SAMHD1 was previously characterized as a dNTPase that protects cells from viral infections. Mutations in SAMHD1 are implicated in cancer development and in a severe congenital inflammatory disease known as Aicardi–Goutières syndrome. The mechanism by which SAMHD1 protects against cancer and chronic inflammation is unknown. Here we show that SAMHD1 promotes degradation of nascent DNA at stalled replication forks in human cell lines by stimulating the exonuclease activity of MRE11. This function activates the ATR–CHK1 checkpoint and allows the forks to restart replication. In SAMHD1-depleted cells, single-stranded DNA fragments are released from stalled forks and accumulate in the cytosol, where they activate the cGAS–STING pathway to induce expression of pro-inflammatory type I interferons. SAMHD1 is thus an important player in the replication stress response, which prevents chronic inflammation by limiting the release of single-stranded DNA from stalled replication forks.
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Goldstone, D. C. et al. HIV-1 restriction factor SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase. Nature 480, 379–382 (2011).
Laguette, N. et al. SAMHD1 is the dendritic- and myeloid-cell-specific HIV-1 restriction factor counteracted by Vpx. Nature 474, 654–657 (2011).
Hrecka, K. et al. Vpx relieves inhibition of HIV-1 infection of macrophages mediated by the SAMHD1 protein. Nature 474, 658–661 (2011).
White, T. E. et al. The retroviral restriction ability of SAMHD1, but not its deoxynucleotide triphosphohydrolase activity, is regulated by phosphorylation. Cell Host Microbe 13, 441–451 (2013).
Cribier, A., Descours, B., Valadão, A. L., Laguette, N. & Benkirane, M. Phosphorylation of SAMHD1 by cyclin A2/CDK1 regulates its restriction activity toward HIV-1. Cell Reports 3, 1036–1043 (2013).
Crow, Y. J. & Manel, N. Aicardi–Goutières syndrome and the type I interferonopathies. Nat. Rev. Immunol. 15, 429–440 (2015).
Yang, Y.-G., Lindahl, T. & Barnes, D. E. Trex1 exonuclease degrades ssDNA to prevent chronic checkpoint activation and autoimmune disease. Cell 131, 873–886 (2007).
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).
Maelfait, J., Bridgeman, A., Benlahrech, A., Cursi, C. & Rehwinkel, J. Restriction by SAMHD1 limits cGAS/STING-dependent innate and adaptive immune responses to HIV-1. Cell Reports 16, 1492–1501 (2016).
Zhao, K. et al. Modulation of LINE-1 and Alu/SVA retrotransposition by Aicardi–Goutières syndrome-related SAMHD1. Cell Reports 4, 1108–1115 (2013).
Lim, Y. W., Sanz, L. A., Xu, X., Hartono, S. R. & Chédin, F. Genome-wide DNA hypomethylation and RNA:DNA hybrid accumulation in Aicardi–Goutières syndrome. eLife 4, e08007 (2015).
Seamon, K. J., Sun, Z., Shlyakhtenko, L. S., Lyubchenko, Y. L. & Stivers, J. T. SAMHD1 is a single-stranded nucleic acid binding protein with no active site-associated nuclease activity. Nucleic Acids Res. 43, 6486–6499 (2015).
Beloglazova, N. et al. Nuclease activity of the human SAMHD1 protein implicated in the Aicardi-Goutieres syndrome and HIV-1 restriction. J. Biol. Chem. 288, 8101–8110 (2013).
Ryoo, J. et al. The ribonuclease activity of SAMHD1 is required for HIV-1 restriction. Nat. Med. 20, 936–941 (2014).
Clifford, R. et al. SAMHD1 is mutated recurrently in chronic lymphocytic leukemia and is involved in response to DNA damage. Blood 123, 1021–1031 (2014).
Rentoft, M. et al. Heterozygous colon cancer-associated mutations of SAMHD1 have functional significance. Proc. Natl Acad. Sci. USA 113, 4723–4728 (2016).
Franzolin, E. et al. The deoxynucleotide triphosphohydrolase SAMHD1 is a major regulator of DNA precursor pools in mammalian cells. Proc. Natl Acad. Sci. USA 110, 14272–14277 (2013).
Zeman, M. K. & Cimprich, K. A. Causes and consequences of replication stress. Nat. Cell Biol. 16, 2–9 (2014).
