The rapid delivery of RNase H variants reveals distinct classes of RNA:DNA hybrids

Abstract Multiple lines of evidence suggest that RNase H1-sensitive RNA:DNA hybrids could disrupt the stability and restart of stalled replication forks. The prolonged over-expression of RNase H1 is the most popular method to manipulate RNA:DNA hybrids in situ but it cannot discriminate between direct and indirect effects resulting from their loss. To overcome this limitation, we developed iCoRD (in Cellulo RNase H Delivery) to rapidly deliver comparable amounts of either Escherichia coli RNase H1 (RnhA) or control proteins in live cells, with a negligible impact on the transcriptome. iCoRD -delivered RnhA recognises RNA:DNA hybrids in situ, is enriched in the vicinity of active replication forks and rescues replication fork progression in different stress conditions. While iCoRD -delivered RnhA had a clear impact on stress-induced, fork-proximal RNA:DNA hybrids and R-ChIP signals, it had no effect on DRIP or S9.6 Cut&Tag signals, even when these were induced by stress. Our results strongly suggest the existence of distinct populations of RNA:DNA hybrids that are best mapped by different methods and are differently sensitive to RNase H1 activity in situ.