Abstract
Reviving Bacillus subtilis spores require the recombinase RecA, the DNA damage checkpoint sensor DisA, and the DNA helicase RadA/Sms to prevent a DNA replication stress. When a replication fork stalls at a template lesion, RecA filaments onto the lesion-containing gap and the fork is remodeled (fork reversal). RecA bound to single-strand DNA (ssDNA) interacts with and recruits DisA and RadA/Sms on the branched DNA intermediates (stalled or reversed forks), but DisA and RadA/Sms limit RecA activities and DisA suppresses its c-di-AMP synthesis. We show that RecA, acting as an accessory protein, activates RadA/Sms to unwind the nascent lagging-strand of the branched intermediates rather than to branch migrate them. DisA limits the ssDNA-dependent ATPase activity of RadA/Sms C13A, and inhibits the helicase activity of RadA/Sms by a protein-protein interaction. Finally, RadA/Sms inhibits DisA-mediated c-di-AMP synthesis and indirectly inhibits cell proliferation, but RecA counters this negative effect. We propose that the interactions among DisA, RecA and RadA/Sms, which are mutually exclusive, contribute to generate the substrate for replication restart, regulate the c-di-AMP pool and limit fork restoration in order to maintain cell survival.
Highlights
Complete, accurate and timely DNA replication is essential to maintain genome integrity and cell proliferation
Our results support a comprehensive role of DisA at the intersection between recombination and replication restart, by regulating RecA and RadA/Sms activities at branched intermediates to prevent fork remodeling that should be pathological during spore revival
When the single genome of an inert mature haploid B. subtilis spore is damaged, unperturbed spore revival requires RecA, RecG, RadA/Sms and DisA, but not functions involved in end resection (as the RecJ singlestrand DNA (ssDNA) exonuclease in concert with a RecQ-like (RecS or RecQ) DNA helicase or the AddAB helicase/nucleases complex) (Vlasic et al, 2014; Raguse et al, 2017)
Summary
Accurate and timely DNA replication is essential to maintain genome integrity and cell proliferation. Replication of DNA containing damaged template bases or DNA distortions can lead to fork reversal ( named fork regression), i.e., the coordinated annealing of the two nascent strands, leading to a structure resembling a Holliday junction (HJ) (Atkinson and McGlynn, 2009; Marians, 2018). This fork remodeling mechanism has emerged as a global and genetically controlled response to aid repair or bypass of DNA damage upon replication stress during the early stage of Bacillus subtilis spore revival (Vlasic et al, 2014; Raguse et al, 2017) as well as in mammalian cells (Branzei and Foiani, 2010; Neelsen and Lopes, 2015; Quinet et al, 2017; Berti et al, 2020). In Escherichia coli, when replication forks encounter template lesions they are skipped, but replication-transcription conflicts mostly trigger fork reversal (Marians, 2018; Wong et al, 2021).
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