Escherichia coli RuvA and RuvB proteins specifically interact with Holliday junctions and promote branch migration.

Abstract

The Escherichia coli ruvA and ruvB genes are involved in DNA repair and in the late step of homologous genetic recombination. We have demonstrated previously that the RuvA-RuvB protein complex in the presence of ATP promotes reabsorption of cruciform structures extruded from a supercoiled plasmid with an inverted repeat sequence. Because the cruciform structure is topologically analogous to the Holliday structure, we have proposed that the role of the RuvA and RuvB proteins in recombination is to promote a strand exchange reaction at the Holliday junction. Here, we studied the specific interaction of the RuvA-RuvB complex with the Holliday structure using synthetic analogs prepared by annealing four oligonucleotides. The affinities of the RuvA protein for synthetic Holliday junctions are much higher (>20-fold) than for duplex DNA, and the affinities of the RuvA protein for the junctions are further enhanced (>4-fold) by the interaction with the RuvB protein. The RuvA-RuvB protein complex in the presence of ATP promotes dissociation of the synthetic Holliday junction with homology in the central core into two halves by catalyzing branch migration to the DNA ends, but it does not affect the structure of the synthetic Holliday junction without the homology. The separation of the synthetic Holliday junction is a result of the activity of the RuvA-RuvB complex that promotes strand exchange and DNA unwinding. Furthermore, RuvA and RuvB promote the strand exchange reaction at the Holliday junctions made by RecA. These results provide further evidence that the RuvA-RuvB complex recognizes the Holliday junction and promotes branch migration in homologous recombination.

The directionality of RuvAB‐mediated branch migration: in vitro studies with three‐armed junctions

“…In previous studies, RuvA and RuvB were shown to Directionality of RuvAB-mediated branch migration promote branch migration of Holliday junctions in vitro (Iwasaki et al 1992;Müller et al 1993;Parsons et al 1992;Parsons & West 1993;Tsaneva et al 1992b). The structure of the RuvAB-Holliday junction complex was analysed by electron microscopy and DNase I protection, which showed that the complex is tripartite, with RuvA sandwiched between two hexameric rings of RuvB (Hiom & West 1995;Parsons et al 1995a).…”

Crystal structure of the Holliday junction DNA in complex with a single RuvA tetramer

“…The DNA oligonucleotides, which were designed to form immobile four-way junctions, were obtained commercially (BEX, Tokyo). Each set of the four oligonucleotides was mixed at an equimolar ratio, and the immobile four-way junctions were prepared by annealing, as described (17). The RuvA tetramer and the synthetic four-way junction were combined in a 2:1 molar ratio and were dialyzed against a buffer containing 20 mM Tris⅐HCl buffer at pH 7.5, 150 mM NaCl, 5% glycerol, and 1 mM EDTA at 4°C.…”

Mutational analysis on structure–function relationship of a Holliday junction specific endonuclease RuvC

“…RuvA and RuvB form a complex that promotes branch migration (Iwasaki et al 1992;Parsons et al 1992;Tsaneva et al 1992). In this reaction, RuvA specifically binds the junction and targets RuvB to it (Iwasaki et al 1992;Parsons et al 1992). RuvB protein forms a hexameric ring (Stasiak et al 1994) and possesses an ATP-dependent helicase activity that provides the driving force for branch migration (Tsaneva et al 1993).…”