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Stalled replication fork protection limits cGAS–STING and P-body-dependent innate immune signalling

Abstract

Protection of stalled replication forks is crucial for cells to respond to replication stress and maintain genome stability. Genome instability and replication stress have been linked to immune activation. Here we show that Abro1 and FANCD2 protect replication forks, which is linked with the restriction of innate immune responses. We reveal that stalled replication fork degradation induced by Abro1 or FANCD2 deficiency leads to accumulation of cytosolic single-stranded DNA and activation of a cGAS–STING-dependent innate immune response that is dependent on DNA2 nuclease. We further show that the increased cytosolic single-stranded DNA contains ribosomal DNA that can bind to cGAS. In addition, Abro1 and FANCD2 limit the formation of replication stress-induced P-bodies, and P-bodies are capable of modulating activation of the innate immune response after prolonged replication stress. Our study demonstrates a connection between replication stress and activation of the innate immune response that may be targeted for therapeutic purpose.

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Fig. 1: Abro1 limits cytosolic ssDNA and replication stress-induced cGAS–STING-dependent innate immune signalling.
Fig. 2: Increased innate immune signalling in Abro1-deficient cells depends on DNA2–WRN.
Fig. 3: Ribosomal DNA fragments accumulate in the cytoplasm in Abro1-deficient cells and are detected by cGAS.
Fig. 4: Replication stress-induced P-bodies are involved in modulating Abro1 deficiency-elicited innate immune responses.
Fig. 5: Stalled replication fork degradation due to FANCD2 deficiency depends on DNA2 and is linked with induction of the innate immune response.
Fig. 6: Abro1 deficiency following replication stress results in increased cytokine secretion and Abro1 or FANCD2 deficiency promotes immune-cell migration.

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. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

References

  1. Chen, Q., Sun, L. & Chen, Z. J. Regulation and function of the cGAS–STING pathway of cytosolic DNA sensing. Nat. Immunol. 17, 1142–1149 (2016).

    CAS  PubMed  Article  Google Scholar 

  2. Li, T. & Chen, Z. J. The cGAS–cGAMP–STING pathway connects DNA damage to inflammation, senescence, and cancer. J. Exp. Med. 215, 1287–1299 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  3. Bakhoum, S. F. & Cantley, L. C. The multifaceted role of chromosomal instability in cancer and its microenvironment. Cell 174, 1347–1360 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  4. Ablasser, A. & Chen, Z. J. cGAS in action: expanding roles in immunity and inflammation. Science 363, eaat8657 (2019).

    CAS  PubMed  Article  Google Scholar 

  5. Ablasser, A. et al. cGAS produces a 2′-5′-linked cyclic dinucleotide second messenger that activates STING. Nature 498, 380–384 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  6. Li, X. D. et al. Pivotal roles of cGAS–cGAMP signaling in antiviral defense and immune adjuvant effects. Science 341, 1390–1394 (2013).

    CAS  PubMed  Article  Google Scholar 

  7. Motwani, M., Pesiridis, S. & Fitzgerald, K. A. DNA sensing by the cGAS–STING pathway in health and disease. Nat. Rev. Genet. 20, 657–674 (2019).

    CAS  PubMed  Article  Google Scholar 

  8. Sun, L., Wu, J., Du, F., Chen, X. & Chen, Z. J. Cyclic GMP–AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science 339, 786–791 (2013).

    CAS  PubMed  Article  Google Scholar 

  9. Wu, J. et al. Cyclic GMP–AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA. Science 339, 826–830 (2013).

    CAS  PubMed  Article  Google Scholar 

  10. Zhang, X. et al. Cyclic GMP–AMP containing mixed phosphodiester linkages is an endogenous high-affinity ligand for STING. Mol. Cell 51, 226–235 (2013).

    CAS  PubMed  Article  Google Scholar 

  11. Mackenzie, K. J. et al. cGAS surveillance of micronuclei links genome instability to innate immunity. Nature 548, 461–465 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  12. Gaillard, H., Garcia-Muse, T. & Aguilera, A. Replication stress and cancer. Nat. Rev. Cancer 15, 276–289 (2015).

