DNA:RNA hybrids form at DNA double-strand breaks in transcriptionally active loci.

The recent discovery of DNA:RNA hybrids, or R-loops, actively forming at DNA double-strand breaks (DSBs) has unlocked fresh insight into how RNA participates in DNA repair. However, the manner of DSB-induced R-loop formation is vital in determining its mechanism of action and is currently under debate. Here, we analyse published DNA:RNA-hybrid sequencing to elucidate the features that determine DSB-induced R-loop formation. We found that pre-existing transcriptional activity was critical for R-loop generation at break sites, suggesting that these RNAs are transcribed prior to break induction. In addition, this appeared to be a specific DSB response at the break, distinct from traditional, co-transcriptionally formed R-loops. We hypothesise that R-loop formation is orchestrated by the damage response at transcriptionally active DSB loci to specifically maintain these genomic regions. Further investigation is required to fully understand how canonical repair processes regulate R-loops at breaks and how they participate in the repair process.