Abstract
Bacillus subtilis diadenylate cyclase DisA converts two ATPs into c-di-AMP, but this activity is suppressed upon interaction with sites of DNA damage. DisA forms a rapid moving focus that pauses upon induction of DNA damage during spore development. We report that DisA pausing, however, was not observed in the absence of the RecO mediator or of the RecA recombinase, suggesting that DisA binds to recombination intermediates formed by RecA in concert with RecO. DisA, which physically interacts with RecA, was found to reduce its ATPase activity without competing for nucleotides or ssDNA. Furthermore, increasing DisA concentrations inhibit RecA-mediated DNA strand exchange, but this inhibition failed to occur when RecA was added prior to DisA, and was independent of RecA-mediated nucleotide hydrolysis or increasing concentrations of c-di-AMP. We propose that DisA may preserve genome integrity by downregulating RecA activities at several steps of the DNA damage tolerance pathway, allowing time for the repair machineries to restore genome stability. DisA might reduce RecA-mediated template switching by binding to a stalled or reversed fork.
Highlights
Natural transformation contributes to the acquisition of genetic diversity and to the restoration of mutated genes, playing a central role in the evolution and the spread of metabolic pathways, pathogenicity traits and antibiotic resistance genes [1,2]
Previous assays have shown that RadA/Sms is crucial, and RecA is essential for natural chromosomal transformation, but in their absence plasmid transformation is marginally affected if at all (Supplementary Material, Annex 2, Figure 2A) [18,42]
Since the DNA uptake machinery takes any DNA with similar efficiency, and competent radA and recA cells are reduced and blocked in chromosomal transfomation, but proficient in plasmid transformation, we concluded that RecA and RadA/Sms are involved in HR rather than in competence development, DNA uptake or controlling the competence exit [18,47], and assumed that another function contributes to chromosomal transformation in the radA context
Summary
Natural transformation contributes to the acquisition of genetic diversity and to the restoration of mutated genes, playing a central role in the evolution and the spread of metabolic pathways, pathogenicity traits and antibiotic resistance genes [1,2]. During Bacillus subtilis natural competence development, DNA replication is halted, a transcriptional program is activated and the membranespecific DNA uptake apparatus is transiently assembled at one of the cell poles [3]. In the ATP·Mg2+ bound form, RecA (RecA·ATP) from natural competent B. subtilis and Streptococcus pneumoniae cells can filament onto ssDNA, but this RecA nucleoprotein filament (or RecA-ssDNA complex) cannot catalyse DNA strand exchange in vitro [10,11,12]. B. subtilis RecA cannot nucleate on the SsbA-ssDNA (or SsbA-ssDNA-SsbB) complexes [7,13] These inabilities are overcome with the help of the two-component mediators (SsbA in concert with DprA or RecO [in the ΔdprA context]). A two-component mediator promotes RecA·ATP nucleation and filament growth onto SsbA- (or SsbA and SsbB)-coated ssDNA with a subsequent activation to catalyse DNA strand exchange [8,9]. The indicated genes and products are of B. subtilis origin
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