Pasero, P. & Vindigni, A. Nucleases acting at stalled forks: how to reboot the replication program with a few shortcuts. Annu. Rev. Genet. 51, 477–499 (2017).
Hashimoto, Y., Ray Chaudhuri, A., Lopes, M. & Costanzo, V. Rad51 protects nascent DNA from Mre11-dependent degradation and promotes continuous DNA synthesis. Nat. Struct. Mol. Biol. 17, 1305–1311 (2010).
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).
Ray Chaudhuri, A. et al. Replication fork stability confers chemoresistance in BRCA-deficient cells. Nature 535, 382–387 (2016).
Langereis, M. A., Rabouw, H. H., Holwerda, M., Visser, L. J. & van Kuppeveld, F. J. M. Knockout of cGAS and STING rescues virus infection of plasmid DNA-transfected cells. J. Virol. 89, 11169–11173 (2015).
Holm, C. K. et al. Influenza A virus targets a cGAS-independent STING pathway that controls enveloped RNA viruses. Nat. Commun. 7, 10680 (2016).
Sirbu, B. M. et al. Analysis of protein dynamics at active, stalled, and collapsed replication forks. Genes Dev. 25, 1320–1327 (2011).
Zellweger, R. et al. Rad51-mediated replication fork reversal is a global response to genotoxic treatments in human cells. J. Cell Biol. 208, 563–579 (2015).
Bétous, R. et al. SMARCAL1 catalyzes fork regression and Holliday junction migration to maintain genome stability during DNA replication. Genes Dev. 26, 151–162 (2012).
Kolinjivadi, A. M. et al. Smarcal1-mediated fork reversal triggers mre11-dependent degradation of nascent DNA in the absence of Brca2 and stable Rad51 nucleofilaments. Mol. Cell 67, 867–881.e7 (2017).
Cruz-García, A., López-Saavedra, A. & Huertas, P. BRCA1 accelerates CtIP-mediated DNA-end resection. Cell Reports 9, 451–459 (2014).
Gunn, A., Bennardo, N., Cheng, A. & Stark, J. M. Correct end use during end joining of multiple chromosomal double strand breaks is influenced by repair protein RAD50, DNA-dependent protein kinase DNA-PKcs, and transcription context. J. Biol. Chem. 286, 42470–42482 (2011).
Daddacha, W. et al. SAMHD1 promotes DNA end resection to facilitate dna repair by homologous recombination. Cell Reports 20, 1921–1935 (2017).
Lee, J. & Dunphy, W. G. The Mre11-Rad50-Nbs1 (MRN) complex has a specific role in the activation of Chk1 in response to stalled replication forks. Mol. Biol. Cell 24, 1343–1353 (2013).
Jazayeri, A., Balestrini, A., Garner, E., Haber, J. E. & Costanzo, V. Mre11-Rad50-Nbs1-dependent processing of DNA breaks generates oligonucleotides that stimulate ATM activity. EMBO J. 27, 1953–1962 (2008).
Härtlova, A. et al. DNA damage primes the type I interferon system via the cytosolic DNA sensor STING to promote anti-microbial innate immunity. Immunity 42, 332–343 (2015).
Shen, Y. J. et al. Genome-derived cytosolic DNA mediates type I interferon-dependent rejection of B cell lymphoma cells. Cell Reports 11, 460–473 (2015).
Ho, S. S. W. et al. The DNA structure-specific endonuclease MUS81 mediates DNA sensor STING-dependent host rejection of prostate cancer cells. Immunity 44, 1177–1189 (2016).
Wolf, C. et al. RPA and Rad51 constitute a cell intrinsic mechanism to protect the cytosol from self DNA. Nat. Commun. 7, 11752 (2016).
Mackenzie, K. J. et al. cGAS surveillance of micronuclei links genome instability to innate immunity. Nature 548, 461–465 (2017).
Bartsch, K. et al. Absence of RNase H2 triggers generation of immunogenic micronuclei removed by autophagy. Hum. Mol. Genet. 26, 3960–3972 (2017).
Garcin, G. et al. High efficiency cell-specific targeting of cytokine activity. Nat. Commun. 5, 3016 (2014).
Lin, Y. L. et al. Feline immunodeficiency virus vectors for efficient transduction of primary human synoviocytes: application to an original model of rheumatoid arthritis. Hum. Gene Ther. 15, 588–596 (2004).
