Rescuing the bacterial replisome at a nick requires recombinational repair and helicase reloading
Year: 2025
Type: article
Abstract: ABSTRACT DNA damage occurs in all cells and must be repaired to maintain genome integrity. Many DNA lesions are targeted for removal by repair systems that excise the damage, thereby generating a temporary single-strand discontinuity in the chromosome. If DNA repair has not been completed prior to a round of genome duplication, the single-strand discontinuity (nick or gap) can be converted to a double-strand break (DSB) by an oncoming replication fork. Because the genomic location of nucleobase damage is stochastic, investigating the fate of replication machinery (replisome) at DNA repair sites with 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 a nick in either leading or lagging strand arrests DNA replication, while the fate of the replicative helicase is distinct and depends upon the strand nicked. Genetic, biochemical, and single cell analyses indicate that replisome/nick encounters generate a single-end DSB which requires recombinational repair to enable PriA-dependent replication restart. Together this work defines the physiologically relevant pathway used by B. subtilis to reinitiate DNA synthesis following replication fork inactivation at a single-strand discontinuity. Highlights Single-strand discontinuities inactivate the bacterial replisome Nicks on the leading or lagging strand template differentially affect fate of the helicase Replication forks are repaired via recombinational repair The single strand binding protein (SSB) acidic tail is essential for replication restart DNA replication restart is PriA-dependent GRAPHICAL ABSTRACT
Source: Nature Communications
Institution: Newcastle University
Language: English
Topic: DNA Repair Mechanisms
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $6,990
Funder: Wellcome Trust
OpenAlex ID: W4416715813