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The rapid delivery of RNase H variants reveals distinct classes of RNA:DNA hybrids.
EMBO J.
Multiple lines of evidence suggest that RNase H1-sensitive RNA:DNA hybrids coulddisrupt the stability and restart of stalled replication forks. The prolongedover-expression of RNase H1 is the most popular method to manipulate RNA:DNAhybrids in situ but it cannot discriminate between direct and indirect effectsresulting from their loss. To overcome this limitation, we developed iCoRD (inCellulo RNase H Delivery) to rapidly deliver comparable amounts of eitherEscherichia coli RNase H1 (RnhA) or control proteins in live cells, with anegligible impact on the transcriptome. iCoRD-delivered RnhA recognises RNA:DNAhybrids in situ, is enriched in the vicinity of active replication forks andrescues replication fork progression in different stress conditions. WhileiCoRD-delivered RnhA had a clear impact on stress-induced, fork-proximal RNA:DNAhybrids 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 theexistence of distinct populations of RNA:DNA hybrids that are best mapped bydifferent methods and are differently sensitive to RNase H1 activity in situ.
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