An E. coli RuvC mutant defective in cleavage of synthetic Holliday junctions.
Escherichia coli RuvC protein is a specific endonuclease that resolves recombination intermediates into viable products. The structural features needed for RuvC activity were investigated by sequencing three ruvC mutations and relating the base pair changes identified to the activity of the mutant proteins. Each of the three mutations is a single base-pair substitution. ruvC51 converts glycine-15 to an aspartic acid residue. The product of ruvC51 was purified and shown to retain the ability to bind junctions, albeit with a slightly reduced affinity. However, it has lost the ability to resolve these structures by symmetrical cleavage. A multicopy ruvC51 plasmid confers sensitivity to UV light in a ruvC+ strain. The ruvC53 allele causes a glycine-17 to serine substitution while ruvC55 produces a stop codon. Neither of these genes produces a stable product. The results suggest that the N-terminal domain of RuvC may be concerned with cleavage of junctions.
Department of Genetics, University of Nottingham, Queens Medical Centre, UK.
G J
GJ
Sharples
R G
RG
Lloyd
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon; D004706:Endodeoxyribonucleases; D004926:Escherichia coli / Q000235:genetics* / Q000528:radiation effects; D029968:Escherichia coli Proteins; D005998:Glycine; D008969:Molecular Sequence Data; D009154:Mutation; D009713:Nucleotidyltransferases / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011938:Rec A Recombinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine; D013329:Structure-Activity Relationship; D019895:Transposases; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D003062:Codon; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C069806:ruvC protein, E coli; D012694:Serine; D009713:Nucleotidyltransferases; D011938:Rec A Recombinases; D019895:Transposases; D004706:Endodeoxyribonucleases; D005998:Glycine