Fig. 2: Replication restart is necessary to resume DNA synthesis following replisome inactivation at a single-strand discontinuity.

A Diagram illustrating genomic loci probed via ChIP-qPCR at either -90° or +90°. The sgRNA targeted Cas9 protein variants to the locus at -90°. Lagging strand and leading strand templates are represented in black and grey lines, respectively (numbers refer to strand polarity). The blue arrow indicates leading strand synthesis and red arrows indicate lagging strand synthesis. B–F ChIP-qPCR analyses of helicase (DnaC) and replication restart proteins (PriA, DnaD, DnaB, DnaI) in strains engineered to express either nCas9 or dCas9. The sgRNAs targeted Cas9 proteins to a locus located at -90° and protein enrichment was normalised to the control locus at +90°. Error bars indicate the standard error of the mean and circles overlaid on bars correspond to three biological replicates. G Diagram illustrating the fate of helicase (red circle) upon encountering a single-strand discontinuity in either the leading or lagging strand template. H MFA using whole genome sequencing in strains engineered to express low levels of sgRNAs and Cas9 proteins (weak nicking system). The frequency of sequencing reads was plotted against genome position. I Genetic system employed to express limiting levels of PriA in strains harbouring the weak nicking system. J Spot-titre analysis of strains engineered to express nCas9 (+ xylose) with either high or limiting PriA. sgRNAs were targeted to a locus at -90°. K MFA using whole genome sequencing in strains engineered to express (+ xylose) nCas9 with either high or limiting PriA. The frequency of sequencing reads was plotted against genome position. Source data are provided as a Source Data file.