GENETIC-RECOMBINATION IN ESCHERICHIA-COLI - RUVC PROTEIN CLEAVES HOLLIDAY JUNCTIONS AT RESOLUTION HOTSPOTS IN-VITRO

The E. coli RuvC protein resolves Holliday junctions during genetic re combination and postreplication repair. Using recombination intermedia tes made by RecA protein, we have identified specific ''hotspots'' for RuvC resolution. Characterization of these sites reveals a common tet ranucleotide sequence, with the consensus 5'-(A)/TTT down arrow(G)/(c) -3'. The correct orientation of the resolution site is required for cl eavage. These observations suggest that the strand bias of this sequen ce will affect the outcome of recombinational crosses by directing res olution to either ''patch'' or ''splice'' recombinant products. Mutati on 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 b iochemically separable events. We propose that efficient RuvC resoluti on requires the translocation of Holliday junctions to specific cleava ge sites, thus providing a biochemical basis for the similar genetic d efects observed in ruvA, ruvB, and ruvC mutants.