    CAS  PubMed  Article  Google Scholar 

  13. Zeman, M. K. & Cimprich, K. A. Causes and consequences of replication stress. Nat. Cell Biol. 16, 2–9 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  14. Berti, M., Cortez, D. & Lopes, M. The plasticity of DNA replication forks in response to clinically relevant genotoxic stress. Nat. Rev. Mol. Cell Biol. 21, 633–651 (2020).

    CAS  PubMed  Article  Google Scholar 

  15. Erdal, E., Haider, S., Rehwinkel, J., Harris, A. L. & McHugh, P. J. A prosurvival DNA damage-induced cytoplasmic interferon response is mediated by end resection factors and is limited by Trex1. Genes Dev. 31, 353–369 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  16. Wolf, C. et al. RPA and Rad51 constitute a cell intrinsic mechanism to protect the cytosol from self DNA. Nat. Commun. 7, 11752 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  17. Bhattacharya, S. et al. RAD51 interconnects between DNA replication, DNA repair and immunity. Nucleic Acids Res. 45, 4590–4605 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  18. Coquel, F., Neumayer, C., Lin, Y. L. & Pasero, P. SAMHD1 and the innate immune response to cytosolic DNA during DNA replication. Curr. Opin. Immunol. 56, 24–30 (2019).

    CAS  PubMed  Article  Google Scholar 

  19. Coquel, F. et al. SAMHD1 acts at stalled replication forks to prevent interferon induction. Nature 557, 57–61 (2018).

    CAS  PubMed  Article  Google Scholar 

  20. Guan, J. et al. MLH1 deficiency-triggered DNA hyperexcision by Exonuclease 1 activates the cGAS–STING pathway. Cancer Cell 39, 109–121 (2021).

    CAS  PubMed  Article  Google Scholar 

  21. 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).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  22. 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).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. 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).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  24. Michl, J., Zimmer, J., Buffa, F. M., McDermott, U. & Tarsounas, M. FANCD2 limits replication stress and genome instability in cells lacking BRCA2. Nat. Struct. Mol. Biol. 23, 755–757 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  25. Xu, S. et al. Abro1 maintains genome stability and limits replication stress by protecting replication fork stability. Genes Dev. 31, 1469–1482 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  26. Luo, Y., Na, Z. & Slavoff, S. A. P-bodies: composition, properties, and functions. Biochemistry 57, 2424–2431 (2018).

    CAS  PubMed  Article  Google Scholar 

  27. Standart, N. & Weil, D. P-bodies: cytosolic droplets for coordinated mRNA storage. Trends Genet. 34, 612–626 (2018).

    CAS  PubMed  Article  Google Scholar 

  28. Youn, J. Y. et al. Properties of stress granule and P-body proteomes. Mol. Cell 76, 286–294 (2019).

    CAS  PubMed  Article  Google Scholar 

  29. Parker, R. & Sheth, U. P bodies and the control of mRNA translation and degradation. Mol. Cell 25, 635–646 (2007).

    CAS  PubMed  Article  Google Scholar 

  30. Anderson, P. Post-transcriptional regulons coordinate the initiation and resolution of inflammation. Nat. Rev. Immunol. 10, 24–35 (2010).

    CAS  PubMed  Article  Google Scholar 

  31. Loll-Krippleber, R. & Brown, G. W. P-body proteins regulate transcriptional rewiring to promote DNA replication stress resistance. Nat. Commun. 8, 558 (2017).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  32. Tkach, J. M. et al. Dissecting DNA damage response pathways by analysing protein localization and abundance changes during DNA replication stress. Nat. Cell Biol. 14, 966–976 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  33. Durkin, S. G. & Glover, T. W. Chromosome fragile sites. Annu Rev. Genet. 41, 169–192 (2007).

    CAS  PubMed  Article  Google Scholar 

  34. Boisvert, F. M., van Koningsbruggen, S., Navascues, J. & Lamond, A. I. The multifunctional nucleolus. Nat. Rev. Mol. Cell Biol. 8, 574–585 (2007).

    CAS  PubMed  Article  Google Scholar 

  35. Weitao, T., Budd, M. & Campbell, J. L. Evidence that yeast SGS1, DNA2, SRS2, and FOB1 interact to maintain rDNA stability. Mutat. Res. 532, 157–172 (2003).

    CAS  PubMed  Article  Google Scholar 

  36. Weitao, T., Budd, M., Hoopes, L. L. & Campbell, J. L. Dna2 helicase/nuclease causes replicative fork stalling and double-strand breaks in the ribosomal DNA of Saccharomyces cerevisiae. J. Biol. Chem. 278, 22513–22522 (2003).

