Fig. 2. TERRA forms R-loops in trans inducing telomere fragility in dependency of RAD51.

a, Detection of telomeric hybrids on dot-blots. Telomeric hybrids were isolated by DNA-RNA immunoprecipitation with S9.6 antibody from cells with long or short telomeres. Cells expressed either the PP7 stem loops or the PP7-15q TERRA and had been transfected with control siRNA (siC), siRNAs against RNaseH1 (RNH1), or with a plasmid that overexpressed RNH1. In vitro treatment with RNaseH1 served as a negative control. Quantification of signals is shown on the right. b, Schematic representation of PP7-15q TERRA expressed from a plasmid and forming R-loops in trans at telomeres 1q, 10q and 13q. Telomeric hybrids arising at the indicated chromosome ends were quantified by qPCR with primers amplifying specific subtelomeric DNA next to the telomeric tracts in cells with long telomeres. c, Telomere fragility induced upon expression of TERRA from a plasmid. The fraction of fragile telomeres was quantified on metaphase chromosomes stained with a telomeric FISH probe. d, Effects of RNaseH1 and RAD51 on telomere fragility. In cells expressing PP7+15qTERRA from a plasmid, telomere fragility was quantified upon depletion (si) or overexpression (OE) of RNaseH1 (RNH1), or depletion of RAD51. e, Detection of endogenous telomeric R-loops in siC, siRNH1 and siRAD51 transfected cells. For all data n=3 biologically independent experiments, data are mean ± s.d. Two-tailed unpaired t test was used to calculate the P values. (*P < 0.05; **P < 0.01; ***P < 0.001). For Fig. c and d the number of metaphases scored are indicated for each condition in Extended Data Fig. 6 b and c.