UvrD controls the access of recombination proteins to blocked replication forks

Abstract

Blocked replication forks often need to be processed by recombination proteins prior to replication restart. In Escherichia coli, the UvrD repair helicase was recently shown to act at inactivated replication forks, where it counteracts a deleterious action of RecA. Using two mutants affected for different subunits of the polymerase III holoenzyme (Pol IIIh), we show here that the anti‐RecA action of UvrD at blocked forks reflects two different activities of this enzyme. A defective UvrD mutant is able to antagonize RecA in cells affected for the Pol IIIh catalytic subunit DnaE. In this mutant, RecA action at blocked forks specifically requires the protein RarA (MgsA). We propose that UvrD prevents RecA binding, possibly by counteracting RarA. In contrast, at forks affected for the Pol IIIh clamp (DnaN), RarA is not required for RecA binding and the ATPase function of UvrD is essential to counteract RecA, supporting the idea that UvrD removes RecA from DNA. UvrD action on RecA is conserved in evolution as it can be performed in E. coli by the UvrD homologue from Bacillus subtilis, PcrA.

Resolving Holliday Junctions with Escherichia coli UvrD Helicase

“…UvrD has been suggested to have a role in replication restart in vivo (15,16,20). At blocked replication forks the creation of a reversed replication fork with a HJ structure has been proposed when the nascent leading and lagging strands anneal.…”

“…Recent studies indicate that UvrD has a direct role in recombination reactions associated with replication fork rescue (13)(14)(15). Specifically, UvrD is required for regression of the nascent leading and lagging strands at a stalled replication fork leading to the formation of a Holliday junction (HJ) 5 (16,17).…”

“…Specifically, UvrD is required for regression of the nascent leading and lagging strands at a stalled replication fork leading to the formation of a Holliday junction (HJ) 5 (16,17). The role played by UvrD has been the subject of intense study because it is now clear that replication fork restart is critical to maintaining genome stability (13,15,16,18). Current models suggest that UvrD removes RecA assembled on single-stranded DNA (ssDNA) at a blocked replication fork to allow replication fork reversal to occur (15).…”

“…The role played by UvrD has been the subject of intense study because it is now clear that replication fork restart is critical to maintaining genome stability (13,15,16,18). Current models suggest that UvrD removes RecA assembled on single-stranded DNA (ssDNA) at a blocked replication fork to allow replication fork reversal to occur (15). This is consistent with in vitro experiments that have shown UvrD to be capable of removing RecA molecules from DNA thereby disrupting a recombination event (9,18,18,19).…”

Promoting and Avoiding Recombination: Contrasting Activities of the Escherichia coli RuvABC Holliday Junction Resolvase and RecG DNA Translocase

“…UvrD has been shown to displace RecA filaments assembled on ssDNA in vitro and is thought to do so in vivo when RecA is loaded on a region of the nascent lagging strand template exposed at a stalled fork through the actions of RecQ helicase and RecJ exonuclease. With no UvrD present, the RecFOR proteins establish a stable RecA filament on the exposed template ( Figure 1C), thus presumably provoking recombination even though this recombination is not essential, as indicated by the fact that inactivating RecA or preventing RecA loading restores viability to uvrD ruv cells (Flores et al 2005;Veaute et al 2005;Lestini and Michel 2007;Magner et al 2007). Although UvrD is thought to reduce such pathology by displacing RecA, it might also limit fork stalling by providing a second helicase motor at the fork to help DnaB drive through obstacles ( Figure 1C).…”