Mutational analysis on structure-function relationship of a holliday junction specific endonuclease RuvC.
BACKGROUND
Escherichia coli RuvC protein is a specific endonuclease that resolves Holliday junctions during homologous recombination. For junction resolution, RuvC undergoes distinct steps such as dimerization, junction-specific binding and endonucleolytic cleavage. The crystal structure of RuvC has been revealed.


RESULTS
To identify functionally important residues, we isolated a large number of mutant ruvC genes created by random mutagenesis and characterized their properties in vivo and in vitro. The mutations which were isolated most frequently were mapped to the four acidic residues constituting the catalytic centre. Amongst the several mutant proteins affected in the dimer interface, only one could not form a dimer. The others were able to form a dimer but were defective in cleavage. F69L and K118R mutant proteins could not cleave the junction, but they were able to form a dimer and bind the junction DNA.


CONCLUSIONS
Random mutagenesis highlighted many structurally and functionally important residues of RuvC, most of which are highly conserved among RuvC homologues. Dimer formation and also conservation of intact interface interactions between the subunits are important for junction binding and subsequent cleavage. Phe-69 and Lys-118 are critically important for the interactions which lead to junction cleavage.
Ichiyanagi
K
K
Department of Molecular Microbiology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka, 565-0871, Japan.
Iwasaki
H
H
Hishida
T
T
Shinagawa
H
H
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000528:radiation effects; D029968:Escherichia coli Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011995:Recombination, Genetic; D013329:Structure-Activity Relationship; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C069806:ruvC protein, E coli; D004247:DNA; D004706:Endodeoxyribonucleases