    PubMed  Article  CAS  Google Scholar 

  37. Garcia-Muse, T. & Aguilera, A. Transcription-replication conflicts: how they occur and how they are resolved. Nat. Rev. Mol. Cell Biol. 17, 553–563 (2016).

    CAS  PubMed  Article  Google Scholar 

  38. Takeuchi, Y., Horiuchi, T. & Kobayashi, T. Transcription-dependent recombination and the role of fork collision in yeast rDNA. Genes Dev. 17, 1497–1506 (2003).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  39. Ayache, J. et al. P-body assembly requires DDX6 repression complexes rather than decay or Ataxin2/2L complexes. Mol. Biol. Cell 26, 2579–2595 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  40. Hubstenberger, A. et al. P-body purification reveals the condensation of repressed mRNA regulons. Mol. Cell 68, 144–157 (2017).

    CAS  PubMed  Article  Google Scholar 

  41. Reislander, T. et al. BRCA2 abrogation triggers innate immune responses potentiated by treatment with PARP inhibitors. Nat. Commun. 10, 3143 (2019).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  42. Heijink, A. M. et al. BRCA2 deficiency instigates cGAS-mediated inflammatory signaling and confers sensitivity to tumor necrosis factor-alpha-mediated cytotoxicity. Nat. Commun. 10, 100 (2019).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  43. Chen, H. et al. cGAS suppresses genomic instability as a decelerator of replication forks. Sci. Adv. 6, eabb8941 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  44. Uggenti, C. et al. cGAS-mediated induction of type I interferon due to inborn errors of histone pre-mRNA processing. Nat. Genet. 52, 1364–1372 (2020).

    CAS  PubMed  Article  Google Scholar 

  45. Boyer, J. A. et al. Structural basis of nucleosome-dependent cGAS inhibition. Science 370, 450–454 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  46. Kujirai, T. et al. Structural basis for the inhibition of cGAS by nucleosomes. Science 370, 455–458 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  47. Michalski, S. et al. Structural basis for sequestration and autoinhibition of cGAS by chromatin. Nature 587, 678–682 (2020).

    CAS  PubMed  Article  Google Scholar 

  48. Pathare, G. R. et al. Structural mechanism of cGAS inhibition by the nucleosome. Nature 587, 668–672 (2020).

    CAS  PubMed  Article  Google Scholar 

  49. Zhao, B. et al. The molecular basis of tight nuclear tethering and inactivation of cGAS. Nature 587, 673–677 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  50. Gasser, S. et al. Sensing of dangerous DNA. Mech. Ageing Dev. 165, 33–46 (2017).

    CAS  PubMed  Article  Google Scholar 

  51. Dhanwani, R., Takahashi, M. & Sharma, S. Cytosolic sensing of immuno-stimulatory DNA, the enemy within. Curr. Opin. Immunol. 50, 82–87 (2018).

    CAS  PubMed  Article  Google Scholar 

  52. Herzner, A. M. et al. Sequence-specific activation of the DNA sensor cGAS by Y-form DNA structures as found in primary HIV-1 cDNA. Nat. Immunol. 16, 1025–1033 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  53. Coppe, J. P., Desprez, P. Y., Krtolica, A. & Campisi, J. The senescence-associated secretory phenotype: the dark side of tumor suppression. Annu. Rev. Pathol. 5, 99–118 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  54. Gorgoulis, V. et al. Cellular senescence: defining a path forward. Cell 179, 813–827 (2019).

    CAS  PubMed  Article  Google Scholar 

  55. Korzeneva, I. B. et al. Human circulating ribosomal DNA content significantly increases while circulating satellite III (1q12) content decreases under chronic occupational exposure to low-dose gamma- neutron and tritium beta-radiation. Mutat. Res. 791–792, 49–60 (2016).

    PubMed  Article  CAS  Google Scholar 

  56. Potapova, T. A. et al. Superresolution microscopy reveals linkages between ribosomal DNA on heterologous chromosomes. J. Cell Biol. 218, 2492–2513 (2019).

