Abstract
The replicative apparatus often encounters blocks to its progression that necessitate removal of the block and reloading of the replication machinery. In Escherichia coli, a major pathway of replication restart involves unwinding of the stalled fork to generate a four-stranded Holliday junction, which can then be cleaved by the RuvABC helicase-endonuclease. This fork regression 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 forked 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 is unrestricted, then RuvAB preferentially unwinds forks in the opposite direction. This is probably related to the greater efficiency of two opposed RuvB hexamers operating across a junction compared with a single hexamer. These data argue against RuvAB acting directly at damaged replication forks and imply that other mechanisms must operate in vivo to catalyze Holliday junction formation.
Highlights
The necessity for an organism to replicate its genome both accurately and efficiently has until recently been attributed to the inherently high fidelity and processivity of the replication machinery
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
We found that RuvAB could unwind fork DNA in the direction necessary for Holliday junction formation, unwinding in the opposing direction was the preferred choice provided the loading of RuvB could freely occur on any of the arms of the fork
Summary
The necessity for an organism to replicate its genome both accurately and efficiently has until recently been attributed to the inherently high fidelity and processivity of the replication machinery. The release of a free duplex end from the stalled fork is thought to be a critical component of replication restart in E. coli Such DNA ends are known to be processed by the helicase/exonuclease RecBCD followed by RecA-mediated strand exchange with an intact homologous duplex to generate a recombination intermediate, a D-loop [20]. RecG can actively unwind forked DNA structures to form Holliday junctions in vitro [24, 25] These findings have led to the proposal that RecG modulates the structure of stalled forks, possibly by a template switching mechanism, to allow PriA binding and concomitant recruitment of the replicative machinery without the need for potentially dangerous chromosome breakage events and subsequent D-loop formation
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