Abstract
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.
Data availability
The data supporting the findings of this study are provided in the paper and its Supplementary information. Original microscopy images are too numerous and large to be uploaded to a public repository but are available upon request from the corresponding author. Source data are provided with this paper.
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Acknowledgements
We thank Fabrizio d’Adda di Fagagna for pBABEneo-HRASV12 construct, Stefano Ferrari for pLXSNneo-CCNE1 construct, Juan Mendez for PRIMPOL antibody, Eva Petermann for BJ-hTert HRASV12ER-TAM cells and Marcel van Vught for RPE1 CE-TetON cells. We thank to Oldrich Benada and Vlada Filimonenko for help with preparation of samples for EM. We also thank the UZH Center for Microscopy and Image Analysis, the Light Microscopy Core Facility of IMG (MEYS - LM2023050, MEYS - CZ.02.1.01/0.0/0.0/18_046/0016045, RVO − 68378050-KAV-NPUI), and the Electron Microscopy Core Facility of IMG (MEYS - LM2023050, ERDF CZ.02.1.01/0.0/0.0/18_046/0016045, CZ.02.01.01/00/23_015/0008205) for support. This work was supported by grants from the Swiss Cancer League (KFS-5484-02-2022 and KFS-6145-08-2024), the Swiss National Science Foundation (310030_214846), the Czech Science Foundation (25-15542S), Sassella Stiftung, and Stiftung zur Krebsbekämpfung. J.D. was supported by the Czech Science Foundation (21-22593X), H.H. was supported by the Czech Science Foundation (25-15199S). A.Z. was supported by the Charles University Grant Agency (GAUK188724).
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A.O., M.D., A.M., M.A., A.Z., K.S., B.B., V.R., C.K. and J.P. performed the experiments and analyzed the data. M.S. and A.O. performed EM experiments and analyzed the data. J.D., L.M., and H.H. contributed to the design of the experiments. P.J. conceived the study, analyzed the data, and wrote the manuscript with contributions from A.O., M.D. and A.M. All authors revised the manuscript.
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Oravetzova, A., Dvorakova, M., Mihai, AI. et al. Distinct mechanisms of replication stress induced by oncogenic RAS and cyclin E1 converge on R-loop-dependent fork reversal. Nat Commun (2026). https://doi.org/10.1038/s41467-026-71353-8
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DOI: https://doi.org/10.1038/s41467-026-71353-8
