R-loops at DNA double-strand breaks (DSBs) play a critical role in homologous recombination repair. An R-loop is a three-stranded nucleic acid structure consisting of an RNA-DNA hybrid and a displaced single-stranded DNA (ssDNA). The ssDNA within an R-loop serves as a recruitment platform for repair factors such as Rad52, which binds to single-stranded regions and initiates the repair process. Recent studies have shown that de novo R-loops are generated at DSBs; however, the mechanisms underlying their formation at break sites remain poorly understood. In this study, using newly developed single-molecule assays, we monitored the real-time co-transcriptional formation of R-loops at DSBs. We found that the probability of R-loop formation (R-loop efficiency) depends strongly on both the type of DSB and the direction of transcription. To elucidate the mechanisms driving R-loop formation at break sites, we quantitatively measured R-loop efficiency using a range of DNA substrates with defined lesions or abnormal structures. These experiments revealed a common principle underlying R-loop generation: RNA polymerases can continue elongation upon encountering single-stranded regions, thereby promoting R-loop formation. Importantly, we observed that co-transcriptional R-loops can also form on discontinuous ssDNA regions, suggesting that RNA polymerases may switch their transcription template to flap structures on the template strand or to 3′ overhangs of the non-template strand. Together, these findings advance our understanding of how RNA polymerases generate R-loops at DSBs and provide new insights into the molecular mechanisms underlying R-loop-mediated DNA repair.
Lim et al. (Sun,) studied this question.