Cleavage of symmetric immobile DNA junctions by Escherichia coli RuvC

The Holliday junction is a key DNA intermediate in the process of genetic r ecombination. It consists of two double-helical domains composed of homolog ous strands that flank a branch point; two of the strands are roughly helic al, and two form the crossover between the helices. RuvC is a Holliday junc tion resolvase that cleaves the helical strands at a symmetric sequence, le ading to the production of two recombinant molecules. We have determined th e position of the cleavage site relative to the crossover point by the use of symmetric immobile junctions; these are DNA molecules containing two cro ssover points, one held immobile by sequence asymmetry and the second a sym metric sequence, but held immobile by torsional coupling to the first junct ion. We have built five symmetric immobile junctions, in which the tetranuc leotide recognition site is moved stepwise relative to the branch point. We have used kinetic analysis of catalysis, gel retardation, and hydroxyl rad ical hypersensitivity to analyze this system. We conclude that the internuc leotide linkage one position 3' to the crossover point is the favored site of cleavage.