RecO and RecR Are Necessary for RecA Loading in Response to DNA Damage and Replication Fork Stress

Abstract

bRecA is central to maintaining genome integrity in bacterial cells. Despite the near-ubiquitous conservation of RecA in eubacteria, the pathways that facilitate RecA loading and repair center assembly have remained poorly understood in Bacillus subtilis. Here, we show that RecA rapidly colocalizes with the DNA polymerase complex (replisome) immediately following DNA damage or damage-independent replication fork arrest. In Escherichia coli, the RecFOR and RecBCD pathways serve to load RecA and the choice between these two pathways depends on the type of damage under repair. We found in B. subtilis that the rapid localization of RecA to repair centers is strictly dependent on RecO and RecR in response to all types of damage examined, including a site-specific double-stranded break and damage-independent replication fork arrest. Furthermore, we provide evidence that, although RecF is not required for RecA repair center formation in vivo, RecF does increase the efficiency of repair center assembly, suggesting that RecF may influence the initial stages of RecA nucleation or filament extension. We further identify singlestranded DNA binding protein (SSB) as an additional component important for RecA repair center assembly. Truncation of the SSB C terminus impairs the ability of B. subtilis to form repair centers in response to damage and damage-independent fork arrest. With these results, we conclude that the SSB-dependent recruitment of RecOR to the replisome is necessary for loading and organizing RecA into repair centers in response to DNA damage and replication fork arrest.

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

“…Two pathways potentially load RecA onto DNA in B. subtilis: RecFOR at single-strand gaps or AddAB at double-strand breaks. Previous studies indicated that RecO is required for RecA localization in response to treatment with DNA-damaging agents in cells grown in minimal medium (16). However, the potential contribution of either pathway to RecA loading in cells grown in rich medium without exogenous damage is unclear.…”

“…AddAB processes double-strand breaks to generate 3= overhangs (22). Previous work suggested that RecO is required to load RecA onto the DNA regardless of the type of damage, even at doublestrand breaks (16). These investigations used RecA-GFP focus formation as a readout for RecA loading in cells growing in minimal medium.…”

“…Depending on the type of DNA break, RecA is loaded onto the DNA (1) by either the RecFOR or AddAB pathway (14)(15)(16)(17). RecFOR recognizes single-strand gaps in the DNA (18), likely through an interaction with the single-stranded DNA binding protein (SSB) (19).…”

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

“…In Bacillus subtilis and Deinococcus radiodurans, the Rec-FOR system is the principal mediator that loads RecA onto ss-DNA. In B. subtilis, recR and recO are required for RecA focus formation on the chromosome in response to replication stress and exogenous DNA damage (17). Loss of RecF does not abolish RecA focus formation during damage but does delay the kinetics, including following a DSB produced by the homing endonuclease I-SceI (17).…”

“…In B. subtilis, recR and recO are required for RecA focus formation on the chromosome in response to replication stress and exogenous DNA damage (17). Loss of RecF does not abolish RecA focus formation during damage but does delay the kinetics, including following a DSB produced by the homing endonuclease I-SceI (17). In contrast, all three components of the RecFOR system, in addition to RecA, are required for B. subtilis spore resistance to ionizing radiation (IR) and desiccation (18).…”