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Bacillus subtilis RarA modulates replication restart.

ABSTRACT: The ubiquitous RarA/Mgs1/WRNIP protein plays a crucial, but poorly understood role in genome maintenance. We show that Bacillus subtilis RarA, in the apo form, preferentially binds single-stranded (ss) over double-stranded (ds) DNA. SsbA bound to ssDNA loads RarA, and for such recruitment the amphipathic C-terminal domain of SsbA is required. RarA is a DNA-dependent ATPase strongly stimulated by ssDNA-dsDNA junctions and SsbA, or by dsDNA ends. RarA, which may interact with PriA, does not stimulate PriA DNA unwinding. In a reconstituted PriA-dependent DNA replication system, RarA inhibited initiation, but not chain elongation. The RarA effect was not observed in the absence of SsbA, or when the host-encoded preprimosome and the DNA helicase are replaced by proteins from the SPP1 phage with similar function. We propose that RarA assembles at blocked forks to maintain genome integrity. Through its interaction with SsbA and with a preprimosomal component, RarA might impede the assembly of the replicative helicase, to prevent that recombination intermediates contribute to pathological DNA replication restart.

SUBMITTER: Carrasco B

PROVIDER: S-EPMC6101539 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Bacillus subtilis RarA modulates replication restart.

Carrasco Begoña B Seco Elena M EM López-Sanz María M Alonso Juan C JC Ayora Silvia S

Nucleic acids research 20180801 14

The ubiquitous RarA/Mgs1/WRNIP protein plays a crucial, but poorly understood role in genome maintenance. We show that Bacillus subtilis RarA, in the apo form, preferentially binds single-stranded (ss) over double-stranded (ds) DNA. SsbA bound to ssDNA loads RarA, and for such recruitment the amphipathic C-terminal domain of SsbA is required. RarA is a DNA-dependent ATPase strongly stimulated by ssDNA-dsDNA junctions and SsbA, or by dsDNA ends. RarA, which may interact with PriA, does not stimul ...[more]

PMID: 29947798

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Project description:UnlabelledEfficient duplication of genomes depends on reactivation of replication forks outside the origin. Replication restart can be facilitated by recombination proteins, especially if single- or double-strand breaks form in the DNA. Each type of DNA break is processed by a distinct pathway, though both depend on the RecA protein. One common obstacle that can stall forks, potentially leading to breaks in the DNA, is transcription. Though replication stalling by transcription is prevalent, the nature of DNA breaks and the prerequisites for replication restart in response to these encounters remain unknown. Here, we used an engineered site-specific replication-transcription conflict to identify and dissect the pathways required for the resolution and restart of replication forks stalled by transcription in Bacillus subtilis. We found that RecA, its loader proteins RecO and AddAB, and the Holliday junction resolvase RecU are required for efficient survival and replication restart after conflicts with transcription. Genetic analyses showed that RecO and AddAB act in parallel to facilitate RecA loading at the site of the conflict but that they can each partially compensate for the other's absence. Finally, we found that RecA and either RecO or AddAB are required for the replication restart and helicase loader protein, DnaD, to associate with the engineered conflict region. These results suggest that conflicts can lead to both single-strand gaps and double-strand breaks in the DNA and that RecA loading and Holliday junction resolution are required for replication restart at regions of replication-transcription conflicts.ImportanceHead-on conflicts between replication and transcription occur when a gene is expressed from the lagging strand. These encounters stall the replisome and potentially break the DNA. We investigated the necessary mechanisms for Bacillus subtilis cells to overcome a site-specific engineered conflict with transcription of a protein-coding gene. We found that the recombination proteins RecO and AddAB both load RecA onto the DNA in response to the head-on conflict. Additionally, RecA loading by one of the two pathways was required for both replication restart and efficient survival of the collision. Our findings suggest that both single-strand gaps and double-strand DNA breaks occur at head-on conflict regions and demonstrate a requirement for recombination to restart replication after collisions with transcription.

Project description:By chance, we discovered a window of extracellular magnesium (Mg2+) availability that modulates the division frequency of Bacillus subtilis without affecting its growth rate. In this window, cells grown with excess Mg2+ produce shorter cells than do those grown in unsupplemented medium. The Mg2+-responsive adjustment in cell length occurs in both rich and minimal media as well as in domesticated and undomesticated strains. Of other divalent cations tested, manganese (Mn2+) and zinc (Zn2+) also resulted in cell shortening, but this occurred only at concentrations that affected growth. Cell length decreased proportionally with increasing Mg2+ from 0.2 mM to 4.0 mM, with little or no detectable change being observed in labile, intracellular Mg2+, based on a riboswitch reporter. Cells grown in excess Mg2+ had fewer nucleoids and possessed more FtsZ-rings per unit cell length, consistent with the increased division frequency. Remarkably, when shifting cells from unsupplemented to supplemented medium, more than half of the cell length decrease occurred in the first 10 min, consistent with rapid division onset. Relative to unsupplemented cells, cells growing at steady-state with excess Mg2+ showed an enhanced expression of a large number of SigB-regulated genes and the activation of the Fur, MntR, and Zur regulons. Thus, by manipulating the availability of one nutrient, we were able to uncouple the growth rate from the division frequency and identify transcriptional changes that suggest that cell division is accompanied by the general stress response and an enhanced demand to sequester and/or increase the uptake of iron, Mn2+, and Zn2+. IMPORTANCE The signals that cells use to trigger cell division are unknown. Although division is often considered intrinsic to the cell cycle, microorganisms can continue to grow and repeat rounds of DNA replication without dividing, indicating that cycles of division can be skipped. Here, we show that by manipulating a single nutrient, namely, Mg2+, cell division can be uncoupled from the growth rate. This finding can be applied to investigate the nature of the cell division signal(s).

Dataset Information

Bacillus subtilis RarA modulates replication restart.

Publications

Bacillus subtilis RarA modulates replication restart.

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