Using recombination intermediates made by RecA protein, we have shown that the Escherichia coli RuvB protein can mediate the branch migration of Holliday junctions in vitro. The reaction is dependent on the presence of > or = 10 mM Mg2+ and stoichiometric amounts of RuvB. The presence of E. coli RuvA protein reduces the requirement for Mg2+ and also the stoichiometric requirement for RuvB (Müller, B., Tsaneva, I. R., and West, S. C. (1993) J. Biol. Chem. 268, 17179-17184). To determine the roles of the two proteins during branch migration, we have investigated the interaction of RuvB with DNA in the absence or presence of RuvA, by (i) gel retardation of protein-DNA complexes, (ii) stimulation of the RuvB ATPase, and (iii) protection of DNA from DNase I. The interaction of RuvB with duplex DNA was Mg(2+)-dependent and correlated with the Mg2+ requirement of the RuvB-mediated branch migration reaction. RuvB also interacted with ssDNA, but the affinity was significantly lower than for duplex DNA. In contrast to RuvB, the interaction of RuvA with duplex DNA occurred in the absence of Mg2+ and was inhibited by Mg2+ in a concentration-dependent manner. At 5 mM Mg2+, RuvA protein facilitated the interaction of RuvB with DNA, leading to the formation of a complex containing RuvA, RuvB, and duplex DNA.