Genetic recombination in E. coli: RuvC protein cleaves Holliday junctions at resolution hotspots in vitro.
The E. coli RuvC protein resolves Holliday junctions during genetic recombination and postreplication repair. Using recombination intermediates made by RecA protein, we have identified specific "hotspots" for RuvC resolution. Characterization of these sites reveals a common tetranucleotide sequence, with the consensus 5'-A/TTT decreases G/C-3'. The correct orientation of the resolution site is required for cleavage. These observations suggest that the strand bias of this sequence will affect the outcome of recombinational crosses by directing resolution to either "patch" or "splice" recombinant products. Mutation of the consensus site in synthetic Holliday junctions abolishes or significantly reduces the efficiency of cleavage, although binding is unaffected, demonstrating that junction recognition and incision are biochemically separable events. We propose that efficient RuvC resolution requires the translocation of Holliday junctions to specific cleavage sites, thus providing a biochemical basis for the similar genetic defects observed in ruvA, ruvB, and ruvC mutants.
Shah
R
R
Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire, England.
Bennett
R J
RJ
West
S C
SC
D001426:Bacterial Proteins / Q000187:drug effects / Q000378:metabolism*; D001483:Base Sequence; D016384:Consensus Sequence; D004269:DNA, Bacterial / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000187:drug effects / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins; D008345:Manganese / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D011995:Recombination, Genetic; D013379:Substrate Specificity
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C069806:ruvC protein, E coli; D008345:Manganese; D004706:Endodeoxyribonucleases