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 (thereafter referred to as 'hybrids') could disrupt the stability and restart of stalled replication forks. The prolonged overexpression 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.
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BibTeX
@misc{bawadi2026rapid,
title = {{The rapid delivery of RNase H variants reveals distinct classes of RNA:DNA hybrids}},
author = {Yara Bawadi and Amandine Basille and Yea-Lih Lin and Connor Kirke and Clara Bourgeay and David Cluet and Laetitia Vachez and Virgile Dervieux and Laura Guiguettaz and Pascal Bernard and Stephan Hamperl and Philippe Pasero and Emiliano P Ricci and Vincent Vanoosthuyse},
howpublished = {HAL},
year = {2026},
month = sep,
doi = {10.64898/2026.09.23.753687},
url = {https://hal.science/hal-05785319v1},
note = {CC BY-NC-ND 4.0},
}
RIS
TY - UNPB
TI - The rapid delivery of RNase H variants reveals distinct classes of RNA:DNA hybrids
AU - Yara Bawadi
AU - Amandine Basille
AU - Yea-Lih Lin
AU - Connor Kirke
AU - Clara Bourgeay
AU - David Cluet
AU - Laetitia Vachez
AU - Virgile Dervieux
AU - Laura Guiguettaz
AU - Pascal Bernard
AU - Stephan Hamperl
AU - Philippe Pasero
AU - Emiliano P Ricci
AU - Vincent Vanoosthuyse
PY - 2026
DA - 2026/09/23
PB - HAL
DO - 10.64898/2026.09.23.753687
UR - https://hal.science/hal-05785319v1
AB - 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 overexpression 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.
ER -