The rapid delivery of RNase H variants reveals distinct classes of RNA:DNA hybrids
Multiple lines of evidence suggest that RNase H1-sensitive RNA:DNA hybrids (thereafter referred to as 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 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 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 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 hybrids that are best mapped by different methods and are differently sensitive to RNase H1 activity in situ.