    PubMed  PubMed Central  Article  Google Scholar 

  57. Ray, A. & Dittel, B. N. Isolation of mouse peritoneal cavity cells. J. Vis. Exp. 28, 1488 (2010).

    Google Scholar 

Download references

Acknowledgements

We thank A. Paulucci and the Department of Genetics Microscope Core Facility for assistance with imaging acquisition, S. Lu (Williams College) for a pilot study as a summer intern and A. D’Andrea (Dana Farber Cancer Institute) for PD20 cells. A.E., X.W., S.X., L.W., S.L. and B.W. were partially supported by the NIH (grant nos CA155025 and CA248088 to B.W.) and Cancer Prevention Research Institute of Texas (CPRIT; grant no. RP180244 to B.W.). Use of the Nikon A1 microscope was made possible via a NIH shared Instrumentation Grant (grant no. 1S10OD024976-01 to the MDACC Department of Genetics).

Author information

Authors and Affiliations

Authors

Contributions

B.W. supervised the project. A.E., X.W. and B.W. designed the study. A.E., X.W., S.X., L.W. and S.L. performed the experiments and prepared the figures. B.W. wrote the manuscript with contributions from the other authors.

Corresponding author

Correspondence to Bin Wang.

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Nature Cell Biology thanks Carina Oliveira Mann, Philippe Pasero and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Abro1 limits cytosolic ssDNA and replication stress-induced cGAS-STING-dependent innate immune response.

(a) Increased cytosolic DNA detected by picogreen in Abro1 KO cells treated with 4 mM HU. Scale bar, 10 μm. (b) Treatment with S1 nuclease eliminates ssDNA staining in immunofluorescence. Cells were fixed, untreated or treated with S1 nuclease before the staining with ssDNA antibody. Scale bar, 10 μm. (c) Isolated cytosolic ssDNA is sensitive to S1 nuclease but not to RNase H, RNase T1 or RNase III. Equal amount of isolated cytosolic ssDNA was used for each condition. After the indicated treatment, the amount of ssDNA is quantified by Qubit ssDNA assay, the amount of total DNA is measured by nanodrop (n = 3 independent experiments). One way Anova was used within each group for the statistics. (d) Detection of cytosolic ssDNA from newly synthesized DNA. Cells were treated as illustrated and BrdU staining was performed under non-denatured condition. Scale bars, 50 μm. Cytosolic BrdU intensity was measured for each cell with mean fluorescence intensity (MFI) and shown as mean ± SD. One way Anova was used for statistics. ‘n’ refers the number of cells analysed across two independent experiments. (e) Re-introduction of Abro1 expression in KO cells reduced pSTAT1 levels in response to HU. GFP-tagged Abro1 was expressed in KO cells. (f) Increased pSTAT1 in Abro1−/− MEFs treated with CPT (25 nM). (g) STING knockdown decreases the elevated pSTAT levels in Abro1 KO cells upon treatment of HU. (h) cGAS knockdown decreases the elevated pSTAT levels in Abro1 −/− MEFs upon treatment of HU. Data shown represent three independent experiments in a, b, f-h and two independent experiments in e.

Source data

Extended Data Fig. 2 Increased innate immune signaling in Abro1-deficient cells depends on DNA2 but not MRE11.

(a) Inhibition of Mre11 does not decrease pSTAT levels in Abro1 KO cells. Cells were treated with 4 mM HU in the absence or presence of Mirin. (b) Inhibition of Mre11 does not decrease cytosolic ssDNA in Abro1 KO cells. Immunofluorescence was performed with ssDNA antibody. Scale bars, 10 μm. Quantification of cytosolic ssDNA (mean fluorescence intensity, MFI) is shown with mean ± SD. One way Anova was used for statistics. ‘n’ refers the number of cells analysed across three independent experiments. (c) Knockdown of RAD51 decreases pSTAT1 levels in Abro1 KO cells. Cells were treated with 4 mM HU. Data shown represent three independent experiments in a and c.

Source data

Extended Data Fig. 3 rDNA fragments accumulate in the cytoplasm of Abro1-deficient cells.

