Abstract
DNA repair genes are generally considered tumor suppressors, as their inactivation is observed in tumors and is associated with carcinogenesis. Mutations in BRCA1 and BRCA2 genes are observed in breast, ovarian, and other cancers. This results in defective homologous recombination DNA repair, as well as in degradation of nascent DNA during replication stress, catalyzed by exonucleases including EXO1 and MRE11. However, most tumors are BRCA pathway-proficient. Here, we show that EXO1 is overexpressed in a significant proportion of tumors. EXO1 overexpression causes the degradation of nascent DNA at both single stranded DNA (ssDNA) gaps and reversed replication forks. Importantly, this degradation occurs efficiently in BRCA-proficient cells, through cooperation with MRE11. This results in increased double strand break formation and hypersensitivity to genotoxic agents. We thus identify increased EXO1 activity as a mechanism of genomic instability similar to BRCA pathway inactivation, but occurring more frequently in tumors compared to BRCA inactivation.
Data availability
All data supporting the findings of this study are available within the paper and its Supplementary Information. Source data are provided with this paper.
References
Hanahan, D. & Weinberg, R. A. Hallmarks of cancer: The next generation. Cell 144, 646–674 (2011).
Mazouzi, A., Velimezi, G. & Loizou, J. I. DNA replication stress: Causes, resolution and disease. Exp. Cell Res. 329, 85–93 (2014).
Zeman, M. K. & Cimprich, K. A. Causes and consequences of replication stress. Nat. Cell Biol. 16, 2–9 (2014).
Gaillard, H., Garcia-Muse, T. & Aguilera, A. Replication stress and cancer. Nat. Rev. Cancer 15, 276–289 (2015).
Berti, M. & Vindigni, A. Replication stress: Getting back on track. Nat. Struct. Mol. Biol. 23, 103–109 (2016).
Ray Chaudhuri, A. et al. Replication fork stability confers chemoresistance in BRCA-deficient cells. Nature 535, 382–387 (2016).
Quinet, A., Tirman, S., Cybulla, E., Meroni, A. & Vindigni, A. To skip or not to skip: choosing repriming to tolerate DNA damage. Mol. Cell 81, 649–658 (2021).
Quinet, A. et al. PRIMPOL-mediated adaptive response suppresses replication fork reversal in BRCA-deficient cells. Mol. Cell 77, 461–474.e469 (2020).
Piberger, A. L. et al. PrimPol-dependent single-stranded gap formation mediates homologous recombination at bulky DNA adducts. Nat. Commun. 11, 5863 (2020).
Taglialatela, A. et al. REV1-Polzeta maintains the viability of homologous recombination-deficient cancer cells through mutagenic repair of PRIMPOL-dependent ssDNA gaps. Mol. Cell 81, 4008–4025.e4007 (2021).
Tirman, S. et al. Temporally distinct post-replicative repair mechanisms fill PRIMPOL-dependent ssDNA gaps in human cells. Mol. Cell 81, 4026–4040.e4028 (2021).
Branzei, D. & Szakal, B. Building up and breaking down: mechanisms controlling recombination during replication. Crit. Rev. Biochem Mol. Biol. 52, 381–394 (2017).
Fu, Y. V. et al. Selective bypass of a lagging strand roadblock by the eukaryotic replicative DNA helicase. Cell 146, 931–941 (2011).
Taylor, M. R. G. & Yeeles, J. T. P. The initial response of a eukaryotic replisome to DNA damage. Mol. Cell 70, 1067–1080.e1012 (2018).
Bainbridge, L. J., Teague, R. & Doherty, A. J. Repriming DNA synthesis: an intrinsic restart pathway that maintains efficient genome replication. Nucleic Acids Res 49, 4831–4847 (2021).
Benureau, Y. et al. Changes in the architecture and abundance of replication intermediates delineate the chronology of DNA damage tolerance pathways at UV-stalled replication forks in human cells. Nucleic Acids Res. 50, 9909–9929 (2022).
Bianchi, J. et al. PrimPol bypasses UV photoproducts during eukaryotic chromosomal DNA replication. Mol. Cell 52, 566–573 (2013).
