Fig. 1: Single-strand discontinuities arrest DNA synthesis and generate DNA damage.

A Diagram illustrating the impact of leading and lagging strand discontinuities on a replication fork, with the bacterial replicative helicase translocating along the lagging strand template. B Genetic system employed to create a site-specific single-strand discontinuity in the B. subtilis chromosome. Black boxes correspond to antibiotic resistance markers, pink to Cas9 variants and green to sgRNA. Chromosome annotations refer to the replication origin (0°), terminus (180°) and sgRNA target sequence (yellow box). C Spot-titre analysis of strains engineered to express (+ xylose) either nCas9 or dCas9. Individual sgRNAs were designed to target the leading or lagging strand template at loci located at either -90° (top panel) or +90° (bottom panel). D MFA using whole genome sequencing of strains engineered to express (+ xylose) either nCas9 or dCas9. Individual sgRNAs were designed to target the leading or lagging strand template at loci located at either -90° (left panel) or +90° (right panel). The frequency of sequencing reads was plotted against genome position. E Fluorescence microscopy images of the DNA damage reporter mNG-RecA in strains engineered to express (+ xylose) either nCas9 or dCas9 (sgRNAs target -90°). Fluorescent RecA organises into bundles in the presence of DNA damage. Green signal corresponds to mNG-RecA fluorescence, grey scale images show corresponding phase contrast images. Scale bar indicates 2.5 µm. F Quantification of fluorescence microscopy images of the DNA damage reporter mNG-RecA in strains engineered to express (+ xylose) either nCas9 or dCas9 (sgRNA target -90°). Detection of spots and bundles corresponds to distinct foci or filament-like structures annotated in (E). Error bars indicate standard deviation and circles overlaid on bars correspond to two biological replicates. Source data are provided as a Source Data file.