Bacillus subtilis PcrA Helicase Removes Trafficking Barriers

Abstract

Bacillus subtilis PcrA interacts with the RNA polymerase and might contribute to mitigate replication–transcription conflicts (RTCs). We show that PcrA depletion lethality is partially suppressed by rnhB inactivation, but cell viability is significantly reduced by rnhC or dinG inactivation. Following PcrA depletion, cells lacking RnhC or DinG are extremely sensitive to DNA damage. Chromosome segregation is not further impaired by rnhB or dinG inactivation but is blocked by rnhC or recA inactivation upon PcrA depletion. Despite our efforts, we could not construct a DrnhC DrecA strain. These observations support the idea that PcrA dismantles RTCs. Purified PcrA, which binds single-stranded (ss) DNA over RNA, is a ssDNA-dependent ATPase and preferentially unwinds DNA in a 3′®5′direction. PcrA unwinds a 3′-tailed RNA of an RNA-DNA hybrid significantly faster than that of a DNA substrate. Our results suggest that a replicative stress, caused by mis-incorporated rNMPs, indirectly increases cell viability upon PcrA depletion. We propose that PcrA, in concert with RnhC or DinG, contributes to removing spontaneous or enzyme-driven R-loops, to counteract deleterious trafficking conflicts and preserve to genomic integrity.

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

“…E. coli BL21(DE3) (pLysS) cells bearing the pCB722 ( ssbA ) , pCB669 ( recO ), pCB1020 ( radA ), pCB1035 ( radA C13A), or pCB906 ( rarA ) plasmid, or E. coli M15 (pREP4) cells bearing the pCB1229 ( pcrA ), or pCB1230 ( pcrA K37A, Walker A mutant variant) plasmid were used to overproduce the SsbA, RecO, RadA/Sms, RadA/Sms C13A, RarA, PcrA, or PcrA K37A proteins, respectively ( Carrasco et al, 2008 ; Manfredi et al, 2008 ; Carrasco et al, 2018 ; Torres et al, 2019 ; Moreno-del Álamo et al, 2021 ). B. subtilis BG214 cells bearing the pBT61 ( recA ) plasmid were used to overproduce RecA ( Gassel and Alonso, 1989 ).…”

“…The UV mimetic 4NQO was from Sigma-Aldrich. Cell sensitivity to chronic 4NQO exposure was determined by growing cultures to OD 560 = 0.4 and plating appropriate dilutions on LB agar plates containing 4NQO (75 nM) or IPTG (500 μM) and 4NQO (75 nM) as described ( Moreno-del Álamo et al, 2021 ). Plates were incubated overnight (16–18 h, 37°C), and the number of CFUs was determined ( Figure 1 ).…”

“…Nevertheless, evidence for strand-specific repair mediated by UvrD Eco /PcrA occurring independently of Mfd-mediated TRC is missing ( Adebali et al, 2017 ; Lindsey-Boltz and Sancar, 2021 ). Third, PcrA and RnhC, which also physically interact with the RNAP, contribute to alleviating RTCs by removing the RNAP damage sensor of the RNA-DNA hybrids and degrading the RNA, respectively ( Delumeau et al, 2011 ; Lang et al, 2017 ; Moreno-del Álamo et al, 2021 ). Fourth, PcrA performs important roles in nudging homologous recombination intermediates toward non-crossover products, and PcrA exhaustion increases the proportion of unsegregated chromosomes by ∼50-fold ( Moreno-Del Alamo et al, 2020 ).…”

“…Third, a two-component mediator (SsbA and RecO) is necessary to activate RecA·ATP to catalyze plasmid-size DSE in vitro (or SsbA, RecO, and RecR in vivo ), whereas DSE is further stimulated by RecA·dATP or RecA Eco ·ATP in the presence of mediators ( Cox, 2007 ; Manfredi et al, 2008 ; Yadav et al, 2012 ; Carrasco et al, 2015 ; Kowalczykowski, 2015 ). Fourth, the negative modulators RecX and RecU, which physically interact with RecA ( Carrasco et al, 2005 ; Cárdenas et al, 2012 ), promote RecA disassembly ( Le et al, 2017 ; Serrano et al, 2018 ), and it is predicted that PcrA, which co-purifies with RecA in tandem affinity purification (Tap-tag) ( Costes et al, 2010 ), may promote RecA disassembly from ssDNA ( Moreno-del Álamo et al, 2021 ). Finally, a Bacilli PcrA monomer translocates along ssDNA in a processive manner, but multiple monomers are required to unwind duplex DNA ( Singleton et al, 2007 ; Lohman et al, 2008 ).…”

β-CASP proteins removing RNA polymerase from DNA: when a torpedo is needed to shoot a sitting duck

“…In Escherichia coli , two replication fork accessory helicases, Rep and UvrD, facilitate efficient replication of double-stranded DNA and affect RNAP dissociation from DNA. Their homolog in Bacillus subtilis , PcrA was proposed to be also involved in suppression of R-loops, three-stranded nucleic acid structures where nascent RNA invades the DNA duplex with potentially inhibitory effects on transcription ( 46 ). The exact molecular basis of the action of these proteins is not known ( 39 ).…”