Abstract
DNA replication is tightly regulated to ensure a single round of chromosome duplication per cell division. DNA licensing restricts origin firing to once-per-cell-cycle while aberrant licensing promotes re-replication and genome instability. Here, we investigate the mechanisms that protect genome integrity following re-replication induced by depletion of the licensing inhibitor Geminin. We find that re-replicating cells require FANCD2 to prevent genome instability. FANCD2 is rapidly recruited to chromatin upon Geminin loss, where it limits unrestrained fork progression and prevents single strand DNA gap accumulation and fork breakage. Genome-wide analyses reveal that upon re-replication, FANCD2 localizes to early origins within highly transcribed regions prone to accumulate R-loops and enriched in early replicating fragile sites. Importantly, reducing transcription and R-loops alleviates re-replication-induced genome fragility, whereas PARP inhibition exacerbates it. Our study uncovers a role for FANCD2 in safeguarding genome integrity during re-replication, offering avenues for selective targeting of cancer cells.
Similar content being viewed by others
Data availability
ChIP-seq data have been deposited in NCBI’s Gene Expression Omnibus with GEO Series accession number GSE285033. A full analysis of the Microscopy data is available at Zenodo (https://doi.org/10.5281/zenodo.18000043). Raw image files and reagents are available from the corresponding author. Source data are provided with this paper.
Code availability
The custom ImageJ/Fiji and Cell Profiler macros used for the analysis of nuclear areas, signal intensity, foci quantification, and colocalization have been deposited in a public GitHub repository [Link: https://github.com/ElenaKarydi/Image-analysis-pipelines/releases/tag/v1.0.0].
References
Rhind, N. & Gilbert, D. M. DNA replication timing. Cold Spring Harb. Perspect. Biol. 5, a010132–a010132 (2013).
Marks, A. B., Fu, H. & Aladjem, M. I. Regulation of replication origins. Adv. Exp. Med Biol. 1042, 43–59 (2017).
Ticau, S. et al. Mechanism and timing of Mcm2-7 ring closure during DNA replication origin licensing. Nat. Struct. Mol. Biol. 24, 309–315 (2017).
Li, J. et al. The human pre-replication complex is an open complex. Cell 186, 98–111.e121 (2023).
Reuter, L. M. et al. MCM2-7 loading-dependent ORC release ensures genome-wide origin licensing. Nat. Commun. 15, 7306 (2024).
Ticau, S., Friedman, L. J., Ivica, N. A., Gelles, J. & Bell, S. P. Single-molecule studies of origin licensing reveal mechanisms ensuring bidirectional helicase loading. Cell 161, 513–525 (2015).
Yang, R., Hunker, O., Wise, M. & Bleichert, F. Multiple mechanisms for licensing human replication origins. Nature (2024).
Symeonidou, I.-E., Taraviras, S. & Lygerou, Z. Control over DNA replication in time and space. FEBS Lett. 586, 2803–2812 (2012).
Fragkos, M., Ganier, O., Coulombe, P. & Méchali, M. DNA replication origin activation in space and time. Nat. Rev. Mol. Cell Biol. 16, 360–374 (2015).
Aparicio, T., Guillou, E., Coloma, J., Montoya, G. & Méndez, J. The human GINS complex associates with Cdc45 and MCM and is essential for DNA replication. Nucleic Acids Res. 37, 2087–2095 (2009).
Ratnayeke, N., Baris, Y., Chung, M., Yeeles, J. T. P. & Meyer, T. CDT1 inhibits CMG helicase in early S phase to separate origin licensing from DNA synthesis. Mol. Cell 83, 26–42.e13 (2023).
Hiraga, S. I. et al. Human RIF1 and protein phosphatase 1 stimulate DNA replication origin licensing but suppress origin activation. EMBO Rep. 18, 403–419 (2017).
Ticau, S., Larry, N. ikola & Gelles, J. & Stephen. Single-Molecule Studies of Origin Licensing Reveal Mechanisms Ensuring Bidirectional Helicase Loading. Cell 161, 513–525 (2015).
Zhang, H. Regulation of DNA replication licensing and re-replication by Cdt1. Int. J. Mol. Sci. 22, (2021).
Kiang, L., Heichinger, C., Watt, S., Bähler, J. & Nurse, P. Specific replication origins promote DNA amplification in fission yeast. J. Cell Sci. 123, 3047–3051 (2010).