Jackson, D. A. & Pombo, A. Replicon clusters are stable units of chromosome structure: evidence that nuclear organization contributes to the efficient activation and propagation of S phase in human cells. J. Cell Biol. 140, 1285–1295 (1998).
Liang, F., Han, M., Romanienko, P. J. & Jasin, M. Homology-directed repair is a major double-strand break repair pathway in mammalian cells. Proc. Natl Acad. Sci. USA 95, 5172–5177 (1998).
Jia, S., Marjavaara, L., Buckland, R., Sharma, S. & Chabes, A. Determination of deoxyribonucleoside triphosphate concentrations in yeast cells by strong anion-exchange high-performance liquid chromatography coupled with ultraviolet detection. Methods Mol. Biol. 1300, 113–121 (2015).
Sannino, V., Pezzimenti, F., Bertora, S. & Costanzo, V. Xenopus laevis as model system to study DNA damage response and replication fork stability. Methods Enzymol. 591, 211–232 (2017).
Hashimoto, Y. & Costanzo, V. Studying DNA replication fork stability in Xenopus egg extract. Methods Mol. Biol. 745, 437–445 (2011).
Hansen, E. C., Seamon, K. J., Cravens, S. L. & Stivers, J. T. GTP activator and dNTP substrates of HIV-1 restriction factor SAMHD1 generate a long-lived activated state. Proc. Natl Acad. Sci. USA 111, E1843–E1851 (2014).
Zadorozhny, K. et al. Fanconi-anemia-associated mutations destabilize Rad51 filaments and impair replication fork protection. Cell Reports 21, 333–340 (2017).
Pinto, C., Kasaciunaite, K., Seidel, R. & Cejka, P. Human DNA2 possesses a cryptic DNA unwinding activity that functionally integrates with BLM or WRN helicases. eLife 5, e18574 (2016).
Matulova, P. et al. Cooperativity of Mus81.Mms4 with Rad54 in the resolution of recombination and replication intermediates. J. Biol. Chem. 284, 7733–7745 (2009).
Marini, V. & Krejci, L. Unwinding of synthetic replication and recombination substrates by Srs2. DNA Repair (Amst.) 11, 789–798 (2012).
We thank N. Laguette, G. Uzé, F. van Kuppeveld and S. Paludan for reagents and cell lines. We also thank J. Cau and the MRI Imaging facility for help with image analysis and the DNA combing facility of Montpellier for silanized coverslips. We thank A. Constantinou, D. Maiorano, B. Pardo, M. Moriel-Carretero and H. Tourrière for discussions and C. Featherstone for professional editing. This work was supported by grants from the Agence Nationale pour la Recherche (ANR) and Institut National du Cancer (INCa) to P.P. and B.L., the Ligue Contre le Cancer (équipe labellisée), Canceropole Grand Sud-ouest and SIRIC Montpellier Cancer to P.P. and the MSDAvenir fund to P.P. and M.B. Work in W.N.’s laboratory is supported by funding from the Medical Research Council and the Institute of Cancer Research. Work in A.C.’s laboratory is supported by the Swedish Cancer Society and the Swedish Research Council. Work in L.K.’s laboratory is supported by the Czech Science Foundation grants (GACR 17-17720S and 13-26629S) and project no. LQ1605 from the National Program of Sustainability II (MEYS CR). M.J.S. was supported by fellowships from CNRS, Region LR and Fondation ARC. F.C. is supported by a fellowship from the French MRES and from Fondation Recherche Médicale. This work was also funded by the Associazione Italiana per Ricerca sul Cancro (AIRC), the European Research Council (ERC) Consolidator Grant 614541, an Armenise–Harvard Foundation career development award and an AICR Worldwide Cancer Research award (13-0026) to V.C.
Nature thanks L. Zou and the other anonymous reviewer(s) for their contribution to the peer review of this work.
