As summarized above, DNA damage can result from the accumulation of diverse RNA-containing structures, including DNA:RNA hybrids. Strikingly, the last decade has also revealed that DNA:RNA hybrids accumulate in
cis at DSBs caused by reactive oxygen species, laser irradiation or site-specific endonucleases, as observed in several distant eukaryotes from yeast to metazoans [
5,
10]. In this context, the hybrids have been proposed to form as a consequence of de novo transcription initiating at DNA ends. Accordingly, RNA synthesis at DSBs is associated with increased recruitment of RNA polymerase II [
76,
77,
78], with reported contributions from RNA polymerase III [
79] or RNA polymerase IV in plants [
80]. In this view, DNA ends bound by MRN (MRE11-RAD50-NBS1) function as promoters and assemble canonical pre-initiation complexes, resulting in bidirectional transcription from the DSB site [
78]. Annealing of the newly synthesized RNA to the 3′ DNA overhang generated by resection then forms double-stranded DNA:RNA duplexes at the break (c). Since hybrid accumulation has been preferentially detected at DSBs located in transcriptionally-active regions [
81], it is also likely that changes in the dynamics of ongoing transcription contribute to the accumulation of canonical three-stranded R-loop structures engaging the pre-existing RNAs (c). There is also evidence that in metazoans, DSBs trigger the formation of small RNA species arising from the processing of double-stranded RNAs, which themselves result from the hybridization of de novo and pre-existing transcripts near the break site [
5]. Interestingly, a novel species of small single-stranded RNAs was recently reported to contribute to DNA repair in human cells [
82]. In line with the accumulation of various types of RNA and DNA:RNA hybrid species, DSBs recruit a growing number of RNA and R-loop processing factors, such as the exosome, the splicing machinery, RNase H2 and Sen1/senataxin [
57,
81,
83,
84,
85]. While the choreography of their respective recruitment will require further investigation, the network of RNA-dependent interactions formed at DSBs has the potential to influence their fate and repair.