Fig. 1: Systematic screens reveal novel sources of DNA:RNA hybrids.
From: DNA lesions can frequently precede DNA:RNA hybrid accumulation
a Principle of hyper-recombination and DNA:RNA hybrid detection assays in systematic screens. Reporter genes are positioned opposite to the unique plasmid replication origin (red dot) and can be replicated in both directions with respect to transcription. b Mutant strains assessed in Screen I (n = 4460) were divided along the x-axis according to recombination indices (left, hyper-rec; right, hypo-rec) and ranked according to their divergence from wt (p-value). Hashed lines indicate cutoff by p = 0.05. c Interaction densities between top-ranked hyper-recombinant strains from Screen I, as assessed by CLIK. Numbers (1,2,3): interaction densities significantly above background; red line: CLIK cutoff34. d Comparison between Screen I and Screen II hyper-recombinant hits. Out of 114 mutants identified in Screen I, only those exhibiting normal fitness in galactose induction conditions (n = 77) were assessed in Screen II. e Gene Ontology analysis for genes whose deletion triggers significant hyper-recombination in both screens I and II (n = 39). f Recombination indices (Screen I) for control strains (****, p = 7.0 × 10−7). g Recombination levels (% LEU+ recombinants, Screen II) for control strains (wt vs tho ****, p = 1.9 × 10−33; wt vs rad52∆ ****, p = 1.4 × 10−18). h DNA:RNA hybrid levels for hyper-rec mutants common to Screen I and II (n = 39; ****, p < 0.0001), as assessed in Screen III. The position of tho and OF processing mutants is indicated. YDL162c deletion overlaps the promoter of the DNA ligase I gene (CDC9) and is considered as a hypomorphic cdc9 allele. i Schematic representation of main players in OF processing. SSE: Structure Specific Endonucleases. The OF RNA primer appears in red. j Quantification of DNA:RNA hybrid foci in MCF7 breast cancer cells expressing the RHINO sensor and treated with the SC13 FEN1 inhibitor (n = 3; total number of cells: control, n = 38, SC13, n = 53; ****, p < 0.0001). For box plots (h, j), boxes extend from the 25th to 75th percentiles, with the median displayed as a line. The whiskers mark 1.5 times the inter-quartile range of the first or third quartile (Tukey’s definition), displaying all the values (h) or outliers (j) as individual points. Statistical tests: b, f, h, j Two-sided Mann–Whitney–Wilcoxon rank sum test; e Hypergeometric test; g Two-sided Fisher exact test. Source data are provided as a Source Data file.