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
a, Western blot analysis of SAMHD1 in HEK293T cells expressing SAMHD1 shRNA (shSAM) or a scrambled control (shScr). b, Cytosolic ssDNA (red) in shScr and shSAM HEK293T cells and in HEK293T cells transfected with TREX1 siRNA (siTREX1). Scale bar, 5 μm. n = 5. c, HL116 cells containing an IFN-stimulated response element–luciferase reporter gene were incubated with culture medium from shScr or shSAM HEK293T cells for 48 h. Mean luciferase activity and s.d. from four independent experiments are shown. d, HeLa cells were transfected for 48 h with control siRNA (siCtrl) or SAMHD1 siRNA (siSAM). Expression of IFNA, IFNG and the IFN-stimulated genes MX1 and ISG15 was quantified by qRT–PCR. Data are representative of three independent experiments. Error bars denote s.d. for a representative triplicate experiment. e, shScr or shSAM HEK293T cells were treated with 4 mM HU for the indicated times and then transferred to fresh medium for a total of 20 h. Culture medium was collected and incubated with HL116 cells for 48 h before luciferase assay. Data shown are representative of three independent experiments. f, Quantification of the mean fluorescence intensity of cytosolic BrdU in the experiment shown in Fig. 1e. Quantification was performed on 250 cells by using CellProfiler. Median BrdU intensity is indicated in red. ****P < 0.0001, Mann–Whitney rank sum test. g, shScr and shSAM HEK293T cells were treated for 2 h with 4 mM HU or for 6 h with 40 μM oligomycin, used here to damage mitochondria. Cells were labelled with the mitochondria-selective dye MitoTracker (Invitrogen). The integrity of mitochondria was assessed by confocal microscopy. Representative images are shown. Scale bar, 5 μm. h, The abundance of mitochondrial COX1 DNA in cytosolic DNA isolated from cells treated as in g was quantified by qPCR and normalized to GAPDH. Mean and s.d. from three independent experiments are shown. i, Levels of STING mRNA in cGAS-knockout, STING-knockout and control HeLa cells transfected with SAMHD1 siRNA were measured by qRT–PCR 48 h after transfection. Mean and s.d. from three independent experiments are shown. j, Levels of cGAS protein in cGAS-knockout, STING-knockout and control HeLa cells transfected with SAMHD1 siRNA were monitored by western blotting 48 h after transfection (n = 3). k, Levels of IRF3 protein in SAMHD1-depleted HeLa cells co-transfected with siRNAs against STING or IFR3 (n = 3). l, HeLa cells were co-transfected for 48 h with siRNAs against SAMHD1 and STING or IRF3 . Levels of ISG15 and MX1 mRNA were analysed by qRT–PCR. Mean and s.d. from three independent experiments are shown. m, Expression levels of STING mRNA in HeLa cells co-transfected with siRNAs against SAMHD1 and STING or IFR3. Mean and s.d. for three independent experiments are shown. n, HeLa cells were co-transfected for 48 h with siRNAs against SAMHD1 and STING or IRF3. They were then treated with 4 mM HU for 8 or 18 h, washed with PBS and further cultured in fresh medium for 18 h. Expression of ISG15 mRNA was quantified by qRT–PCR. Data are representative of three independent experiments. Mean and s.d. correspond to triplicates of a representative experiment. o, HEK293 cells (±STING siRNA) and CRISPR–Cas9-mediated STING-knockout THP-1 cells were transfected with SAMHD1 siRNA. Expression of MX1 mRNA was quantified by qRT–PCR. Mean and s.d. from three independent experiments are shown. Source data
a, EdU (red) and SAMHD1 (green) foci in HeLa cells expressing HA-tagged SAMHD1 (SAMHD1–HA) or GFP–HA. Immunofluorescence microscopy was performed as indicated in Fig. 2a. Scale bar, 5 μm. n = 2. b, HeLa cells were labelled with 10 μM EdU for 10 min then processed for iPOND analysis. Proteins associated with nascent DNA were analysed by mass spectrometry. The number of peptides from SAMHD1 and other factors found associated with nascent DNA is indicated. The table summarizes the data from two independent experiments. c, Measurement of intracellular dNTP pools in shScr and shSAM HEK293T cells (two independent experiments). d, Production of recombinant His–xSamhd1 (see Supplementary Information) and characterization of the antibody raised against this protein. e, xSamhd1 associates with nascent DNA. Xenopus sperm DNA was incubated in Xenopus egg extract for 45 min then nascent DNA was labelled for 10 min with 40 μM biotin-16-dUTP in the absence or presence of 5 μM aphidicolin (Aph). Nascent chromatin was isolated on streptavidin beads and analysed by western blotting for the proteins indicated (see Methods). A representative experiment is shown (n = 2). f, Samhd1 binds chromatin in response to DSBs and aphidicolin. Left, Xenopus egg extracts were treated with the indicated doses of aphidicolin (μM), topotecan (T, 100 μM) or EcoRI (U μl−1). Chromatin-bound proteins were then analysed by western blotting. Right, histograms show relative signal intensity of Samhd1 on chromatin. Data are mean and s.d. from four independent experiments. Source data
