RuvAB is essential for replication forks reversal in certain replication mutants

Abstract

Inactivated replication forks may be reversed by the annealing of leading-and lagging-strand ends, resulting in the formation of a Holliday junction (HJ) adjacent to a DNA double-strand end. In Escherichia coli mutants deficient for double-strand end processing, resolution of the HJ by RuvABC leads to fork breakage, a reaction that we can directly quantify. Here we used the HJ-specific resolvase RusA to test a putative role of the RuvAB helicase in replication fork reversal (RFR). We show that the RuvAB complex is required for the formation of a RusA substrate in the polymerase III mutants dnaEts and holD, affected for the Pol III catalytic subunit and clamp loader, and in the helicase mutant rep. This finding reveals that the recombination enzyme RuvAB targets forks in vivo and we propose that it directly converts forks into HJs. In contrast, RFR occurs in the absence of RuvAB in the dnaNts mutant, affected for the processivity clamp of Pol III, and in the priA mutant, defective for replication restart. This suggests alternative pathways of RFR.

Formation of a Stable RuvA Protein Double Tetramer Is Required for Efficient Branch Migration in Vitro and for Replication Fork Reversal in Vivo

“…In cells carrying the temperature-sensitive replication mutant dnaEts, RuvA is required for RFR (24). In the strain JJC3723, RFR can be measured by re-introducing RuvA on a plasmid and measuring the linearized DNA formed in this recB mutant by RuvC cleavage of reversed forks (29).…”

“…In E. coli, the rusA gene encodes a Holliday junction resolvase carried on a cryptic prophage, but this gene in not normally expressed (8,29). The E. coli strain JJC2761 (⌬ruvABC rus-1) does not encode RuvABC, but the rus-1 mutation results in rusA expression, and the cells survive UV damage as RusA resolves Holliday junctions formed by recombination-mediated repair (24,29). We used the JJC2761 strain as a tool to test the effect of RuvA2 KaP on Holliday junction resolution by RusA in vivo.…”

“…RuvAB could reverse model replication forks in vitro if RuvB was only allowed to form one hexameric ring on the parental duplex of the fork (25). It has been speculated that in vivo the asymmetric binding of a single RuvA tetramer onto a three-armed fork may result in asymmetric loading of a single RuvB hexamer onto the parental duplex (24). Alternatively, cellular factors may force RuvA to load RuvB in this manner.…”

“…Both pathways result in PriA-dependent replication restart (23). Intriguingly, RuvAB are actually required for replication fork reversal to occur in dnaEts, holD Q10am and rep E. coli mutants (24). Yet in vitro, RuvAB preferentially unwinds synthetic replication forks in a direction that is opposite the direction for fork reversal (25).…”

Regression of Replication Forks Stalled by Leading-strand Template Damage

“…Conclusions about which protein acts when vary, depending on the type of damage that is used to arrest the replication fork. RuvAB-catalyzed RFR has been invoked in the rescue of replication forks stalled by either denaturation of temperature-sensitive components of the DNA polymerase III holoenzyme (Pol III HE), the cellular replicase (23) (the ␣ and subunits (24)), or in mutants lacking the Rep DNA helicase (24), which causes slower replication fork progression (25) because of reduced ability to clear proteins from in front of the replication fork (26). RecG-catalyzed fork regression has been suggested to occur in UV-irradiated cells after replication forks stall, primarily because they encounter an RNA polymerase that has stalled at a leading-strand cyclopyrimidine dimer (CPD) (27,28), whereas RecA-catalyzed regression is proposed to occur after stalling forks by denaturation of a thermosensitive replication fork DNA helicase, DnaB (29,30), and also possibly after UV irradiation (31).…”