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Title
Japanese: 
English:Identification of four acidic amino acids that constitute the catalytic center of the RuvC Holliday junction resolvase 
Author
Japanese: Saito, A., Saito, A., 岩崎博史, Ariyoshi, M., Morikawa, K., Shinagawa, H..  
English: Saito, A., Saito, A., Hiroshi Iwasaki, Ariyoshi, M., Morikawa, K., Shinagawa, H..  
Language English 
Journal/Book name
Japanese:Proceedings of the National Academy of Sciences of the United States of America 
English:Proceedings of the National Academy of Sciences of the United States of America 
Volume, Number, Page Vol. 92    No. 16    pp. 7470-7474
Published date Aug. 1995 
Publisher
Japanese: 
English: 
Conference name
Japanese: 
English: 
Conference site
Japanese: 
English: 
Official URL http://www.scopus.com/inward/record.url?eid=2-s2.0-0029142820&partnerID=MN8TOARS
 
DOI https://doi.org/10.1073/pnas.92.16.7470
Abstract 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.

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