Replication Restart after Replication-Transcription Conflicts Requires RecA in Bacillus subtilis

Abstract

Efficient 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.

Importance: Head-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.

FIG 1

(A) CFU arising from exponentially growing cultures at an OD₆₀₀ of 0.3 were enumerated in the presence (+) and absence (−) of transcription (Trx) from P_xis-lacZ. (B) Plating efficiencies (transcription-specific survival) were determined by enumerating the ratio of CFU arising from cultures of strains expressing P_xis-lacZ to that from strains repressed for transcription of the construct in a given mutant background. Data shown are averages from 8 to 12 biological replicates per strain. Error bars represent standard error of the mean (A) or standard deviations (B).

FIG 2

(A) RecA-GFP focus formation was quantified by microscopy in the absence (−) and presence (+) of transcription (Trx) of the P_xis-lacZ gene. (B) Relative association of RecA with the head-on gene-containing region was measured by ChIP-qPCR in the absence and presence of transcription of the P_xis-lacZ gene. Data shown represent averages from 6 to 16 biological replicates. Error bars represent standard error of the mean.

FIG 3

(A) CFU arising from exponentially growing cultures at an OD₆₀₀ of 0.3 were enumerated in the presence (+) and absence (−) of transcription (Trx) from P_xis-lacZ. (B) Plating efficiencies (transcription-specific survival) were determined by enumerating the ratio of CFU arising from cultures of strains expressing P_xis-lacZ to that from strains repressed for transcription of the construct in a given mutant background. Data shown are averages from 8 to 14 biological replicates per strain. Error bars represent standard error of the mean (A) or standard deviation (B).

FIG 4

Relative association of DnaD with the head-on gene-containing region was measured by ChIP-qPCR in the absence (−) and presence (+) of transcription (Trx) of the P_xis-lacZ gene. Data shown represent averages from 12 biological replicates. Error bars represent standard error of the mean.

FIG 5

Model for restart upon replication-transcription collision in B. subtilis. RecFOR may recognize single-stranded DNA at stalled forks, leading to RecA loading, recombination, and restart. Alternatively, double-strand breaks in the DNA at stalled forks may be processed by AddAB, leading to RecA loading, recombination, and restart.