Abstract
Ubiquitous RarA AAA+ ATPases play crucial roles in the cellular response to blocked replication forks in pro- and eukaryotes. Here, we provide evidence that absence of RarA reduced the viability of ΔrecA, ΔrecO, and recF15 cells during unperturbed growth. The rarA gene was epistatic to recO and recF genes in response to H2O2- or MMS-induced DNA damage. Conversely, the inactivation of rarA partially suppressed the HR defect of mutants lacking end-resection (ΔaddAB, ΔrecJ, ΔrecQ, ΔrecS) or branch migration (ΔruvAB, ΔrecG, ΔradA) activity. RarA contributes to RecA thread formation, that are thought to be the active forms of RecA during homology search. The absence of RarA reduced RecA accumulation, and the formation of visible RecA threads in vivo upon DNA damage. When ΔrarA was combined with mutations in genuine RecA accessory genes, RecA accumulation was further reduced in ΔrarA ΔrecU and ΔrarA ΔrecX double mutant cells, and was blocked in ΔrarA recF15 cells. These results suggest that RarA contributes to the assembly of RecA nucleoprotein filaments onto single-stranded DNA, and possibly antagonizes RecA filament disassembly.
Highlights
During DNA replication, the replisomes encounter obstacles that can block their progression, and replication impairment is recognized as an important source of genetic instability (Kuzminov, 1995; Haber, 2015; Gaillard and Aguilera, 2016)
We show that lack of RarA reduces cell viability in the RecA mediators (recO) and recA and in less extent in the recF15 context in the absence of DNA damage, but these single and double mutant strains are sensitive to H2O2- or methyl methane sulfonate (MMS)-induced DNA lesions
homologous recombination (HR) is the ultimate step for error-free repair of a double strand break (DSB) and for promoting the re-establishment of replication forks during vegetative growth
Summary
During DNA replication, the replisomes encounter obstacles that can block their progression, and replication impairment is recognized as an important source of genetic instability (Kuzminov, 1995; Haber, 2015; Gaillard and Aguilera, 2016). In addition to RarA, SsbA interacts with various recombination (RecQ, RecS, RecJ, RecG, RecO, RecD2, SbcC, and SbcE) and replication (PriA, DnaG, and DnaE) proteins, of which RecS, RecD2, SbcE, and DnaE are absent in E. coli cells (Costes et al, 2010) These data suggest a role of RarA in recombination-dependent DNA replication, RarA might follow different avenues in distantly related bacteria or depending on the type of DNA damage (Stanage et al, 2017; Carrasco et al, 2018; Romero et al, 2019a,b). These data suggest that RarA may facilitate RecA filament growth and might counteract negative mediators RecX and/or RecU
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