Excessive reactive oxygen species induce transcription-dependent replication stress

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.

MutSβ-MutLβ-FANCJ axis mediates the restart of DNA replication after fork stalling at cotranscriptional G4/R-loops

“…Our data implicating FANCJ in G4 unwinding during the restart of R-loop–stalled forks are consistent with the previous observation of a FANCJ requirement for unrestrained DNA synthesis in HLTF-deficient cells exposed to hydroxyurea (HU) ( 46 ), since HU-induced replication fork stalling is known to be caused by R-loops ( 47 ). Moreover, abrogation of HU-induced fork reversal by HLTF depletion has been shown to trigger replication restart via the MUS81-LIG4-ELL axis ( 47 ).…”

RNase H2 degrades toxic RNA:DNA hybrids behind stalled forks to promote replication restart

“…RNase H could therefore target different structures at HU‐arrested forks and at replication barriers such as those induced by CPT and RTS1 . This view is consistent with the fact that different fork restart mechanisms operate in the presence of HU and CPT and that slow forks are particularly sensitive to TRCs (Chappidi et al , 2020; Andrs et al , 2023).…”

“…To investigate the requirement for RNase H1 and RNase H2 in cells exposed to replication stress, we first compared the growth of wild type (WT), rnh1Δ , rnh201Δ , and rnh1Δ rnh201Δ cells in the presence of two genotoxic agents, MMS and HU. MMS blocks replication forks by alkylating DNA and HU slows down DNA synthesis by depleting dNTP pools and inducing an oxidative stress (Koc et al , 2004; Poli et al , 2012; Somyajit et al , 2017; Andrs et al , 2023). All four strains grew at the same rate in the absence of drugs.…”

“…Indeed, unlike HU, aphidicolin completely blocks fork progression when used at high concentration and would therefore prevent the conversion of R‐loops into toxic RNA:DNA hybrids. The view that deleterious consequences on fork structure arise from the encounter of slow‐moving forks and transcription complexes is supported by a recent study showing that HU promotes R‐loop formation at TRCs and drives fork reversal (Andrs et al , 2023). In RNase H‐deficient cells, dealing with these conflicts would be particularly challenging when slow forks encounter multiple RNAPII complexes and R‐loops in a head‐on orientation, as it is the case at TTS (Skourti‐Stathaki et al , 2011; Promonet et al , 2020).…”

Transcription–replication conflicts underlie sensitivity to PARP inhibitors

“…Recent observations by others are consistent with the TRC mechanism proposed here. Depletion of TIMELESS induces formation of R-loops 43 , consistent with induction of TRCs; PARP1 binds to R-loops 44 ; PARP inhibitors enhance replication fork speed 45 and HR repairs DNA damage induced by TRCs 33 , 34 .…”