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Updated: Aug 4, 2025

Quantifying Replication Stress in Ovarian Cancer Cells Using Single-Stranded DNA Immunofluorescence
Published on: February 10, 2023
Excessive reactive oxygen species induce transcription-dependent replication stress
Martin Andrs1,2, Henriette Stoy2, Barbora Boleslavska1,3
1Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic.
Abstract:
Elevated levels of reactive oxygen species (ROS) reduce replication fork velocity by causing dissociation of the TIMELESS-TIPIN complex from the replisome. Here, we show that ROS generated by exposure of human cells to the ribonucleotide reductase inhibitor hydroxyurea (HU) promote replication fork reversal in a manner dependent on active transcription and formation of co-transcriptional RNA:DNA hybrids (R-loops). The frequency of R-loop-dependent fork stalling events is also increased after TIMELESS depletion or a partial inhibition of replicative DNA polymerases by aphidicolin, suggesting that this phenomenon is due to a global replication slowdown. In contrast, replication arrest caused by HU-induced depletion of deoxynucleotides does not induce fork reversal but, if allowed to persist, leads to extensive R-loop-independent DNA breakage during S-phase. Our work reveals a link between oxidative stress and transcription-replication interference that causes genomic alterations recurrently found in human cancer.
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