Fig. 4: Replication forks stall and reverse while facing a TRC in B. subtilis.
From: Direct visualization of transcription-replication conflicts reveals post-replicative DNA:RNA hybrids
a, Model system and experimental workflow; red boxes indicate the area from which linear and replicating conflict (RI) regions were extracted for further analysis. gDNA, genomic DNA. b, qPCR–DRIP analysis of accumulation of DNA:RNA hybrids within the conflict region in WT and Δrnhc mutant strains with or without IPTG induction and with or without in vitro RNase H digestion. Data were normalized to a negative locus and are shown as mean ± s.d., n = 3 independent biological replicates. Significance was determined by ordinary one-way ANOVA, followed by Sidak test. c–e, Native EM analysis of RIs extracted from the RI region, marked in Extended Data Figure 5a (data from additional replicates of this experiment are provided in Extended Data Figure 5b–g). c, Fragment lengths of all imaged RIs from one replicate. The two numbers on top indicate the number of RI within the expected size range (top number, dark dots) and the number of total RIs imaged (in parentheses, all dots). d, Top, alignment of selected RIs, according to daughter strand length. Daughter strands are indicated in green, and parental strands in gray. Reversed forks are labeled in pink and black, with pink marking length of the regressed arm and black the length of the parental strand prior to reversal. Bottom, representative electron micrographs of normal (I, II, IV) and reversed (III, V, VI) forks. Scale bars, 500 nm. e, Fork reversal frequency, shown as mean ± s.d., n = 3 independent biological replicates (the corresponding fragment maps for the additional replicates can be found in Extended Data Figure 5e,g). Numbers below the graph indicate the total number of molecules analyzed in all three experiments. Significance was determined by ordinary one-way ANOVA, followed by Sidak test.
