Single-Molecule Fluorescence Imaging of Reco Localization and Dynamics in Bacillus Subtilis

In all organisms, the high fidelity of DNA replication is essential for maintenance of chromosome integrity. DNA damage can be caused by polymerase errors or by external factors (e.g., X-rays or mutagenic chemicals). Thus, the cell has evolved a number of repair mechanisms to respond to different types of damage. In B. subtilis, repair of double-strand breaks (DSBs) in the DNA occurs through RecA-mediated homologous recombination. The mechanism by which RecA finds DSBs in vivo is not well described, but is believed to involve the proteins RecF, RecO, and RecR. This role for RecO in DSB repair in B. subtilis is distinct from its role in E. coli but analogous to that of Rad52 in eukaryotes, making B. subtilis an excellent model system for studying cellular response to DNA damage. Previously, bulk fluorescence studies have shown that RecO forms foci after the induction of double-strand breaks. However, RecO in undamaged cells can only be visualized when overexpressed, leaving questions about its true localization at wild type expression levels. Here, we have created cells in which PAmCherry-RecO is natively expressed from the RecO promoter as the only RecO source. We use single-molecule fluorescence microscopy in live B. subtilis to show that RecO rapidly forms foci following treatment with the DNA damaging agent phleomycin, but is diffuse throughout the cell under non-damaging conditions. This result suggests that, unlike several other proteins involved in DNA repair, RecO is not associated with the replisome prior to DSB recognition. Future work will examine the previously reported role of single-strand binding protein (SSB) in recruitment of RecO to DSBs.