RETRACTED: RecA-Promoted, RecFOR-Independent Progressive Disassembly of Replisomes Stalled by Helicase Inactivation

Abstract

In all organisms, replication impairment is a recognized source of genomic instability, raising an increasing interest in the fate of inactivated replication forks. We used Escherichia coli strains with a temperature-inactivated replicative helicase (DnaB) and in vivo single-molecule microscopy to quantify the detailed molecular processing of stalled replication forks. After helicase inactivation, RecA binds to blocked replication forks and is essential for the rapid release of hPol III. The entire holoenzyme is disrupted little by little, with some components lost in few minutes, while others are stable in 70% of cells for at least 1 hr. Although replisome dissociation is delayed in a recA mutant, it is not affected by RecF or RecO inactivation. RecFOR are required for full RecA filaments formation, and we propose that polymerase clearance can be catalyzed by short, RecFOR-independent RecA filaments. Our results identify a function for the universally conserved, central recombination protein RecA.

RecA acts as a switch to regulate polymerase occupancy in a moving replication fork

“…Although RecFOR promote efficient exchange of RecA for SSB at blocked forks (refs. 25-27 and references therein), shorter RecA-filaments are formed in absence of RecFOR and are proposed to bind lagging-strand ssDNA produced during fork advance (24). Extension of RecA filaments toward the replisome destabilizes the Pol III*-replisome, facilitating its dissociation, consistent with our earlier in vitro observations that RecA dislodges Pol III* from a primed site (23).…”

“…Our earlier studies on the effect of RecA on Pol III*-β have also demonstrated that RecA can disassemble the replicase from DNA (23). Furthermore, a recent in vivo study using single-molecule techniques finds that RecA indeed binds to DNA at the replication fork, and favors disassembly of the Pol III replisome (24).…”

“…A surprising finding is that RecA strongly inhibits leadingstrand advance by the Pol III*-replisome, and this finding is supported by a recent in vivo study that visualizes RecA binding and dissociation of Pol III (24). The precise mechanism underlying RecA inhibition could occur in any of several possible ways.…”

“…Specifically, only 3-5 nt are needed for RecA, whereas 35-65 nt are required for each SSB tetramer (45)(46)(47)(48). This second possibility has also been suggested by a recent study that directly visualizes fluorescent RecA at the fork in live cells even in the absence of RecFOR (24). As the fork advances, generating lagging ssDNA, RecA may get the first chance to bind ssDNA until the ssDNA achieves a long enough length to bind SSB.…”

“…Hence, one possibility is that RecA competes with SSB, indirectly inhibiting the Pol III*-replisome. Recently, it has been shown in vivo that RecA induces Pol III dissociation from the fork in a RecF, RecO, and RecR (RecFOR)-independent manner (24). Although RecFOR promote efficient exchange of RecA for SSB at blocked forks (refs.…”