(a) Co-staining of ssDNA and nucleolin in Abro1 KO cells treated with HU (4 mM, 4 h). Scale bars, 10 μm. (b) Leptomycin B (LMB) blocks accumulation of cytosolic ssDNA. Cells incubated with LMB for 8 h followed by 4 h continuous incubation with or without addition of 4 mM HU were stained with antibody to ssDNA. Scale bars, 10 μm. (c) pSTAT1 levels were reduced when cells were treated with LMB. (d) Detection of rDNA in the cytoplasm of Abro1 KO cells by slot blot with biotin-labelled rDNA probe. ‘Nuclear DNA’ (from 2 × 105 cells) and ‘Cytosolic DNA’ (‘1x’ from 5 × 105 cells, ‘2x’ from 1 × 106 cells) were loaded. Band intensity (a.u.) is quantified using ImageJ. (e) Knockdown DNA2 reduces cytosolic rDNA in the cytoplasm of Abro1 KO cells by slot blot with biotin-labelled rDNA 18S probe. ‘Nuclear DNA’ (from 2 × 105 cells) and ‘Cytosolic DNA’ (from 5 × 105 cells) were loaded. Band intensity (a.u.) is quantified using ImageJ. (f) Detection of rDNA by FISH with biotin-labelled rDNA 18S probe. Scale bars, 10 μm. (g) Treatment with RNAPI inhibitor, CX5461, reduces cytosolic ssDNA accumulation in Abro1 KO cells treated with HU (4 mM, 4 h). CX-5461 (1 μM) was added for 1 h before the end of the HU treatment. IF staining was carried out with ssDNA antibody. Scale bars, 10 μm. Data shown represent three independent experiments in a, d-g and two independent experiments in b, c. (h) Treatment with CX5461 reduces the upregulation of IL6 and CXCL10 in Abro1 KO cells treated with HU (4 mM, 16 h). CX-5461 (1 μM) was added for 1 h before the end of the HU treatment. Relative fold change was quantified and shown with mean ± SD (n = 3 independent experiments). Two way Anova was used for statistics.

Source data

Extended Data Fig. 4 Cytosolic rDNA fragments are detected by cGAS.

(a) Nuclear and cytosolic fraction used in slot blots were confirmed by western blot using antibodies to Lamin (nuclear) or GAPDH (cytosolic). (b) Western blot of IgG and cGAS immunoprecipitates with antibodies to cGAS and ssDNA. Immunoprecipitatation was carried out from the cytoplasmic fraction after cell fractionation. (c) Cytosolic rDNA is detected bound to cGAS by slot blot with biotin-labelled rDNA 18S probe. ‘Nuclear DNA’ (from 2 × 105 cells); ‘Cytosolic DNA’ (from 5 × 105 cells), or ‘cGAS bound DNA’ (prepared from 1.25 × 106 cells) were loaded. Band intensity (a.u.) is quantified using ImageJ. Data shown represent three independent experiments in a-c. (d) Detection of cGAS bound rDNA by qPCR using 18S-1 primers and cGAS bound DNA extracted from cGAS IP. Cells were untreated or treated with HU (4 mM, 4 h). Relative fold change was shown with mean ± SD (n = 3 biological replicates). One way Anova was used for statistics. (e) rDNA PCR fragments activates cGAS in cGAMP synthesis in vitro. Salmon sperm DNA (100 ng) or rDNA PCR products using indicated primers and genomic DNA as a template (28S (145 ng), 18S-1 (151 ng), 18S-2 (88 ng) rDNA PCR products) were used in the reaction with recombinant cGAS. The amount of cGAMP was measured by ELISA and shown with mean from 2 independent experiments.

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Extended Data Fig. 5 Replication stress induced P-bodies are involved in modulating Abro1 deficiency-elicited innate immune response.

(a) GFP-Abro1 forms cytoplasmic foci in cells treated with various replication stressing agents including HU (4 mM), camptothecin (CPT, 100 nM), aphidicholin (APH, 0.1 uM), or cisplatin (5 uM) at 4 h after treatment. IF staining was carried out with anti-GFP antibody. Scale bars, 10 μm. (b) Increased P-bodies in Abro1-/- MEFs. Cells were untreated or treated with 4 mM HU. Number of P-bodies per cell was quantified and shown with mean ± SD. ‘n’ refers the number of cells analysed across three independent experiments. One way Anova was used for statistics. (c) Knockdown of DCP1a or DDX6 disrupts P-bodies formation and cytoplasmic GFP-Abro1 foci upon HU treatment (4 mM, 4 h). DCP1a antibody was used for the staining. (d) Knockdown of DCP1a or DDX6 does not reduce rDNA accumulation in the cytoplasm in Abro1 KO cells. FISH was carried out with cells transfected with indicated siRNAs, either untreated or treated with HU (4 mM, 4 h), and biotin-labelled rDNA 28S probe. Scale bars, 10 μm. (e) DCP1a knockdown decreases pSTAT1 levels in Abro1 KO cells treated with HU (4 mM). (f) DCP1a knockdown or DCP1a/STING double knockdown decreases pSTAT1 levels in Abro1 KO cells treated with HU (4 mM). Data shown represent three independent experiments in a, c-f.