Thomer, M., May, N. R., Aggarwal, B. D., Kwok, G. & Calvi, B. R. Drosophila double-parkedis sufficient to induce re-replication during development and is regulated by cyclin E/CDK2. Development 131, 4807–4818 (2004).
Davidson, I. F., Li, A. & Blow, J. J. Deregulated replication licensing causes DNA fragmentation consistent with head-to-tail fork collision. Mol. Cell 24, 433–443 (2006).
Liontos, M. et al. Deregulated overexpression of hCdt1 and hCdc6 promotes malignant behavior. Cancer Res. 67, 10899–10909 (2007).
Galanos, P. et al. Chronic p53-independent p21 expression causes genomic instability by deregulating replication licensing. Nat. Cell Biol. 18, 777–789 (2016).
Muñoz, S. et al. In Vivo DNA Re-replication Elicits Lethal Tissue Dysplasias. Cell Rep. 19, 928–938 (2017).
Petropoulos, M. et al. Cdt1 overexpression drives colorectal carcinogenesis through origin overlicensing and DNA damage. J. Pathol. 259, 10–20 (2023).
Petropoulou, C. Cdt1 and Geminin in cancer: markers or triggers of malignant transformation? Front. Biosci. 4485 (2008).
Vanderdys, V. et al. The neddylation inhibitor pevonedistat (MLN4924) suppresses and radiosensitizes head and neck squamous carcinoma cells and tumors. Mol. Cancer Therapeutics 17, 368–380 (2018).
Yoshimura, C. et al. TAS4464, a highly potent and selective inhibitor of NEDD8-activating enzyme, suppresses neddylation and shows antitumor activity in diverse cancer models. Mol. Cancer Ther. 18, 1205–1216 (2019).
Pozo, P. & Cook, J. Regulation and Function of Cdt1; A Key Factor in Cell Proliferation and Genome Stability. Genes 8, 2 (2016).
De Marco, V. et al. Quaternary structure of the human Cdt1-Geminin complex regulates DNA replication licensing. Proc. Natl. Acad. Sci. USA 106, 19807–19812 (2009).
Dimaki, M. et al. Cell cycle-dependent subcellular translocation of the human DNA licensing inhibitor geminin. J. Biol. Chem. 288, 23953–23963 (2013).
McGarry, T. J. & Kirschner, M. W. Geminin, an inhibitor of DNA replication, is degraded during mitosis. Cell 93, 1043–1053 (1998).
Tada, S., Li, A., Maiorano, D., Méchali, M. & Blow, J. J. Repression of origin assembly in metaphase depends on inhibition of RLF-B/Cdt1 by geminin. Nat. Cell Biol. 3, 107–113 (2001).
Zhu, W. & Depamphilis, M. L. Selective killing of cancer cells by suppression of geminin activity. Cancer Res 69, 4870–4877 (2009).
Shreeram, S., Sparks, A., Lane, D. P. & Blow, J. J. Cell type-specific responses of human cells to inhibition of replication licensing. Oncogene 21, 6624–6632 (2002).
Petropoulos, M., Champeris Tsaniras, S., Taraviras, S. & Lygerou, Z. Replication licensing aberrations, replication stress, and genomic instability. Trends Biochem. Sci. 44, 752–764 (2019).
Champeris Tsaniras, S. et al. Geminin ablation in vivo enhances tumorigenesis through increased genomic instability. J. Pathol. 246, 134–140 (2018).
Kalogeropoulou, A. et al. Intrinsic neural stem cell properties define brain hypersensitivity to genotoxic stress. Stem Cell Rep. 17, 1395–1410 (2022).
Karantzelis, N. et al. Small molecule inhibitor targeting CDT1/geminin protein complex promotes DNA damage and cell death in cancer cells. Front. Pharmacol. 13, (2022).
Pappas, K. et al. BRCA2 reversion mutation-independent resistance to PARP inhibition through impaired DNA prereplication complex function. Proc. Natl. Acad. Sci. USA 122, e2426743122 (2025).
Semlow, D. R. & Walter, J. C. Mechanisms of vertebrate DNA interstrand cross-link repair. Annu. Rev. Biochem. 90, 107–135 (2021).
