<i>Bacillus subtilis</i>RecO and SsbA are crucial for RecA-mediated recombinational DNA repair

Carrasco, Begoña; Yadav, Tribhuwan; Serrano, Ester; Alonso, Juan C.

doi:10.1093/nar/gkv545

Cited by 38 publications

(72 citation statements)

References 89 publications

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“…The requirement for RecR in the AdnAB pathway implies that AdnAB does not have intrinsic mediator activity, in contrast to E. coli RecBCD, which does have such activity. This finding is similar to recent data from B. subtilis showing that RecO, and possibly RecR, are required for RecA loading and strand exchange, even in the presence of AddAB (49). However, our data also indicate that the combination of ⌬recO and ⌬recF abolishes HR, thus phenocopying loss of RecR.…”

Section: Figsupporting

confidence: 92%

RecF and RecR Play Critical Roles in the Homologous Recombination and Single-Strand Annealing Pathways of Mycobacteria

2015

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Mycobacteria encode three DNA double-strand break repair pathways: (i) RecA-dependent homologous recombination (HR), (ii) Ku-dependent nonhomologous end joining (NHEJ), and (iii) RecBCD-dependent single-strand annealing (SSA). Mycobacterial HR has two presynaptic pathway options that rely on the helicase-nuclease AdnAB and the strand annealing protein RecO, respectively. Ablation of adnAB or recO individually causes partial impairment of HR, but loss of adnAB and recO in combination abolishes HR. RecO, which can accelerate annealing of single-stranded DNA in vitro, also participates in the SSA pathway. The functions of RecF and RecR, which, in other model bacteria, function in concert with RecO as mediators of RecA loading, have not been examined in mycobacteria. Here, we present a genetic analysis of recF and recR in mycobacterial recombination. We find that RecF, like RecO, participates in the AdnAB-independent arm of the HR pathway and in SSA. In contrast, RecR is required for all HR in mycobacteria and for SSA. The essentiality of RecR as an agent of HR is yet another distinctive feature of mycobacterial DNA repair. N ature has evolved mechanisms to repair the structurally diverse DNA lesions that arise during chromosomal replication and from exogenous mutagens. Double-strand breaks (DSBs) are especially deleterious, insofar as a single chromosomal DSB can be lethal to a cell if not repaired prior to cell division. The major mechanisms of DSB repair are conserved, at least in their general principles, from bacteria to humans. This parallel is especially relevant for mycobacteria, which implement the same three distinct DSB repair pathways operative in eukarya: homologous recombination (HR), nonhomologous end joining (NHEJ), and singlestrand annealing (SSA). Mycobacterial NHEJ relies on the end-binding protein Ku, DNA ligase LigD (a multifunctional ligase-polymerase-3=-phosphoesterase), DNA ligase LigC1, and two additional polymerases (PolD1 and PolD2) (1-9). The SSA pathway of mycobacteria repairs DSBs that arise between repeats, with consequent deletion of the DNA between the repeats. SSA is independent of the strand exchange protein RecA but requires the helicase-nuclease RecBCD and the strand-annealing protein RecO (9,10). IMPORTANCE This study clarifies the molecular requirements for homologous recombination in mycobacteria. Specifically, we demonstrate that RecF and RecR play important roles in both theThe central agent of homology search in the HR pathways of all organisms is a strand exchange protein that polymerizes on the single-strand DNA (ssDNA) resulting from end resection at a DSB. In bacteria, the strand exchange protein is RecA; in eukarya, it is Rad51. RecA nucleation on ssDNA is regulated in order to prevent inappropriate nucleoprotein filament formation, which can be toxic (11). The presence of single-strand binding protein (SSB) on ssDNA prevents RecA loading unless a "mediator" protein or protein complex catalyzes the exchange of SSB for RecA to create the RecA nucleoprotein filam...