a, Western blot analysis of SAMHD1 protein in SAMHD1-depleted HeLa cells (using shSAM) and in SAMHD1-depleted HEK293T cells expressing an shRNA-resistant, full-length SAMHD1 (SAM) under the control of a doxycycline-inducible promoter. Expression of SAMHD1 was analysed 72 h after induction with doxycycline. b, Control (shScr) and SAMHD1-depeleted (shSAM) HEK293T cells were seeded in 24-well plates. Cell number was determined by using trypan blue exclusion and haemocytometry. Mean and s.d. are shown from three independent experiments. c, SAMHD1 is required for normal S-phase progression. shScr and shSAM HEK293T cells were pulse-labelled with EdU for 20 min and chased with thymidine for 4 h before flow cytometry analysis. The arrowhead indicates the EdU-labelled cell population that completed DNA replication and came back to G1 phase. The percentage of cells in G2/M phase is indicated. n = 2. d, shScr and shSAM HeLa cells were labelled sequentially for 20 min with IdU and CldU and the length of CldU tracks (n = 150) was analysed by DNA fibre spreading. Median track lengths are indicated in red. e, Representative images of stretched DNA fibres. Red, IdU; green, CldU; blue, DNA. The green channel is shown separately for clarity. Scale bar, 4 µm. f, DNA fibres from shScr and shSAM HEK293T cells sequentially labelled for 20 min with IdU and CldU were either stretched on glass slides (DNA fibre spreading; n = 190) or combed on silanised coverslips (DNA combing; n = 130) and then analysed by immunofluorescence microscopy. The length distribution of CldU tracks is shown. Median track lengths are indicated in red. g, DNA fibres from cells treated as in f were stretched by DNA combing and the distance between CldU tracks, which is indicative of the density of active origins, was determined for five independent experiment. Median distances are indicated. Whiskers correspond to 10–90 percentiles. ****P < 0.0001, Mann–Whitney rank sum test. h, SAMHD1-depleted cells show increased spontaneous fork arrest. The ratio of the shortest to the longest CldU track from cells treated as in d was calculated for each pair of divergent sister replication forks and the percentage of sister forks showing a ratio of less than 0.6 is shown (n = 75). Error bars indicate s.d. from three independent experiments. P < 0.05, Mann–Whitney rank sum test. Source data
a, Map of SAMHD1 protein domains indicating the positions of the mutations analysed in this study. The level of phospho-SAMHD1 (T592) in HEK293T cells collected by FACS in G1, S and G2/M phases was determined with a phospho-specific antibody. Levels of wild-type and mutant SAMHD1 were also analysed by western blotting after induction of the genes with doxycycline (Dox) for 72 h. n = 4. b, SAMHD1-depleted HEK293T cells were complemented with the phosphomimetic (T592E) or non-phosphorylatable (T592A) mutants of SAMHD1. Intracellular dNTP pools were quantified as described in Methods. Median and s.d. are shown for five independent experiments. c, Wild-type SAMHD1 or the K312A and Y315A mutant forms were expressed in SAMHD1-depleted (shSAM) HEK293T cells and the cells were labelled sequentially with IdU and CldU for 15 min each. The lengths of the CldU tracks (n = 180) were measured on spread DNA fibres. d, Immortalized B cells from an SAMHD1−/− patient with a homozygous Q548X mutation or a healthy donor (WT) were labelled with IdU and CldU and the lengths of CldU tracks (n = 240) were measured as in c. ****P < 0.0001, Mann–Whitney rank sum test. Source data
a, Control (shScr) and shSAM HEK293T cells were labelled with IdU for 15 min and then exposed to 4 mM HU for 30, 60 or 120 min in the presence of CldU. The lengths of the IdU tracks (n = 230) were measured on spread DNA fibres. b, shScr and shSAM HEK293T cells were sequentially labelled for 15 min with IdU and for 15 min with CldU. Then, they were either collected immediately or treated for 2 h with 4 mM HU before DNA fibre analysis. The lengths of the IdU and CldU tracks (n = 160) were plotted as the ratio of CldU to IdU. c, shScr and shSAM HEK293T cells were treated as in b, except that HU was replaced with 1 μM aphidicolin (n = 160). d, shScr and shSAM HEK293T cells were incubated for 120 min with a balanced mix of nucleosides (+dN) and then labelled with IdU and CldU in the presence of 4 mM HU, as indicated. The lengths of IdU tracks (n = 160) were measured on spread DNA fibres. e, shScr and shSAM HEK293T