Badra Fajardo, N., Taraviras, S. & Lygerou, Z. Fanconi anemia proteins and genome fragility: unraveling replication defects for cancer therapy. Trends Cancer 8, 467–481 (2022).
Zhu, W., Chen, Y. & Dutta, A. Rereplication by depletion of geminin is seen regardless of p53 status and activates a G2/M Checkpoint. Mol. Cell. Biol. 24, 7140–7150 (2004).
O’Connell, M. J., Raleigh, J. M., Verkade, H. M. & Nurse, P. Chk1 is a wee1 kinase in the G2 DNA damage checkpoint inhibiting cdc2 by Y15 phosphorylation. Embo J. 16, 545–554 (1997).
Sørensen, C. S. & Syljuåsen, R. G. Safeguarding genome integrity: the checkpoint kinases ATR, CHK1 and WEE1 restrain CDK activity during normal DNA replication. Nucleic Acids Res. 40, 477–486 (2012).
Panagopoulos, A., Taraviras, S., Nishitani, H. & Lygerou, Z. CRL4(Cdt2): coupling genome stability to ubiquitination. Trends Cell Biol. 30, 290–302 (2020).
Lee, J. & Zhou, P. DCAFs, the missing link of the CUL4-DDB1 ubiquitin ligase. Mol. Cell 26, 775–780 (2007).
Kawasaki, T. et al. BCL2L2 is a probable target for novel 14q11.2 amplification detected in a non-small cell lung cancer cell line. Cancer Sci. 98, 1070–1077 (2007).
Natsume, T., Kiyomitsu, T., Saga, Y. & Kanemaki, M. T. Rapid protein depletion in human cells by auxin-inducible degron tagging with short homology donors. Cell Rep. 15, 210–218 (2016).
Thakur, B. L., Ray, A., Redon, C. E. & Aladjem, M. I. Preventing excess replication origin activation to ensure genome stability. Trends Genet. 38, 169–181 (2022).
Ceccaldi, R., Sarangi, P. & D’Andrea, A. D. The Fanconi anaemia pathway: new players and new functions. Nat. Rev. Mol. Cell Biol. 17, 337–349 (2016).
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).
Kais, Z. et al. FANCD2 maintains fork stability in BRCA1/2-deficient tumors and promotes alternative end-joining DNA repair. Cell Rep. 15, 2488–2499 (2016).
Liu, W. et al. FANCD2 and RAD51 recombinase directly inhibit DNA2 nuclease at stalled replication forks and FANCD2 acts as a novel RAD51 mediator in strand exchange to promote genome stability. Nucleic Acids Res. 51, 9144–9165 (2023).
Alexander, J. L., Barrasa, M. I. & Orr-Weaver, T. L. Replication fork progression during re-replication requires the DNA damage checkpoint and double-strand break repair. Curr. Biol. 25, 1654–1660 (2015).
Lin, J. J. & Dutta, A. ATR Pathway Is the Primary Pathway for Activating G2/M Checkpoint Induction After Re-replication. J. Biol. Chem. 282, 30357–30362 (2007).
Zhu, W. & Dutta, A. An ATR- and BRCA1-mediated Fanconi anemia pathway is required for activating the G2/M checkpoint and DNA damage repair upon rereplication. Mol. Cell Biol. 26, 4601–4611 (2006).
Lossaint, G. et al. FANCD2 binds MCM proteins and controls replisome function upon activation of s phase checkpoint signaling. Mol. Cell 51, 678–690 (2013).
Neelsen, K. J. et al. Deregulated origin licensing leads to chromosomal breaks by rereplication of a gapped DNA template. Genes Dev. 27, 2537–2542 (2013).
Luis et al. ATR prohibits replication catastrophe by preventing global exhaustion of RPA. Cell 155, 1088–1103 (2013).
Maya-Mendoza, A. et al. High speed of fork progression induces DNA replication stress and genomic instability. Nature 559, 279–284 (2018).
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).
Machacova, Z., Chroma, K., Lukac, D., Protivankova, I. & Moudry, P. DNA polymerase α-primase facilitates PARP inhibitor-induced fork acceleration and protects BRCA1-deficient cells against ssDNA gaps. Nat. Commun. 15, 7375 (2024).
Thompson, E. L. et al. FANCI and FANCD2 have common as well as independent functions during the cellular replication stress response. Nucleic Acids Res. 45, 11837–11857 (2017).
