Excessive reactive oxygen species induce transcription-dependent replication stress
Martin Andrš, Henriette Stoy, Barbora Boleslavska, Nagaraja Chappidi, Radhakrishnan Kanagaraj, Zuzana Naščáková, Shruti Menon, Satyajeet Rao, Anna Oravetzova, Jana Dobrovolná, Kalpana Surendranath, Massimo Lopes, Pavel Janščák
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.