Transcriptional repression accounts for RNA:DNA hybrids accumulation at DNA Double Strand breaks

RNA:DNA hybrids accumulate at the vicinity of DNA double-strand breaks (DSBs) and were shown to regulate homologous recombination repair. The mechanism that elicits the formation of these non-canonical RNA:DNA structures is yet unclear although they were proposed to arise consequently to RNA Polymerase II or III loading at the break site followed by DSB-induced de novo transcription. Here, we found that none of these RNA polymerases are recruited at DSBs. Rather, strand-specific R-loop mapping revealed that RNA:DNA hybrids are mainly generated from the hybridization of pre-existing pre-mRNA to the 3' overhang left by DNA end resection. We further identified the H3K4me3 reader Spindlin 1 to be essential for RNA:DNA hybrids accumulation at DSBs, through its role in mediating transcriptional repression in cis to DSBs. Altogether, we provide evidence that RNA:DNA hybrids accumulate at DSBs occurring in transcribing loci as a result of DSB-induced transcriptional shut-down.