PcrA Dissociates RecA Filaments and the SsbA and RecO Mediators Counterbalance Such Activity

Abstract

PcrA depletion is lethal in wild-type Bacillus subtilis cells. The PcrA DNA helicase contributes to unwinding RNA from the template strand, backtracking the RNA polymerase, rescuing replication-transcription conflicts, and disassembling RecA from single-stranded DNA (ssDNA) by poorly understood mechanisms. We show that, in the presence of RecA, circa one PcrA/plasmid-size circular ssDNA (cssDNA) molecule hydrolyzes ATP at a rate similar to that on the isolated cssDNA. PcrA K37A, which poorly hydrolyses ATP, fails to displace RecA from cssDNA. SsbA inhibits and blocks the ATPase activities of PcrA and RecA, respectively. RecO partially antagonizes and counteracts the negative effect of SsbA on PcrA- and RecA-mediated ATP hydrolysis, respectively. Conversely, multiple PcrA molecules are required to inhibit RecA·ATP-mediated DNA strand exchange (DSE). RecO and SsbA poorly antagonize the PcrA inhibitory effect on RecA·ATP-mediated DSE. We propose that two separable PcrA functions exist: an iterative translocating PcrA monomer strips RecA from cssDNA to prevent unnecessary recombination with the mediators SsbA and RecO balancing such activity; and a PcrA cluster that disrupts DNA transactions, as RecA-mediated DSE.

Processing of stalled replication forks in Bacillus subtilis

“…The RecD2 helicase, translocating in the 5′→3′ direction, removes RecA bound to the ssDNA region present in collapsed forks to facilitate in vitro replication restart (Ramos et al 2022 ). Similarly, PcrA translocating in the 3′→5′ direction actively disassembles RecA from ssDNA to prevent unnecessary recombination, with SsbA and RecO counterbalancing such activity (Park et al 2010 , Carrasco et al 2022 ).…”

“…PcrA has a template-clearing role at RTCs, but there is no information on whether PcrA works as an accessory replication fork DNA helicase. PcrA physically interacts with and actively displaces RecA from ssDNA (Park et al 2010 , Carrasco et al 2022 ). PcrA interacts with and backtracks a paused RNAP, and unwinds the RNA moiety of an R-loop, but it fails to remodel a stalled fork (Soultanas et al 2000 , Delumeau et al 2011 , Sanders et al 2017 , Torres and Alonso 2021 , Carrasco et al 2022 ).…”

“…PcrA physically interacts with and actively displaces RecA from ssDNA (Park et al 2010 , Carrasco et al 2022 ). PcrA interacts with and backtracks a paused RNAP, and unwinds the RNA moiety of an R-loop, but it fails to remodel a stalled fork (Soultanas et al 2000 , Delumeau et al 2011 , Sanders et al 2017 , Torres and Alonso 2021 , Carrasco et al 2022 ). In E. coli , three accessory DNA helicases (Rep, UvrD, and DinG), which act at stalled replication forks, remove the protein barriers without fork remodeling (Guy et al 2009 , Boubakri et al 2010 , Hawkins et al 2019 , Syeda et al 2019 ).…”

“…Second, RecA interacts with DisA, RadA/Sms, RecG, LexA, RecU, RecX, RarA, RecD2, PcrA, RNAP, RnhC, and DinG (Carrasco et al 2005 , 2022 , 2023 , Groban et al 2005 , Cañas et al 2008 , Torres et al 2019a , c , Ramos et al 2022 ). Among these protein interactions, it can be highlighted that RarA, RecD2, and DnaE are part of both SsbA and RecA interactomes, and that RarA physically interacts with PriA (Carrasco et al 2018 ).…”

“…First, RecA forms foci that are not converted into threads at RTCs or in response to a low UV dose (1 J/m 2 ) (Simmons et al 2007 , Million-Weaver et al 2015 ). Second, RecA interacts with many proteins, among them with DisA or RadA/Sms, which inhibit the ATPase and the DNA strand exchange activities of RecA, perhaps to prevent RecA from engaging in unnecessary homology search and strand exchange (Torres et al 2019a , c , Carrasco et al 2022 , 2023 ) Third, upon artificial inversion of the rrnIHG operons, that strongly compromises (>1000-fold) the plating efficiency in LB medium (Srivatsan et al 2010 , Huang et al 2023 ), PcrA overexpression improves viability of recA + cells, but not of Δ recA cells. Contrarily, upon rrnIHG inversion, overexpression of RnhC does not improve the viability of recA + cells grown on LB, but slightly increases viability of Δ recA cells (Yeesin 2019 ).…”