Raghunandan, M., Chaudhury, I., Kelich, S. L., Hanenberg, H. & Sobeck, A. FANCD2, FANCJ and BRCA2 cooperate to promote replication fork recovery independently of the Fanconi Anemia core complex. Cell Cycle 14, 342–353 (2015).
Chen, X., Bosques, L., Sung, P. & Kupfer, G. M. A novel role for non-ubiquitinated FANCD2 in response to hydroxyurea-induced DNA damage. Oncogene 35, 22–34 (2016).
Zhou, B. et al. Comprehensive, integrated, and phased whole-genome analysis of the primary ENCODE cell line K562. Genome Res. 29, 472–484 (2019).
Madireddy, A. et al. FANCD2 facilitates replication through common fragile sites. Mol. Cell 64, 388–404 (2016).
Okamoto, Y. et al. FANCD2 protects genome stability by recruiting RNA processing enzymes to resolve R-loops during mild replication stress. FEBS J. 286, 139–150 (2019).
Picard, F. et al. The spatiotemporal program of DNA replication is associated with specific combinations of chromatin marks in human cells. PLoS Genet. 10, e1004282 (2014).
Chen, Y.-H. et al. Transcription shapes DNA replication initiation and termination in human cells. Nat. Struct. Mol. Biol. 26, 67–77 (2019).
Buratowski, S. Progression through the RNA polymerase II CTD cycle. Mol. Cell 36, 541–546 (2009).
Abe, K., Schauer, T. & Torres-Padilla, M. E. Distinct patterns of RNA polymerase II and transcriptional elongation characterize mammalian genome activation. Cell Rep. 41, 111865 (2022).
Local, A. et al. Identification of H3K4me1-associated proteins at mammalian enhancers. Nat. Genet. 50, 73–82 (2018).
Shoaib, M. et al. Histone H4K20 methylation mediated chromatin compaction threshold ensures genome integrity by limiting DNA replication licensing. Nat. Commun. 9, (2018).
Macheret, M. & Halazonetis, T. D. Intragenic origins due to short G1 phases underlie oncogene-induced DNA replication stress. Nature 555, 112–116 (2018).
Audit, B. et al. Open chromatin encoded in DNA sequence is the signature of ‘master’ replication origins in human cells. Nucleic Acids Res. 37, 6064–6075 (2009).
Cayrou, C. et al. The chromatin environment shapes DNA replication origin organization and defines origin classes. Genome Res. 25, 1873–1885 (2015).
Hansen, R. S. et al. Sequencing newly replicated DNA reveals widespread plasticity in human replication timing. Proc. Natl. Acad. Sci. USA 107, 139–144 (2010).
Barlow, J. H. et al. Identification of early replicating fragile sites that contribute to genome instability. Cell 152, 620–632 (2013).
Mortusewicz, O., Herr, P. & Helleday, T. Early replication fragile sites: where replication–transcription collisions cause genetic instability. EMBO J. 32, 493–495 (2013).
Pang, B., de Jong, J., Qiao, X., Wessels, L. F. A. & Neefjes, J. Chemical profiling of the genome with anti-cancer drugs defines target specificities. Nat. Chem. Biol. 11, 472–480 (2015).
Bayona-Feliu, A., Barroso, S., Muñoz, S. & Aguilera, A. The SWI/SNF chromatin remodeling complex helps resolve R-loop-mediated transcription–replication conflicts. Nat. Genet. 53, 1050–1063 (2021).
Fu, H. et al. Dynamics of replication origin over-activation. Nat. Commun. 12, 3448 (2021).
Lin, J. J., Milhollen, M. A., Smith, P. G., Narayanan, U. & Dutta, A. NEDD8-targeting drug MLN4924 Elicits DNA rereplication by stabilizing Cdt1 in S phase, triggering checkpoint activation, apoptosis, and senescence in cancer cells. Cancer Res. 70, 10310–10320 (2010).
Green, B. M., Finn, K. J. & Li, J. J. Loss of DNA replication control is a potent inducer of gene amplification. Science 329, 943–946 (2010).
Finn, K. J. & Li, J. J. Single-stranded annealing induced by re-initiation of replication origins provides a novel and efficient mechanism for generating copy number expansion via non-allelic homologous recombination. PLoS Genet. 9, e1003192 (2013).
