Résumé
SUMMARY The over-expression of RNase H1 enables the progression of replication forks under stress and safeguards genome stability. The most common interpretation of these observations is that RNase H1 over-expression removes R-loops, whose formation would hinder replication fork progression. Here we challenge this model using an innovative strategy to rapidly deliver ready-made Escherichia coli RNase H1 (RnhA) in live cells. We show that RnhA is enriched in the vicinity of active replication forks and that it rescues replication fork progression in different stress conditions. However, RnhA had no impact on R-loops mapped by the DRIP or Cut&Tag methods. Our results reveal the existence of distinct populations of RNA:DNA hybrids that are differently sensitive to RnhA and invites new interpretations of well-established observations. HIGHLIGHTS Rapid delivery of R-loop sensors and regulators in live cells Rapid delivery of RnhA rescues replication fork progression in stress conditions Rapid delivery of RnhA does not impact R-loops DRIP detects RNA:DNA species insensitive to RNase H1 in cells