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bioRxiv · 10.1101/2025.02.28.640380

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

Winterhalter, C.·Fenyk, S.·Murray, H.
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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. HighlightsO_LISingle-strand discontinuities inactivate the bacterial replisome C_LIO_LINicks on the leading or lagging strand template differentially affect fate of the helicase C_LIO_LIReplication forks are repaired via recombinational repair C_LIO_LIThe single strand binding protein (SSB) acidic tail is essential for replication restart C_LIO_LIDNA replication restart is PriA-dependent C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/640380v2_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@10734e0org.highwire.dtl.DTLVardef@208eddorg.highwire.dtl.DTLVardef@183794aorg.highwire.dtl.DTLVardef@901628_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Winterhalter, C., Fenyk, S., Murray, H.. 2025-03-02. Rescuing bacterial genome replication: essential functions to repair a double-strand break and restart DNA synthesis. https://doi.org/10.1101/2025.02.28.640380

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