INTERACTIONS BETWEEN RUVA AND RUVC AT HOLLIDAY JUNCTIONS - INHIBITIONOF JUNCTION CLEAVAGE AND FORMATION OF A RUVA-RUVC-DNA COMPLEX

The RuvAB and RuvC enzymes of Escherichia coli define a molecular path way for the resolution of Holliday intermediates in recombination and DNA repair. They bind specifically to Holliday junctions, and catalyse their branch migration and cleavage, respectively. In a RuvA(B)-junct ion complex, the Holliday structure is held in an open (square planar) configuration on the concave surface of a 4-fold symmetrical tetramer of RuvA, whereas in a RuvC-junction complex it is folded in an altern ative arrangement as part of the cleavage reaction. Genetic studies ha ve shown that the activity of RuvC in vivo depends on RuvAB, which sug gests that the two enzymes act in concert, with junction cleavage by R uvC following from branch migration by RuvAB. We have investigated how RuvC can take over a junction from RuvAB to cleave the DNA. We show t hat RuvA inhibits junction cleavage by RuvC, probably by sandwiching t he junction between two tetramers. The extent of inhibition depends on the reaction kinetics of RuvA binding relative to RuvC binding and cl eavage. The presence of RuvB and the concentration of Mg2+ both have a significant effect on cleavage in the presence of RuvA. However, a no vel protein-DNA complex can be formed when junction DNA is incubated w ith both RuvA and RuvC. Its mobility is consistent with a RuvC dimer b inding to a junction held in an open configuration on the surface of a RuvA tetramer. We suggest that this arrangement provides RuvC with th e means to scan the junction during the RuvAB-mediated branch migratio n reaction for DNA sequences that it can cleave. We further suggest th at recognition of the target may provide a trigger for dissociating Ru vA, allowing the junction to be folded and cleaved by RuvC. (C) 1996 A cademic Press Limited.