G-quadruplex binders cause DNA damage by inducing R-loops in human cancer cells

G-Quadruplexes (G4s) and R-loops are non-B DNA structures that can regulate transcription and replication. G4s are formed from four guanine residues that are held together in the same plane by Hoogsteen hydrogen bonds and further stabilized by the presence of monovalent cations. R-loops are triple-strand structures that contain an RNA-DNA hybrid and displaced single-stranded DNA. One of the most important features that influence these DNA structures is the GC content. Indeed, R-loop structures can be favoured by a high guanine density in the non-template DNA strand and this is specifically due to the higher thermodynamic stability of RNA-DNA hybrid. R-loops and G4s are generally regarded as highly deleterious, indeed the structures can block both transcription and DNA replication, creating replicative stress and potentially causing DNA damage. Here, we used immunofluorescence analysis in order to identify the increase of G4s and R-loops in cancer cells treated with specific G4 binders at long and very short time. At long time, the increase of these two non-B DNA structures triggers genomic DNA damage as established by the formation of γH2AX foci and other markers of cellular DNA damage response. Interestingly, stable and transient overexpression of RNaseH, an enzyme that specifically removes R-loop structures, induce a rescue of G4 binder-induced DNA damage and genome instability. Our study provides the first direct evidence of a mechanistic link between G4s and R-loops in human cancer cells.