Résumé
Transcription-replication conflicts represent a major source of genomic instability but the mechanisms that underlie these conflicts remain poorly understood. Part of the problem could come from non-B DNA structures called R-loops, which are formed of an RNA:DNA hybrid and a displaced ssDNA loop. RNA:DNA hybrids could directly interfere with DNA replication by acting as roadblocks. Alternatively, these structures could form at stalled forks as a consequence of fork arrest and interfere with restart. To discriminate between these two possibilities, we monitored DNA replication in budding yeast and human cells devoid of RNase H activity. RNase H1 and 2 remove RNA:DNA hybrids, ribonucleosides monophosphate wrongly incorporated in the genome and RNA primers during Okazaki fragments maturation. Loss of RNase H activity sensitizes cells to genotoxic agents and is associated with genome instability. Using DNA combing, we found that rnh1Δ rnh201Δ yeast mutants fail to restart replication after MMS‐induced replication stress.Failure to restart replication is associated with reduced RPA loading at HU and MMS‐arrested forks, indicating that RNase H promotes the resection of nascent DNA at stalled forks. In human cells, we also found that RNase H2 depletion blocks resection of nascent DNA. Interestingly, this defect could be rescued by transcription inhibition and degradation of RNA polymerases II. Altogether, these data suggest that in the absence of RNase H, cotranscriptional R-loops are converted into toxic RNA:DNA hybrids upon fork passage, which interfere with fork remodeling events involved in fork restart.