cells were treated for 2 h with 4 mM HU, or not treated, and intracellular dNTP pools were measured and expressed relative to intracellular rNTP pools in two independent experiments. f, shScr and shSAM HEK293T cells were transfected for 48 h with an siRNA against CtIP (siCtIP) or a control (siCtrl) and then labelled with IdU and CldU in the presence of 4 mM HU, as indicated. The lengths of the CldU and IdU tracks (n = 160) were measured on spread DNA fibres and plotted as the ratio of CldU to IdU track lengths. g, shScr and shSAM HEK293T cells were transfected with siRNA against SMARCAL1 or with a siCtrl for 48 h and then labelled with IdU and CldU in the presence of 4 mM HU, as indicated. The lengths of the CldU and IdU tracks (n = 200) were measured on spread DNA fibres and plotted as the ratio of CldU to IdU. h, Wild-type SAMHD1, the dNTPase-deficient mutant K312A or dNTPase-proficient mutant Y315A were expressed in shSAM HEK293T cells and the cells were labelled with IdU for 15 min and then with CldU in the presence of 4 mM HU for 2 h, as indicated. The lengths of the IdU tracks (n = 140) were measured on spread DNA fibres. i, Immortalized B cells from a SAMHD1−/− patient with a homozygous Q548X mutation or a healthy donor (WT) were labelled with IdU and CldU in the presence of HU, as in h. The lengths of the IdU tracks (n = 200) were measured on spread DNA fibres. In a–i, median track lengths are indicated in red. *P < 0.05, ****P < 0.0001, Mann–Whitney rank sum test. Source data
a, Analysis of DNA end resection at the level of individual DNA fibres with the SMART assay. Control (shScr) and SAMHD1-depleted (shSAM) HEK293T cells were grown for 24 h in the presence of BrdU to label genomic DNA and DSBs were induced with 5 μg ml−1 bleocin for 1 h. Cells were then washed and collected at the indicated times. DNA fibres were spread on glass slides and BrdU was detected without DNA denaturation. Representative images (three independent experiments) of BrdU tracks (red) 1 h after bleocin removal are shown. Scale bar, 5 μm. b, Quantification of BrdU track lengths (n = 200) in shScr and shSAM HEK293T cells treated with bleocin as in a. Median track lengths are indicated in red. ****P < 0.0001, Mann–Whitney rank sum test. c, Schematic of the U2OS single-strand annealing cell assay for DNA DSB repair. These cells carry a reporter vector in which an I-SceI site has been incorporated into a GFP gene, such that single-strand annealing-mediated repair events result in GFP fluorescence. d, U2OS single-strand annealing cells were transfected with siRNAs against SAMHD1, CtIP, or both, or with a control scrambled siRNA (Scr). They were then transfected with a plasmid expressing HA-tagged I-SceI under the control of a CMV promoter. Percentages of GFP-positive cells were quantified by flow cytometry and were normalized to the control cells. Error bars denote s.d. of three independent experiments. e, Expression of SAMHD1, CtIP and HA-tagged I-SceI in the experiment shown in d were monitored by western blotting (n = 3). f, Xenopus sperm DNA was incubated in Xenopus egg extract in the presence of 0.05 U μl−1 of EcoRI for the indicated times then the chromatin was purified and analysed by western blotting for the indicated proteins. A representative experiment is shown (n = 3). Source data
a, Various fluorescently labelled DNA substrates (ssDNA, dsDNA, dsDNA with a 5′ overhang, forked DNA and reversed fork DNA of the nucleotide lengths indicated) were incubated with a range of concentrations of purified SAMHD1 and the formation of protein–DNA complexes was determined by electrophoretic mobility shift assay (EMSA) as described in the Supplementary Information. Representative gel shift images are shown (n = 3). b, Quantification of the EMSA in a. c, SAMHD1 stimulates the nuclease activity of yeast MRE11, but not that of bacterial ExoIII and human FEN1 and DNA2. Error bars denote s.d. of three independent experiments. d, e, SAMHD1 binds MRE11 and RPA as monitored by microscale thermophoresis assay. Error bars denote s.d. of three independent experiments. f, Co-immunoprecipitation of Samhd1 and RPA from Xenopus egg extracts. Western blots are shown of the input egg extract (XE), the proteins immunoprecipitated (IP) by a control antibody (IgG), and an antibody against Samhd1 (αSAM), and the proteins that remained unbound (flow through, FT). A representative experiment is shown (n = 2). g, Co-immunoprecipitation of Samhd1 and CtIP from Xenopus egg extracts, as in f and described in the Supplementary Information. A representative experiment is shown (n = 2). Source data
Extended Data Fig. 8 SAMHD1 is required to recruit MRE11 and RPA to stalled forks and to activate the replication checkpoint.
