Evidence that phenylalanine 69 in Escherichia coli RuvC resolvase forms a stacking interaction during binding and destabilization of a Holliday junction DNA substrate.
Escherichia coli RuvC resolvase is a specific endonuclease that recognizes and cleaves Holliday junctions formed during homologous recombination and recombinational repair. This study examines the phenotype of RuvC mutants with amino acid substitutions at phenylalanine 69 (F69L, F69Y, F69W, and F69A), a catalytically important residue that faces the catalytic center of the enzyme. F69Y, but not the other three mutants, almost fully complements the UV sensitivity of a DeltaruvC strain and substantially resolves synthetic Holliday junctions in vitro. In the presence of 100 mm NaCl, RuvC F69A and F69L are defective in junction binding, but F69Y and F69W retain near wild-type binding activity during a gel shift binding assay. KMnO(4) was used to probe synthetic Holliday junction DNA in a complex with wild-type and mutant RuvC; F69A and F69L did not induce disruption of base pairing at the crossover to the same extent as wild-type RuvC. Thus, the aromatic ring of Phe-69 is involved in DNA binding, probably via a stacking interaction with a nucleotide base, and this interaction may induce a structural change in junction DNA that is required to form a catalytically competent complex.
Yoshikawa
M
M
Department of Molecular Microbiology, Research Institute for Microbial Diseases, Osaka University, and PRESTO, Japan Science and Technology Corporation, Suita, Osaka, 565-0871, Japan.
Iwasaki
H
H
Shinagawa
H
H
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001483:Base Sequence; D002384:Catalysis; D004252:DNA Mutational Analysis; D004260:DNA Repair; D004305:Dose-Response Relationship, Drug; D004307:Dose-Response Relationship, Radiation; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D008027:Light; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010641:Phenotype; D010649:Phenylalanine / Q000737:chemistry* / Q000502:physiology*; D011196:Potassium Permanganate / Q000494:pharmacology; D011485:Protein Binding; D011995:Recombination, Genetic; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C069806:ruvC protein, E coli; D011196:Potassium Permanganate; D010649:Phenylalanine; D004706:Endodeoxyribonucleases