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

Abstract

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.

Fig. 1. DSB-induced R-loops form in a transcription-dependent manner.

Boxplots (top) and metagenes (bottom) of R-loop generation at ASISI-induced double-strand breaks. a The 99 breaks are grouped by their repair pathway preference (determined by Aymard et al.). b Breaks are grouped by their genomic location status. c Breaks are grouped by their relative transcriptional activity (determined by Aymard et al.). DRIP read coverage was calculated and normalised to the total read number; then the log2 fold change of broken (+4OHT) over unbroken (−4OHT) was calculated. Statistics were calculated using a directional, unpaired Wilcoxon signed-rank non-parametric test, *p < 0.05, **p < 0.01.

Fig. 2. Transcriptional activity is a central driving force behind DSB-induced R-loop formation.

a Genome browser plots of ASISI-induced double-strand breaks with and without break induction where R-loop generation correlates with transcriptional activity. Two individual sites are shown; one with high transcriptional activity (top) and one with low transcriptional activity (bottom). b Same as a, except at sites where R-loop generation does not correlate with transcriptional activity. c Correlation plot of relative transcriptional activity against the break-induced R-loop generation at each of the ASISI-induced double-strand break sites. Correlation and p values determined by Pearson correlation testing. d Heatmap of break-induced R-loop generation around each ASISI-induced double-strand break site with the sites ordered from the highest (top) to the lowest (bottom) transcriptional activity.