UNWINDING OF CLOSED CIRCULAR DNA BY THE ESCHERICHIA-COLI RUVA AND RUVB RECOMBINATION REPAIR PROTEINS

The RuvA and RuvB proteins of Escherichia coli promote the branch migration of Holliday junctions during genetic recombination and the recombinational repair of damaged DNA. Using a topological assay that measures the underwinding of covalently closed duplex DNA, we find that RuvA and RuvB promote the transient unwinding of relaxed or supercoiled DNA. Detection of unwinding by RuvAB requires the presence of Am and a non-hydrolysable ATP analogue (ATP gamma S), and was not observed in the presence of ATP or ATP gamma S alone. These results indicate that RuvAB catalyse the unwinding and rewinding of duplex DNA via an intermediate that can be stabilised by the presence a non-hydrolysable cofactor. At elevated concentrations of Mg2+ (12 to 30 mM), which are known to favour RuvB binding to DNA without the need for RuvA, RuvB protein alone promotes DNA unwinding. These results show that RuvB protein, an ATPase that forms hexameric ring structures that encircle the DNA, is directly responsible for the DNA unwinding activity exhibited by RuvAB. From these results, we propose that branch migration of Holliday junctions by RuvAB occurs by the passage of double-stranded DNA through the RuvAB complex, in a reaction coupled to transient DNA unwinding.