a, Top, control (shScr) and SAMHD1-depleted (shSAM) HEK293T cells were labelled with EdU for 10 min then grown without EdU for a further 2 h in the presence of 4 mM HU. Chromatin-bound MRE11 foci were detected by confocal microscopy and compared to EdU foci. Representative confocal images are shown. Scale bar, 5 μm. Bottom, the co-localization of EdU and MRE11 foci was quantified by using the JACoP plugin of ImageJ (n = 55). Whiskers indicate the 10th and 90th percentiles. ****P < 0.0001, Mann–Whitney rank sum test. b, Top, foci (arrowheads) of ssDNA (red) in the nuclei (blue) of HU-treated shScr and shSAM HEK293T cells labelled for 24 h with 10 μM BrdU. Representative images from two independent experiments are shown. Asterisks indicate BrdU-labelled cytosolic ssDNA. Bottom, quantification of nuclear and cytosolic BrdU foci per cell as in a. c, Top, shScr and shSAM HEK293T cells were incubated with 10 μM EdU for 10 min and then treated for 2 h with 4 mM HU. RPA foci (green) were detected by using an anti-RPA1 antibody after CSK extraction. Bottom, mean fluorescence intensity of chromatin-bound RPA1 was quantified from 70 EdU-positive cells by using CellProfiler. ****P < 0.0001, Mann–Whitney rank sum test. Scale bar, 5 μm. d, Immunoblots of CHK1 phosphorylated on S345 (p-CHK1), γ-H2AX phosphorylated on S139, and CHK2 phosphorylated on T68 (p-CHK2) in shScr and shSAM HEK293T cells treated for 60 min with 0.25 mM HU or 1 μM CPT (n = 3). e, Expression of CHK1 and CHK2 proteins after HU or CPT treatment as indicated in d. f, shScr and shSAM HEK293T cells, and shSAM HEK293T cells expressing full-length SAMHD1 (shSAM + SAMHD1) were tested for their ability to phosphorylate CHK1 on S345 upon exposure to 0.25 or 1 mM HU for 2 h. The fold of induction was normalized to untreated cells by quantifying the bands on the blots and calculating the ratios of p-CHK1 to SAMHD1 (n = 3). g, shSAM HEK293T cells expressing the non-phosphorylatable mutant T592A or the phosphomimetic mutant T592E of SAMHD1 were treated with 0.25 mM or 1 mM HU for 2 h. The amounts of p-CHK1 and SAMHD1 were analysed by immunoblotting and quantified as described in f; n = 2. h, shSAM HEK293T cells expressing the exonuclease positive (K312A) or negative (Y315A) mutants of SAMHD1 were treated with the indicated doses of HU for 2 h. The amounts of p-CHK1 and SAMHD1 were analysed by immunoblotting and quantified as described in f; n = 2. i, Depletion of Samhd1 from Xenopus egg extracts impairs the phosphorylation of Chk1 in response to EcoRI (0.05 U μl−1), aphidicolin (20 μM) or etoposide (Eto, 30 μM; n = 3). j, Samhd1 and Mre11 are required to activate Chk1 in Samhd1-depleted or mock-depleted Xenopus egg extracts treated with 0.05 U μl−1 of EcoRI. Mre11 was inhibited with 100 μM mirin; this experiment was performed once. k, CHK1 activation in Samhd1-depleted Xenopus egg extracts upon formation of DSBs by addition of 0.05 U μl−1 EcoRI can be restored by the addition of approximately 25 nM of recombinant His–xSamhd1; a representative experiment is shown (n = 2). l, DNA fibre analysis of fork restart in shScr and shSAM HEK293T cells treated for 18 h with 10 µM mirin and for 2 h with 1 μM CPT. Fork restart (that is, formation of red and green tracks) was monitored 30 min after CPT removal (n = 70; three independent experiments). ****P < 0.0001, Mann–Whitney test. Source data
Extended Data Fig. 9 Depletion of RECQ1 prevents accumulation of cytosolic ssDNA in SAMHD1-depleted cells.
