Bacillus subtilis RarA acts at the interplay between replication and repair-by-recombination

Romero, Hector; Torres, Rubén; Hernández-Tamayo, Rogelio; Carrasco, Begoña; Ayora, Silvia; Graumann, Peter L.; Alonso, Juan C.

doi:10.1016/j.dnarep.2019.03.010

Cited by 6 publications

(15 citation statements)

References 83 publications

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“…The combination of rarA with addAB, recS, recQ or recJ (impaired in alternative end resection pathways), recX (negative modulator) or recD2 (a putative branch migration translocase) yielded similar or only slightly reduced (<1.4-fold) viability relative to rec + cells (Supplementary Figure S1). Similarly, rarA polY1 and rarA polY2 double mutants have a viability similar than the single mutant strains (Romero et al, 2019b).…”

Section: Experimental Systemmentioning

confidence: 88%

“…The MMS and H 2 O 2 drugs were chosen for our analysis (further explanation in Supplementary Annex S1). Our previous work showed that RarA single mutants are very sensitive to H 2 O 2induced lesions, but in the absence of RarA, cells remain recombination proficient and apparently are as capable of repairing MMS-induced DNA lesions as wt cells (Romero et al, 2019b), showing that RarA deals differently with the effect of the two drugs.…”

Section: Experimental Systemmentioning

confidence: 99%

“…Previous assays have indicated a poorly understood role for bacterial RarA in homologous recombination (HR). A Bacillus subtilis null rarA ( rarA) mutant strain renders cells very sensitive to H 2 O 2 , but not to methyl methane sulfonate (MMS) or to UV radiation-mimetic compound 4nitroquinoline-1-oxide (Romero et al, 2019b). In contrast, an Escherichia coli rarA strain remains as capable of repairing UV-induced DNA damage as wild-type (wt or rec + ) cells (Barre et al, 2001;Shibata et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

“…In both bacteria, E. coli and B. subtilis, the viability under unperturbed conditions of rarA recA cells is significantly lower than that of the recA control (Shibata et al, 2005;Romero et al, 2019b). Since the recA gene is not epistatic with functions involved in base or nucleotide excision repair, but the E. coli or B. subtilis rarA gene is epistatic to recA in response to DNA damage (Shibata et al, 2005;Romero et al, 2019b), we assume that RarA is a genuine repair-by-recombination protein.…”

Section: Introductionmentioning

confidence: 99%

“…However, in vitro studies suggested that RarA Eco may contribute to replication fork rescue by creating a flap on the lagging strand, so that the replicative helicase and its associated replisome could continue chain elongation without the need for replisome disassembly and replication restart (Stanage et al, 2017). In B. subtilis cells, inhibition of the replicative DNA polymerase PolC, by the specific inhibitor p-hydroxyphenylazo-uracil (HPUra), confines the RarA molecules toward the collapsed replication forks in vivo (Romero et al, 2019b). In this bacterium it was shown that B. subtilis RarA-mVenus (RarA-YFP) transiently colocalizes with the DnaX-CFP protein, and it alternates between static and dynamic states.…”

Section: Introductionmentioning

confidence: 99%

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Bacillus subtilis RarA Acts as a Positive RecA Accessory Protein

et al. 2020

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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 recA, recO, and recF15 cells during unperturbed growth. The rarA gene was epistatic to recO and recF genes in response to H 2 O 2-or MMS-induced DNA damage. Conversely, the inactivation of rarA partially suppressed the HR defect of mutants lacking end-resection (addAB, recJ, recQ, recS) or branch migration (ruvAB, recG, radA) 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 rarA was combined with mutations in genuine RecA accessory genes, RecA accumulation was further reduced in rarA recU and rarA recX double mutant cells, and was blocked in rarA 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.

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Section: Experimental Systemmentioning

confidence: 88%

Section: Experimental Systemmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Bacillus subtilis RarA Acts as a Positive RecA Accessory Protein

et al. 2020

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Single molecule tracking reveals functions for RarA at replication forks but also independently from replication during DNA repair in Bacillus subtilis

Romero

Rösch

Hernández-Tamayo

et al. 2019

Sci Rep

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RarA is a widely conserved protein proposed to be involved in recombination-dependent replication. We present a cell biological approach to identify functional connections between RarA and other proteins using single molecule tracking. We found that 50% of RarA molecules were static, mostly close to replication forks and likely DNA-bound, while the remaining fraction was highly dynamic throughout the cells. RarA alternated between static and dynamic states. Exposure to H2O2 increased the fraction of dynamic molecules, but not treatment with mitomycin C or with methyl methanesulfonate, which was exacerbated by the absence of RecJ, RecD2, RecS and RecU proteins. The ratio between static and dynamic RarA also changed in replication temperature-sensitive mutants, but in opposite manners, dependent upon inhibition of DnaB or of DnaC (pre)primosomal proteins, revealing an intricate function related to DNA replication restart. RarA likely acts in the context of collapsed replication forks, as well as in conjunction with a network of proteins that affect the activity of the RecA recombinase. Our novel approach reveals intricate interactions of RarA, and is widely applicable for in vivo protein studies, to underpin genetic or biochemical connections, and is especially helpful for investigating proteins whose absence does not lead to any detectable phenotype.

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Processing of stalled replication forks in Bacillus subtilis

Carrasco,

Torres,

Moreno-del Álamo

et al. 2023

FEMS Microbiology Reviews

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Accurate DNA replication and transcription elongation are crucial for preventing the accumulation of unreplicated DNA and genomic instability. Cells have evolved multiple mechanisms to deal with impaired replication fork progression, challenged by both intrinsic and extrinsic impediments. The bacterium Bacillus subtilis, which adopts multiple forms of differentiation and development, serves as an excellent model system for studying the pathways required to cope with replication stress to preserve genomic stability. This review focuses on the genetics, single molecule choreography and biochemical properties of the proteins that act to circumvent the replicative arrest allowing the resumption of DNA synthesis. The RecA recombinase, its mediators (RecO, RecR, RadA/Sms) and modulators (RecF, RecX, RarA, RecU, RecD2, PcrA), repair licensing (DisA), fork remodelers (RuvAB, RecG, RecD2, RadA/Sms, PriA), Holliday junction resolvase (RecU), nucleases (RnhC, DinG), and translesion synthesis DNA polymerases (PolY1 and PolY2) are the key functions required to overcome a replication stress, provided that the fork does not collapse.

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Bacillus subtilis RarA acts at the interplay between replication and repair-by-recombination

Cited by 6 publications

References 83 publications

Bacillus subtilis RarA Acts as a Positive RecA Accessory Protein

Bacillus subtilis RarA Acts as a Positive RecA Accessory Protein

Single molecule tracking reveals functions for RarA at replication forks but also independently from replication during DNA repair in Bacillus subtilis

Processing of stalled replication forks in Bacillus subtilis

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