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Extended Data Fig. 6 Stalled replication fork degradation due to FANCD2- but not BRCA2-deficiency is linked with induction of innate immune response.

(a) pSTAT1 levels in BRCA2 knockdown cells at different times after treatment with HU (4 mM). (b) Knockdown of FANCD2 but not BRCA2 leads to cytosolic ssDNA accumulation by immunofluorescence staining with ssDNA antibody. Scale bars, 10 μm. (c) BRCA2 knockdown does not lead to HU-induced cytosolic ssDNA accumulation by quantification of Mean fluorescence intensity (MFI) with mean ± SD. One way Anova was used for statistics. (d) Picogreen staining of BRCA2 knockdown does not show HU-induced cytosolic DNA accumulation. Scale bars, 10 μm. Data shown represent three independent experiments in a, b, d, e. (e) FANCD2 knockdown leads to increased pSTAT1 in MEFs. (f) BRCA2 knockdown does not induce an increase of P-bodies. Number of P-bodies per cell was quantified and shown with mean ± SD. One way Anova was used for statistics. (g) Mirin restores fork protection in FANCD2 depleted cells. FANCD2-deficient PD20 cells expressing empty vector or FANCD2 gene were used and treated as illustrated. IdU/CIdU ratio was quantified with mean ± SD. One-way Anova was used for statistics. (h) Knockdown of DNA2 decreases P-bodies number in FANCD2-deficient cells. Number of P-bodies per cell was quantified and shown with mean ± SD. One-way Anova was used for statistics. (i) Cytosolic rDNA fragment accumulation in FANCD2-deficient cells detected by FISH with biotin-labelled rDNA 18S probe. Scale bars, 10 μm. Mean fluorescence intensity was quantified with mean ± SD. One-way Anova was used for statistics. ‘n’ refers the number of cells analysed across three independent experiments in c, f-i.

Source data

Extended Data Fig. 7 Abro1 deficiency upon replication stress results in increased cytokines secretion.

Realtime qPCR of indicated cytokine gene expression in Abro1 WT or KO U2OS cells treated with HU is quantified and shown with mean from 2 independent experiments.

Source data

Extended Data Fig. 8 cGAS localization in Abro1- or FANCD2-deficient cells.

(a) cGAS rarely colocalizes with P-bodies. Abro1 WT or KO cells untreated or treated with HU (4 mM, 4 h) were stained with antibodies to cGAS and DCP1a. Images with DAPI, cGAS and DCP1 staining were collected by Nikon A1-Confocal using ×60 objective (left panel). The scare bar=10 μm. NIH Elements AR software was used to analyse co-localization of cGAS and DCP1 (right panel). Red lines represent cGAS signal and green lines represent DCP1 signal. Occasionally, cGAS (red) signal can be detected within DCP1a foci (green) as shown in ‘1’ for each sample. In majority of the P-bodies (green), cGAS signal (red) can not be detected as shown in ‘2’. (b) Cytosolic and nuclear cGAS levels in Abro1 KO cells. Cell fractionation was carried out with cells untreated or treated with HU (4 mM, 4 h). (k) Immunofluorescence of cGAS and DCP1a in FANCD2-deficient cells. Co-staining of cGAS and DCP1a was carried out with cells treated with indicated siRNAs untreated or treated with HU (4 mM, 4 h). (d) Cytosolic and nuclear cGAS levels in FANCD2 depleted cells. Cell fractionation was carried out with cells untreated or treated with HU (4 mM, 4 h). Data shown represent three independent experiments in a-d.

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Emam, A., Wu, X., Xu, S. et al. Stalled replication fork protection limits cGAS–STING and P-body-dependent innate immune signalling. Nat Cell Biol 24, 1154–1164 (2022). https://doi.org/10.1038/s41556-022-00950-8

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