a, RECQ1, CtIP, BLM, DNA2 and WRN proteins remaining 48 h after transfection with the corresponding siRNAs (n = 2). b, SAMHD1-depleted (shSAM) HEK293T cells were transfected with the indicated siRNAs for 48 h and then treated with 4 mM HU for 2 h. Cytosolic ssDNA was visualized by immunofluorescence microscopy. Representative confocal microscopy images are shown (n = 2). Scale bar, 5 μm. c, Depletion of RecQ1 prevents the accumulation of cytosolic ssDNA in untreated shSAM cells. Cytosolic ssDNA was detected by immunofluorescence microscopy. Representative images are shown (n = 3). Scale bar, 5 μm. d, Mean fluorescence intensity of cytosolic ssDNA in the cells c was quantified by using CellProfiler (n = 130). ****P < 0.0001, Mann–Whitney rank sum test. e, RECQ1 and SAMHD1 proteins remaining after siRNA transfection, as analysed by western blotting (n = 3). f, shScr and shSAM HEK293T cells were transfected with siRNA against RECQ1 (siRECQ1) for 48 h. Expression of IFNB mRNA was quantified by qRT–PCR. Data shown are representative of three independent experiments. Error bars denote s.d. of triplicates. g, HeLa cells were transfected with siRNAs as indicated. They were cultured in the absence or presence of 4 mM HU for 8 h and then without HU for a total of 20 h before mRNA extraction. Expression of IFNA mRNA was analysed by qRT–PCR and normalized to GADPH mRNA. Data shown are representative of three independent experiments. Error bars denote s.d. of triplicates. h, shScr and shSAM HEK293T cells were transfected with siRNA against RECQ1 for 48 h. The cells were sequentially labelled for 15 min with IdU and for 15 min with CldU. Then, they were either collected immediately or treated for 2 h with 4 mM HU before DNA fibre analysis. The lengths of the IdU and CldU tracks (n = 200) were plotted as the ratio of CldU to IdU. Median values are indicated in red. ****P < 0.0001, Mann–Whitney rank sum test. Source data
Extended Data Fig. 10 SAMHD1 depletion promotes accumulation of cytosolic nascent DNA under replication stress.
a, SAMHD1-depleted HEK293T cells were labelled for 2 h with IdU and then for 2 h with CldU in the presence of 4 mM HU. They were then chased with thymidine for the indicated times and cytosolic IdU (red) and CldU (green) were visualized by confocal immunofluorescence microscopy. Representative images are shown. Scale bar, 5 µm. b, Quantification of the cytosolic IdU and CldU signals in a by using CellProfiler (n = 300). c, DNA fibre analysis showing the increasing length of CldU tracks in HU-treated SAMHD1-depleted HEK293T cells. Median track lengths (n = 190) are indicated in red. d, Model of the role of SAMHD1 at stalled replication forks. In SAMHD1-proficient cells (top), phosphorylation of SAMHD1 by the cyclin A (CycA)-CDK contributes to the MRE11-dependent resection of stalled replication forks and activates the ATR–CHK1 pathway at RPA-coated ssDNA, together with the DNA repair enzyme TopBP1 and the 9-1-1 (Rad9–Hus1–Rad1) complex. In SAMHD1-deficient cells (bottom), nascent DNA is displaced by the RECQ1 helicase and cleaved by an endonuclease, such as MRE11. The resulting ssDNA fragments accumulate in the cytosol and activate the type I IFN response. Source data
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Coquel, F., Silva, M., Técher, H. et al. SAMHD1 acts at stalled replication forks to prevent interferon induction. Nature 557, 57–61 (2018). https://doi.org/10.1038/s41586-018-0050-1
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