RuvAB-mediated branch migration does not involve extensive DNA opening within the RuvB hexamer

The Escherichia coil RuvA and RuvB proteins promote the branch migration of Holliday junctions during the late stages of homologous recombination and DNA repair (reviewed in [1]). Biochemical and structural studies of the Ruv AB-Holliday junction complex have shown that RuvA binds directly to the Hol liday junction [2-6] and acts as a specificity factor that promotes the tar geting of RuvB [7,8], a hexameric ring protein that drives branch migration [9-11]. Electron microscopic visualisation of the RuvAB complex revealed t hat RuvA is flanked by two RuvB hexamers, which bind DNA arms that lie diam etrically opposed across the junction [8], ATP-dependent branch migration o ccurs as duplex DNA is pumped out through the centre of each ring. Because RuvB possesses well conserved helicase motifs and RuvAB exhibits a 5'-3' DN A helicase activity in vitro [12], the mechanism of branch migration is tho ught to involve DNA opening within the RuvB ring, which provides a single s trand for the unidirectional translocation of the protein along DNA. We hav e investigated whether the RuvB ring can translocate along duplex DNA conta ining a site directed interstrand psoralen crosslink. Surprisingly, we foun d that the crosslink failed to inhibit branch migration, We interpret these data as evidence against a base-by-base tracking model and suggest that ex tensive DNA opening within the RuvB ring is not required for DNA translocat ion by RuvB.