Formation of co-transcriptional R-loops underlies replication fork stalling upon head-on transcriptionreplication encounters. Here, we demonstrate that RAD51-dependent replication fork reversal induced by R-loops is followed by the restart of semiconservative DNA replication mediated by RECQ1 and RECQ5 helicases, MUS81/EME1 endonuclease, RAD52 strand-annealing factor, the DNA ligase IV (LIG4)/XRCC4 complex, and the non-catalytic subunit of DNA polymerase d, POLD3. RECQ5 disrupts RAD51 filaments assembled on stalled forks after RECQ1-mediated reverse branch migration, preventing a new round of fork reversal and facilitating fork cleavage by MUS81/EME1. MUS81-dependent DNA breaks accumulate in cells lacking RAD52 or LIG4 upon induction of R-loop formation, suggesting that RAD52 acts in concert with LIG4/XRCC4 to catalyze fork religation, thereby mediating replication restart. The resumption of DNA synthesis after R-loop-associated fork stalling also requires active transcription, the restoration of which depends on MUS81, RAD52, LIG4, and the transcription elongation factor ELL. These findings provide mechanistic insights into transcription-replication conflict resolution. the paper; M.L. and A.P. revised and modified the paper.

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.

R-loops are three-stranded structures generated by annealing of nascent transcripts to the template DNA strand, leaving the non-template DNA strand exposed as a single-stranded loop. Although R-loops play important roles in physiological processes such as regulation of gene expression, mitochondrial DNA replication, or immunoglobulin class switch recombination, dysregulation of the R-loop metabolism poses a threat to the stability of the genome. A previous study in yeast has shown that the homologous recombination machinery contributes to the formation of R-loops and associated chromosome instability. On the contrary, here, we demonstrate that depletion of the key homologous recombination factor, RAD51, as well as RAD51 inhibition by the B02 inhibitor did not prevent R-loop formation induced by the inhibition of spliceosome assembly in human cells. However, we noticed that treatment of cells with B02 resulted in RAD51-dependent accumulation of R-loops in an early G1 phase of the cell cycle accompanied by a decrease in the levels of chromatin-bound ORC2 protein, a component of the pre-replication complex, and an increase in DNA synthesis. Our results suggest that B02-induced R-loops might cause a premature origin firing.