Muñoz, S. et al. RAD51 restricts DNA over-replication from re-activated origins. Embo J. 43, 1043–1064 (2024).
Liu, Z., Jiang, H., Lee, S. Y., Kong, N. & Chan, Y. W. FANCM promotes PARP inhibitor resistance by minimizing ssDNA gap formation and counteracting resection inhibition. Cell Rep. 43, 114464 (2024).
Alexander, J. L. & Orr-Weaver, T. L. Replication fork instability and the consequences of fork collisions from rereplication. Genes Dev. 30, 2241–2252 (2016).
Blow, J. J., Gillespie, P. J., Francis, D. & Jackson, D. A. Replication origins in Xenopus egg extract Are 5-15 kilobases apart and are activated in clusters that fire at different times. J. Cell Biol. 152, 15–25 (2001).
Nathanailidou, P. et al. Aberrant replication licensing drives Copy Number Gains across species (Cold Spring Harbor Laboratory, 2020).
Panneerselvam, J. et al. Basal level of FANCD2 monoubiquitination is required for the maintenance of a sufficient number of licensed-replication origins to fire at a normal rate. Oncotarget 5, 1326–1337 (2014).
Luebben, S. W., Kawabata, T., Johnson, C. S., O’Sullivan, M. G. & Shima, N. A concomitant loss of dormant origins and FANCC exacerbates genome instability by impairing DNA replication fork progression. Nucleic Acids Res. 42, 5605–5615 (2014).
Alcón, P. et al. FANCD2-FANCI is a clamp stabilized on DNA by monoubiquitination of FANCD2 during DNA repair. Nat. Struct. Mol. Biol. 27, 240–248 (2020).
Alcón, P. et al. FANCD2–FANCI surveys DNA and recognizes double- to single-stranded junctions. Nature 632, 1165–1173 (2024).
Chaudhury, I., Sareen, A., Raghunandan, M. & Sobeck, A. FANCD2 regulates BLM complex functions independently of FANCI to promote replication fork recovery. Nucleic Acids Res. 41, 6444–6459 (2013).
Higgs, M. R. et al. Histone methylation by SETD1A protects nascent DNA through the nucleosome chaperone activity of FANCD2. Mol. Cell 71, 25–41.e26 (2018).
Sato, K. et al. FANCI-FANCD2 stabilizes the RAD51-DNA complex by binding RAD51 and protects the 5’-DNA end. Nucleic Acids Res. 44, 10758–10771 (2016).
Pentzold, C. et al. FANCD2 binding identifies conserved fragile sites at large transcribed genes in avian cells. Nucleic Acids Res. 46, 1280–1294 (2018).
Debatisse, M. & Rosselli, F. A journey with common fragile sites: From S phase to telophase. Genes Chromosomes Cancer 58, 305–316 (2019).
Dellino, G. I. et al. Genome-wide mapping of human DNA-replication origins: levels of transcription at ORC1 sites regulate origin selection and replication timing. Genome Res 23, 1–11 (2013).
Karnani, N., Taylor, C. M., Malhotra, A. & Dutta, A. Genomic study of replication initiation in human chromosomes reveals the influence of transcription regulation and chromatin structure on origin selection. Mol. Biol. Cell 21, 393–404 (2010).
Chen, Y. H. et al. Transcription shapes DNA replication initiation and termination in human cells. Nat. Struct. Mol. Biol. 26, 67–77 (2019).
Mei, L., Kedziora, K. M., Song, E. A., Purvis, J. E. & Cook, J. G. The consequences of differential origin licensing dynamics in distinct chromatin environments. Nucleic Acids Res 50, 9601–9620 (2022).
Liang, Z. et al. Binding of FANCI-FANCD2 complex to RNA and R-loops stimulates robust FANCD2 monoubiquitination. Cell Rep. 26, 564–572.e565 (2019).
García-Rubio, M. L. et al. The Fanconi anemia pathway protects genome integrity from R-loops. PLoS Genet. 11, e1005674 (2015).
Jones, R. M. et al. Increased replication initiation and conflicts with transcription underlie Cyclin E-induced replication stress. Oncogene 32, 3744–3753 (2013).
Gómez, M. & Antequera, F. Overreplication of short DNA regions during S phase in human cells. Genes Dev. 22, 375–385 (2008).
