Bacillus subtilis PcrA Couples DNA Replication, Transcription, Recombination and Segregation

María Moreno-Del Alamo,Juan Carlos Alonso,Rubén Torres,Candela Manfredi,José A Ruiz-Masó,Gloria Del Solar

doi:10.3389/fmolb.2020.00140

María Moreno-Del Alamo, Juan Carlos Alonso + Show 4 more

Open Access

https://doi.org/10.3389/fmolb.2020.00140

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Abstract

Bacillus subtilis PcrA abrogates replication-transcription conflicts in vivo and disrupts RecA nucleoprotein filaments in vitro. Inactivation of pcrA is lethal. We show that PcrA depletion lethality is suppressed by recJ (involved in end resection), recA (the recombinase), or mfd (transcription-coupled repair) inactivation, but not by inactivating end resection (addAB or recQ), positive and negative RecA modulators (rarA or recX and recU), or genes involved in the reactivation of a stalled RNA polymerase (recD2, helD, hepA, and ywqA). We also report that B. subtilis mutations previously designated as recL16 actually map to the recO locus, and confirm that PcrA depletion lethality is suppressed by recO inactivation. The pcrA gene is epistatic to recA or mfd, but it is not epistatic to addAB, recJ, recQ, recO16, rarA, recX, recU, recD2, helD, hepA, or ywqA in response to DNA damage. PcrA depletion led to the accumulation of unsegregated chromosomes, and this defect is increased by recQ, rarA, or recU inactivation. We propose that PcrA, which is crucial to maintain cell viability, is involved in different DNA transactions.

Highlights

Homologous recombination is the major pathway to circumvent a replicative stress, a replication fork collapse and for the elimination of DNA double-strand breaks (DSBs) induced by endogenous or exogenous stress
To understand the role of PcrA in rich medium exponentially growing B. subtilis cells, we have studied the genetic linkage of PcrA depletion (Merrikh et al, 2015) with mutations in genes acting at the presynaptic and synaptic stages, as well as in genes that contribute to bypass RTCs or facilitate RNAP backtracking or removal
We show that PcrA depletion lethality is suppressed by recJ, recO16, or recU) and synaptic ( (recA) inactivation, but not by addAB, recQ, rarA, recX, or recU inactivation when cells are grown in rich medium (Figures 1A,B)

Summary

Introduction

Homologous recombination is the major pathway to circumvent a replicative stress, a replication fork collapse and for the elimination of DNA double-strand breaks (DSBs) induced by endogenous or exogenous stress. Super-family 1 (SF1) DNA helicases, which are conserved motor proteins that couple nucleoside triphosphate hydrolysis to the unwinding of duplex DNA, play crucial roles in repair-by-recombination and in coping with replication-transcription conflicts (RTCs) (Wu and Hickson, 2006; Singleton et al, 2007). The prototype of bacterial SF1 helicases that translocate with 3′ → 5′ direction is UvrD (Singleton et al, 2007; Dillingham, 2011). This enzyme shares a significant degree of structural similarity with Rep, which is restricted to the γ-Proteobacteria Class, PcrA and yeast Srs DNA helicases (Wu and Hickson, 2006; Marini and Krejci, 2010). In vitro studies reveal that PcrA and UvrD interact with the RNA polymerase (RNAP), Rep interacts with the replicative DNA helicase (DnaB in Proteobacteria) and Srs physically interacts with Rad and with the PCNA sliding clamp

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