Action of RuvAB at replication fork structures

The replicative apparatus often encounters blocks to its progression that n ecessitate removal of the block and reloading of the replication machinery. In Escherichia coli, a major pathway of replication restart involves unwin ding of the stalled fork to generate a four-stranded Holliday junction, whi ch can then be cleaved by the RuvABC helicase-endonuclease. This fork regre ssion may be catalyzed by RecG but is thought to occur even in its absence. Here we test whether RuvAB helicase can also catalyze the unwinding of for ked DNA to form Holliday junctions. We find that fork DNA is unwound in the direction required for Holliday junction formation only if the loading of RuvB is restricted to the parental duplex DNA arm. If the binding of RuvB i s unrestricted, then RuvAB preferentially unwinds forks in the opposite dir ection. This is probably related to the greater efficiency of two opposed R uvB hexamers operating across a junction compared with a single hexamer. Th ese data argue against RuvAB acting directly at damaged replication forks a nd imply that other mechanisms must operate in vivo to catalyze Holliday ju nction formation.