recombination, Genetic; Ribonuclease H - chemistry
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.92.16.7470

Escherichia coli RuvC protein is a specific endonuclease that resolves Holliday junctions during homologous recombination. Since the endonucleolytic activity of RuvC requires a divalent cation and since 3 or 4 acidic residues constitute the catalytic centers of several nucleases that require a divalent cation for the catalytic activity, we examined whether any of the acidic residues of RuvC were required for the nucleolytic activity. By site-directed mutagenesis, we constructed a series of ruvC mutant genes with similar amino acid replacements in 1 of the 13 acidic residues. Among them, the mutant genes with an alteration at Asp-7, Glu-66, Asp-138, or Asp-141 could not complement UV sensitivity of a ruvC deletion strain, and the multicopy mutant genes showed a dominant negative phenotype when introduced into a wild-type strain. The products of these mutant genes were purified and their biochemical properties were studied. All of them retained the ability to form a dimer and to bind specifically to a synthetic Holliday junction. However, they showed no, or extremely reduced, endonuclease activity specific for the junction. These 4 acidic residues, which are dispersed in the primary sequence, are located in close proximity at the bottom of the putative DNA binding cleft in the three-dimensional structure. From these results, we propose that these 4 acidic residues constitute the catalytic center for the Holliday junction resolvase and that some of them play a role in coordinating a divalent metal ion in the active center.