Rescuing bacterial genome replication: essential functions to repair a double-strand break and restart DNA synthesis

Many antibiotics indirectly generate reactive oxygen species (ROS) that can damage bacterial genomes. Oxidised nucleobases become genotoxic when they are targeted for repair through excision, generating a single-strand discontinuity that can be converted to a double-strand break (DSB) by an oncoming replication fork. Because the genomic location of nucleobase oxidation is stochastic, investigating the fate of DNA replication machinery (replisome) at single-strand discontinuities has been limited. Here we have addressed this issue by expressing Cas9 nickases in Bacillus subtilis to create site specific single-strand discontinuities in a bacterial chromosome. We find that nicks in either leading or lagging strand arrest bacterial replication fork progression and generate a DSB that requires repair using homologous recombination to allow replication restart. These discoveries provoke reassessment of the fundamental mechanism of bacterial homologous recombination and provide insights to the development of alternative antimicrobials by identifying a specific pathway that can potentiate ROS-dependent bacterial killing.