RecO is critical for the assembly of RecA-mediated DNA repair complexes in Bacil
In all domains of life, the ability to repair DNA is critical to maintaining genomic stability. Double-strand breaks (DSB’s) and replication fork arrest requires a series of recombinational (rec) proteins that are recruited to the site of damage to facilitate repair. RecA protein is central to maintaining genome integrity. RecA stabilizes stalled replication forks, plays a regulator role in the SOS-transcriptional response to DNA damage, and induces strand exchange during homologous recombination. Previously, we showed that the DNA damage-dependent organization of RecA-GFP into foci requires ongoing DNA replication. However, the mechanism that governs the recruitment of RecA-GFP in response to DNA replication stress has remained unclear. Since it is known that RecO contributes to the loading of RecA onto ssDNA in vitro, we asked if the recO gene was necessary to observe the organization of RecA-GFP into foci following DNA damage in vivo. Consistent with previous studies, we show that cells challenged with the DNA damaging agent mitomycin C (MMC) showed an increase in the percentage of RecA-GFP foci. In striking contrast, we found that RecA-GFP focus formation was almost completely blocked in cells deficient for recO. Upon complementation of RecO function by expressing recO+ from an ectopic locus, we observed the complete restoration in the percentage of cells with RecA-GFP foci in response to MMC. With these data, we suggest that RecO is responsible for recruitment of RecA to sites of replication fork stress. Moreover, we present additional results that define the dependence of RecA on other DNA repair proteins for organization into repair complexes.