Ensuring productive resolution by the junction-resolving enzyme RuvC: large enhancement of the second-strand cleavage rate

RuvC is the principal junction-resolving enzyme of Escherichia coli, cleaving four-way DNA junctions created in homologous recombination. It binds with structural specificity to DNA junctions as a dimer, whereupon each subunit cleaves a phosphodiester bond of diametrically disposed strands. To generate a productive resolution event, these cleavages must be symmetrically located with respect to the point of strand exchange, and in the context of a branch-migrating junction, this requires near-simultaneous cleavage by the two subunits. Using a supercoil-stabilized cruciform as a substrate, we have analyzed the kinetics of strand cleavage. Coordinated bilateral cleavage is not essential in RuvC action, because a heterodimer comprising active and inactive subunits is active in unilateral cleavage. However, in operational terms, fully active RuvC appears to introduce simultaneous cleavages of two strands, because the rate of second-strand cleavage is accelerated by a factor of almost 150 relative to the first. We suggest that relief of strain following the first cleavage could lead to acceleration of subsequent cleavage, and show that DNA junctions rendered more flexible by the presence of strand breaks or bulges are subject to faster cleavage by RuvC. Cleavage of one strand of a junction generated in situ by the action of RuvC can accelerate cleavage at an intrinsically poor site by a factor of 500. Very large rate enhancement of second-strand cleavage by RuvC is likely to be essential to ensure productive resolution of a junction that is being actively branch migrated by the RuvAB machinery.