Mouron, S. et al. Repriming of DNA synthesis at stalled replication forks by human PrimPol. Nat. Struct. Mol. Biol. 20, 1383–1389 (2013).
Schlacher, K. et al. Double-strand break repair-independent role for BRCA2 in blocking stalled replication fork degradation by MRE11. Cell 145, 529–542 (2011).
Schlacher, K., Wu, H. & Jasin, M. A distinct replication fork protection pathway connects Fanconi anemia tumor suppressors to RAD51-BRCA1/2. Cancer Cell 22, 106–116 (2012).
Taglialatela, A. et al. Restoration of Replication Fork Stability in BRCA1- and BRCA2-Deficient Cells by Inactivation of SNF2-Family Fork Remodelers. Mol. Cell 68, 414–430.e418 (2017).
Dungrawala, H. et al. RADX Promotes Genome Stability and Modulates Chemosensitivity by Regulating RAD51 at Replication Forks. Mol. Cell 67, 374–386.e375 (2017).
Keijzers, G. et al. Human exonuclease 1 (EXO1) regulatory functions in DNA replication with putative roles in cancer. Int. J. Mol. Sci. 20, 74 (2018).
Prakash, R., Zhang, Y., Feng, W. & Jasin, M. Homologous recombination and human health: the roles of BRCA1, BRCA2, and associated proteins. Cold Spring Harb. Perspect. Biol. 7, a016600 (2015).
Spies, M. & Fishel, R. Mismatch repair during homologous and homeologous recombination. Cold Spring Harb. Perspect. Biol. 7, a022657 (2015).
Dhoonmoon, A., Nicolae, C. M. & Moldovan, G. L. The KU-PARP14 axis differentially regulates DNA resection at stalled replication forks by MRE11 and EXO1. Nat. Commun. 13, 5063 (2022).
Cantor, S. B. Revisiting the BRCA-pathway through the lens of replication gap suppression: “Gaps determine therapy response in BRCA mutant cancer”. DNA repair 107, 103209 (2021).
Cong, K. et al. Replication gaps are a key determinant of PARP inhibitor synthetic lethality with BRCA deficiency. Mol. Cell 81, 3128–3144.e3127 (2021).
Panzarino, N. J. et al. Replication gaps underlie BRCA deficiency and therapy response. Cancer Res. 81, 1388–1397 (2021).
Kang, Z. et al. BRCA2 associates with MCM10 to suppress PRIMPOL-mediated repriming and single-stranded gap formation after DNA damage. Nat. Commun. 12, 5966 (2021).
Berti, M. et al. Human RECQ1 promotes restart of replication forks reversed by DNA topoisomerase I inhibition. Nat. Struct. Mol. Biol. 20, 347–354 (2013).
Ray Chaudhuri, A. et al. Topoisomerase I poisoning results in PARP-mediated replication fork reversal. Nat. Struct. Mol. Biol. 19, 417–423 (2012).
Simoneau, A., Xiong, R. & Zou, L. The trans cell cycle effects of PARP inhibitors underlie their selectivity toward BRCA1/2-deficient cells. Genes Dev. 35, 1271–1289 (2021).
Thakar, T. et al. Lagging strand gap suppression connects BRCA-mediated fork protection to nucleosome assembly through PCNA-dependent CAF-1 recycling. Nat. Commun. 13, 5323 (2022).
Jackson, L. M. & Moldovan, G. L. Mechanisms of PARP1 inhibitor resistance and their implications for cancer treatment. NAR Cancer 4, zcac042 (2022).
Thakar, T. & Moldovan, G. L. The emerging determinants of replication fork stability. Nucleic Acids Res 49, 7224–7238 (2021).
Jackson, L. M. et al. Loss of MED12 activates the TGFbeta pathway to promote chemoresistance and replication fork stability in BRCA-deficient cells. Nucleic Acids Res 49, 12855–12869 (2021).
Thakar, T. et al. Ubiquitinated-PCNA protects replication forks from DNA2-mediated degradation by regulating Okazaki fragment maturation and chromatin assembly. Nat. Commun. 11, 2147 (2020).