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Section: Figsupporting

confidence: 92%

RecF and RecR Play Critical Roles in the Homologous Recombination and Single-Strand Annealing Pathways of Mycobacteria

2015

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“…In their ATP-bound form, these recombinases can nucleate on protein-free ssDNA, but they cannot catalyze DSE in the absence of accessory factors (inactive RecA) ( 10 , 32 – 34 ). Unlike RecA Eco ·ATP ( 11 – 13 ) or RecA·dATP ( 32 , 35 ), RecA·ATP cannot nucleate or polymerize in the SsbA– or SsbB–ssDNA complexes ( 10 , 16 , 17 , 36 ). The presence of DprA reverses the negative effect of SsbA or SsbB on RecA filament growth, and DprA–SsbA are necessary and sufficient to activate RecA·ATP to catalyze bidirectional DSE, with greater efficiency in the 5′→3′ direction ( 10 , 17 ).…”

Section: Introductionmentioning

confidence: 99%

Bacillus subtilis RecA with DprA–SsbA antagonizes RecX function during natural transformation

Serrano

Kawamura

et al. 2017

Nucleic Acids Research

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Bacillus subtilis DprA and RecX proteins, which interact with RecA, are crucial for efficient chromosomal and plasmid transformation. We showed that RecA, in the rATP·Mg2+ bound form (RecA·ATP), could not compete with RecX, SsbA or SsbB for assembly onto single-stranded (ss)DNA, but RecA·dATP partially displaced these proteins from ssDNA. RecX promoted reversible depolymerization of preformed RecA·ATP filaments. The two-component DprA–SsbA mediator reversed the RecX negative effect on RecA filament extension, but not DprA or DprA and SsbB. In the presence of DprA–SsbA, RecX added prior to RecA·ATP inhibited DNA strand exchange, but this inhibition was reversed when RecX was added after RecA. We propose that RecA nucleation is more sensitive to RecX action than is RecA filament growth. DprA–SsbA facilitates formation of an active RecA filament that directly antagonizes the inhibitory effects of RecX. RecX and DprA enable chromosomal transformation by altering RecA filament dynamics. DprA–SsbA and RecX proteins constitute a new regulatory network of RecA function. DprA–SsbA contributes to the formation of an active RecA filament and directly antagonizes the inhibitory effects of RecX during natural transformation.

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“…High expression levels at recJ and rrnO are unsurprising given that their products are a single-strand DNA-specific exonuclease involved in recombinational DNA repair and genome maintenance, and the 23S rRNA respectively. Disruption of recJ is reported to increase susceptibility to killing by DNA damaging agents (Carrasco et al, 2015). This should be considered if recJ is chosen as insertion locus for strains with applications where this may be problematic, for instance in long continuous fermentations.…”

Section: Additional Partsmentioning

confidence: 99%

BacilloFlex: A modular DNA assembly toolkit forBacillus subtilissynthetic biology

Wicke

Radford

Verrone

et al. 2017

Preprint

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Bacillus subtilis is a valuable industrial production platform for proteins, a bacterial model for cellular differentiation and its endospores have been proposed as a vehicle for vaccine delivery. As such B. subtilis is a major synthetic biology chassis but, unlike Escherichia coli, lacks a standardized toolbox for genetic manipulation. EcoFlex is a versatile modular DNA assembly toolkit for E. coli synthetic biology based on Golden Gate cloning. Here we introduce BacilloFlex, an extension of the EcoFlex assembly standard to B. subtilis. Transcription units flanked by sequences homologous to loci in the B. subtilis genome were rapidly assembled by the EcoFlex standard and subsequently chromosomally integrated. At present, BacilloFlex includes a range of multi-functional B. subtilis specific parts with applications including metabolic engineering, biosensors and spore surface display. We hope this work will form the foundation of a widely adopted cloning standard for B. subtilis facilitating collaboration and the sharing of parts.

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Bacillus subtilisRecO and SsbA are crucial for RecA-mediated recombinational DNA repair

Cited by 38 publications

References 89 publications

RecF and RecR Play Critical Roles in the Homologous Recombination and Single-Strand Annealing Pathways of Mycobacteria

RecF and RecR Play Critical Roles in the Homologous Recombination and Single-Strand Annealing Pathways of Mycobacteria

Bacillus subtilis RecA with DprA–SsbA antagonizes RecX function during natural transformation

BacilloFlex: A modular DNA assembly toolkit forBacillus subtilissynthetic biology

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