Distinct mechanisms of replication stress induced by oncogenic RAS and cyclin E1 converge on R-loop-dependent fork reversal.

Activated oncogenes elicit genomic instability by inducing DNA replication stress. Here we show that replication fork reversal and chromosome mis-segregation induced by oncogenic RAS (HRASV12) or cyclin E1 overexpression are largely caused by co-transcriptional RNA:DNA hybrids (R-loops) formed during S-phase. Furthermore, we demonstrate that replication stress induced by HRASV12, but not cyclin E1, is driven by reactive oxygen species (ROS) in a manner dependent on the replisome-associated ROS sensor peroxiredoxin 2 (PRDX2) and is linked to PRDX2-mediated release of the fork acceleration factor TIMELESS from the replisome. Inhibition of fork reversal in cells overexpressing HRASV12 or cyclin E1 induces unrestrained DNA synthesis mediated by the MUS81 endonuclease and the primase-polymerase PRIMPOL, thereby promoting proper chromosome segregation in mitosis. These results establish PRIMPOL repriming as part of the MUS81-dependent replication restart mechanism that operates at sites of R-loop-mediated transcription-replication conflicts to maintain genomic stability. Furthermore, our data indicate that, despite their protective role during S-phase, persistent reversed forks impair chromosome segregation in mitosis, potentially leading to DNA breaks and chromosomal rearrangements.