Hale, A., Dhoonmoon, A., Straka, J., Nicolae, C. M. & Moldovan, G. L. Multi-step processing of replication stress-derived nascent strand DNA gaps by MRE11 and EXO1 nucleases. Nat. Commun. 14, 6265 (2023).
Nusawardhana, A., Pale, L. M., Nicolae, C. M. & Moldovan, G. L. USP1-dependent nucleolytic expansion of PRIMPOL-generated nascent DNA strand discontinuities during replication stress. Nucleic Acids Res. 52, 2340–2354 (2024).
Liu, J. et al. An integrated TCGA pan-cancer clinical data resource to drive high-quality survival outcome analytics. Cell 173, 400–416.e411 (2018).
Gao, J. et al. Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Sci. Signal 6, pl1 (2013).
Liu, J. & Zhang, J. Elevated EXO1 expression is associated with breast carcinogenesis and poor prognosis. Ann. Transl. Med 9, 135 (2021).
Liu, Z. et al. EXO1’s pan-cancer roles: Diagnostic, prognostic, and immunological analyses through bioinformatics. Discov. Oncol. 16, 310 (2025).
Muthuswami, M. et al. Breast tumors with elevated expression of 1q candidate genes confer poor clinical outcome and sensitivity to Ras/PI3K inhibition. PLoS One 8, e77553 (2013).
Wang, J., Liu, F., Heng, J. & Li, G. Identification of EXO1 as a potential biomarker associated with prognosis and tumor immune microenvironment for specific human cancers. Mamm. Genome 36, 262–279 (2025).
Zhou, C. S. et al. Exonuclease 1 (EXO1) is a potential prognostic biomarker and correlates with immune infiltrates in lung adenocarcinoma. Onco Targets Ther. 14, 1033–1048 (2021).
Rakha, E. A., Reis-Filho, J. S. & Ellis, I. O. Basal-like breast cancer: A critical review. J. Clin. Oncol. 26, 2568–2581 (2008).
Holstege, H. et al. BRCA1-mutated and basal-like breast cancers have similar aCGH profiles and a high incidence of protein truncating TP53 mutations. BMC Cancer 10, 654 (2010).
Kwei, K. A., Kung, Y., Salari, K., Holcomb, I. N. & Pollack, J. R. Genomic instability in breast cancer: Pathogenesis and clinical implications. Mol. Oncol. 4, 255–266 (2010).
Duijf, P. H. G. et al. Mechanisms of genomic instability in breast cancer. Trends Mol. Med 25, 595–611 (2019).
Schleicher, E. M., Galvan, A. M., Imamura-Kawasawa, Y., Moldovan, G. L. & Nicolae, C. M. PARP10 promotes cellular proliferation and tumorigenesis by alleviating replication stress. Nucleic Acids Res 46, 8908–8916 (2018).
Khatib, J. B., Dhoonmoon, A., Moldovan, G. L. & Nicolae, C. M. PARP10 promotes the repair of nascent strand DNA gaps through RAD18 mediated translesion synthesis. Nat. Commun. 15, 6197 (2024).
Vaisman, A. & Woodgate, R. Translesion DNA polymerases in eukaryotes: what makes them tick?. Crit. Rev. Biochem Mol. Biol. 52, 274–303 (2017).
Anand, J. et al. Roles of trans-lesion synthesis (TLS) DNA polymerases in tumorigenesis and cancer therapy. NAR Cancer 5, zcad005 (2023).
Chen, X., Paudyal, S. C., Chin, R. I. & You, Z. PCNA promotes processive DNA end resection by Exo1. Nucleic Acids Res 41, 9325–9338 (2013).
Tran, P. T., Erdeniz, N., Dudley, S. & Liskay, R. M. Characterization of nuclease-dependent functions of Exo1p in Saccharomyces cerevisiae. DNA repair 1, 895–912 (2002).
Bester, A. C. et al. Nucleotide deficiency promotes genomic instability in early stages of cancer development. Cell 145, 435–446 (2011).
