Identification of four acidic amino acids that constitute the catalytic center of the RuvC Holliday junction resolvase.
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.
Saito
A
A
Department of Molecular Microbiology, Osaka University, Japan.
Iwasaki
H
H
Ariyoshi
M
M
Morikawa
K
K
Shinagawa
H
H
D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D004269:DNA, Bacterial / Q000235:genetics; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D029968:Escherichia coli Proteins; D005816:Genetic Complementation Test; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D011487:Protein Conformation; D011995:Recombination, Genetic; D016914:Ribonuclease H / Q000737:chemistry
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C069806:ruvC protein, E coli; D001224:Aspartic Acid; D018698:Glutamic Acid; D008345:Manganese; D004706:Endodeoxyribonucleases; D016914:Ribonuclease H; D008274:Magnesium