Tsantoulis, P. K. et al. Oncogene-induced replication stress preferentially targets common fragile sites in preneoplastic lesions. A genome-wide study. Oncogene 27, 3256–3264 (2008).
Brison, O. et al. Mistimed origin licensing and activation stabilize common fragile sites under tight DNA-replication checkpoint activation. Nat. Struct. Mol. Biol. 30, 539–550 (2023).
Arbi, M. et al. GemC1 controls multiciliogenesis in the airway epithelium. EMBO Rep. 17, 400–413 (2016).
Kilgas, S., Kiltie, A. E. & Ramadan, K. Immunofluorescence microscopy-based detection of ssDNA foci by BrdU in mammalian cells. STAR Protoc. 2, 100978 (2021).
Mäkelä, R. et al. Ex vivo analysis of DNA repair targeting in extreme rare cutaneous apocrine sweat gland carcinoma. Oncotarget 12, 1100–1109 (2021).
Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).
Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).
Ramírez, F. et al. deepTools2: a next generation web server for deep-sequencing data analysis. Nucleic Acids Res. 44, W160–W165 (2016).
Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).
Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).
Ernst, J. et al. Mapping and analysis of chromatin state dynamics in nine human cell types. Nature 473, 43–49 (2011).
Rice, P., Longden, I. & Bleasby, A. EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet. 16, 276–277 (2000).
Robinson, J. T. et al. Integrative genomics viewer. Nat. Biotechnol. 29, 24–26 (2011).
Bayona-Feliu, A. et al. The chromatin network helps prevent cancer-associated mutagenesis at transcription-replication conflicts. Nat. Commun. 14, 6890 (2023).
Kent, W. J. et al. The human genome browser at UCSC. Genome Res. 12, 996–1006 (2002).
Acknowledgements
We thank the Advanced Light Microscopy facility at the Medical School of the University of Patras for their support with experiments and the members of our groups for insightful discussions. We are also grateful to Dr. Raphael Ceccaldi for sharing the PD20 cells, Dr. Bert van de Kooij for sharing the FANCD2-KO and corrected U2OS cells and Dr. Vassilis Roukos for scientific advice in the generation of the mAID-Geminin HCT116 cells. This study was supported by research funding from the European Union Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No. 722729, the European Union Horizon Europe (2021-2027), “ESPERANCE” ERA Chair program (GA 101087215), the Hellenic Foundation for Research and Innovation (H.F.R.I.) under the “2nd Call for H.F.R.I. Research Projects to support Faculty Members & Researchers” (Project Number: 2728) and the project TAEDR-0539180 implemented within the framework of “Actions in interdisciplinary scientific areas with special interests for the connection with the productive fabric”, Greece 2.0 - National Recovery and Resilience Plan to Z.L. N.B.F. received fellowships from the Federation of European Biochemical Societies (FEBS) and the Operational Programme «Human Resources Development, Education and Lifelong Learning 2014- 2020» (IKY). This work was also funded by grants from the Spanish Agencia Estatal de Investigación (PID2022-138251NB-I00 funded by MCIN/AEI/10.13039/501100011033 “ERDF A way of making Europe”) and the Caixa Research Foundation (LCF/PR/HR22/52420014) to A.A. The publication fees of this manuscript have been financed by the Research Council of the University of Patras.
Author information
Authors and Affiliations
Contributions
Conceptualization, N.B.F., and Z.L.; Methodology, N.B.F., A.B.F., B.G.G., J.K.R., S.T., A.A. and Z.L.; Investigation, N.B.F., E.K., M.A. and A.K.; Data analysis, N.B.F., E.K., A.B.F. and O.P.; Writing of original draft, review & editing, N.B.F., B.G.G., A.A. and Z.L.; Supervision, S.T., A.A. and Z.L.
Corresponding author
Ethics declarations
Competing interests
Juha K. Rantala is the founder of Misvik Biology Oy. All other authors of this manuscript declare that they have no competing interests.
Peer review
Peer review information
Nature Communications thanks Corrado Santocanale and the other anonymous reviewer(s) for their contribution to the peer review of this work. A peer review file is available.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Source data
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
About this article
Cite this article
Badra-Fajardo, N., Karydi, E., Bayona-Feliu, A. et al. FANCD2 restrains fork progression and prevents fragility at early origins upon re-replication. Nat Commun (2026). https://doi.org/10.1038/s41467-026-68966-4
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41467-026-68966-4