Dasari, S. & Tchounwou, P. B. Cisplatin in cancer therapy: Molecular mechanisms of action. Eur. J. Pharm. 740, 364–378 (2014).
Makovec, T. Cisplatin and beyond: molecular mechanisms of action and drug resistance development in cancer chemotherapy. Radio. Oncol. 53, 148–158 (2019).
Morocz, M., Gali, H., Rasko, I., Downes, C. S. & Haracska, L. Single cell analysis of human RAD18-dependent DNA post-replication repair by alkaline bromodeoxyuridine comet assay. PLoS One 8, e70391 (2013).
Martins, D. J., Tirman, S., Quinet, A. & Menck, C. F. M. Detection of post-replicative gaps accumulation and repair in human cells using the DNA fiber assay. J. Vis. Exp. 180, e63448 (2022).
Roy, S., Luzwick, J. W. & Schlacher, K. SIRF: Quantitative in situ analysis of protein interactions at DNA replication forks. J. cell Biol. 217, 1521–1536 (2018).
Mijic, S. et al. Replication fork reversal triggers fork degradation in BRCA2-defective cells. Nat. Commun. 8, 859 (2017).
Poole, L. A. & Cortez, D. Functions of SMARCAL1, ZRANB3, and HLTF in maintaining genome stability. Crit. Rev. Biochem Mol. Biol. 52, 696–714 (2017).
Bozic, I. et al. Accumulation of driver and passenger mutations during tumor progression. Proc. Natl. Acad. Sci. USA 107, 18545–18550 (2010).
Kumar, S. et al. Passenger mutations in more than 2,500 cancer genomes: Overall molecular functional impact and consequences. Cell 180, 915–927.e916 (2020).
McFarland, C. D. et al. The damaging effect of passenger mutations on cancer progression. Cancer Res. 77, 4763–4772 (2017).
Wodarz, D., Newell, A. C. & Komarova, N. L. Passenger mutations can accelerate tumour suppressor gene inactivation in cancer evolution. J. R. Soc. Interface 15, 20170967 (2018).
Murai, J. & Pommier, Y. BRCAness, homologous recombination deficiencies, and synthetic lethality. Cancer Res 83, 1173–1174 (2023).
Clements, K. E. et al. Loss of E2F7 confers resistance to poly-ADP-ribose polymerase (PARP) inhibitors in BRCA2-deficient cells. Nucleic Acids Res. 46, 8898–8907 (2018).
Nicolae, C. M. et al. The ADP-ribosyltransferase PARP10/ARTD10 interacts with proliferating cell nuclear antigen (PCNA) and is required for DNA damage tolerance. J. Biol. Chem. 289, 13627–13637 (2014.
Acknowledgements
We would like to thank Dr. Clare Sample and Cole Burgess for technical support and advice, as well as the Penn State College of Medicine Advanced Light Microscopy (RRID:SCR-022526) and Flow Cytometry (RRID:SCR_021134) core facilities. This work was supported by: NIH F31CA294862 (to AN), NIH R01ES026184 and NIH R01GM134681 (to GLM), NIH R01CA244417 (to CMN), as well as the Four Diamonds Transformative Patient-Oriented Cancer Research Project 4D01_2024_1002 (to GLM and CMN). The content is solely the responsibility of the authors and does not necessarily represent the official views of Four Diamonds. This manuscript is the result of funding in whole or in part by the National Institutes of Health (NIH). It is subject to the NIH Public Access Policy. Through acceptance of this federal funding, NIH has been given a right to make this manuscript publicly available in PubMed Central upon the Official Date of Publication, as defined by NIH.
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A.N., C.M.N., and G.L.M. designed the experiments; A.N. and C.M.N. performed the experiments; A.N. and G.L.M. wrote the manuscript.
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Nusawardhana, A., Nicolae, C.M. & Moldovan, GL. The nuclease EXO1 promotes genomic instability by degrading nascent DNA in BRCA-proficient cells. Nat Commun (2026). https://doi.org/10.1038/s41467-026-69981-1
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DOI: https://doi.org/10.1038/s41467-026-69981-1
