Abstract
Accurate repair of DNA double-stranded breaks by homologous recombination preserves genome integrity and inhibits tumorigenesis. Cyclic GMP–AMP synthase (cGAS) is a cytosolic DNA sensor that activates innate immunity by initiating the STING–IRF3–type I IFN signalling cascade1,2. Recognition of ruptured micronuclei by cGAS links genome instability to the innate immune response3,4, but the potential involvement of cGAS in DNA repair remains unknown. Here we demonstrate that cGAS inhibits homologous recombination in mouse and human models. DNA damage induces nuclear translocation of cGAS in a manner that is dependent on importin-α, and the phosphorylation of cGAS at tyrosine 215—mediated by B-lymphoid tyrosine kinase—facilitates the cytosolic retention of cGAS. In the nucleus, cGAS is recruited to double-stranded breaks and interacts with PARP1 via poly(ADP-ribose). The cGAS–PARP1 interaction impedes the formation of the PARP1–Timeless complex, and thereby suppresses homologous recombination. We show that knockdown of cGAS suppresses DNA damage and inhibits tumour growth both in vitro and in vivo. We conclude that nuclear cGAS suppresses homologous-recombination-mediated repair and promotes tumour growth, and that cGAS therefore represents a potential target for cancer prevention and therapy.
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The authors declare that data supporting the findings of this study are available within the manuscript and Supplementary Information.
References
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).
Wu, J. et al. Cyclic GMP–AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA. Science 339, 826–830 (2013).
Harding, S. M. et al. Mitotic progression following DNA damage enables pattern recognition within micronuclei. Nature 548, 466–470 (2017).
Mackenzie, K. J. et al. cGAS surveillance of micronuclei links genome instability to innate immunity. Nature 548, 461–465 (2017).
Civril, F. et al. Structural mechanism of cytosolic DNA sensing by cGAS. Nature 498, 332–337 (2013).
Petersen, D. L. et al. A novel BLK-induced tumor model. Tumour Biol. 39, https://www.doi.org/10.1177/1010428317714196 (2017).
Kosugi, S., Hasebe, M., Tomita, M. & Yanagawa, H. Systematic identification of cell cycle-dependent yeast nucleocytoplasmic shuttling proteins by prediction of composite motifs. Proc. Natl Acad. Sci. USA 106, 10171–10176 (2009).
Goldfarb, D. S., Corbett, A. H., Mason, D. A., Harreman, M. T. & Adam, S. A. Importin α: a multipurpose nuclear-transport receptor. Trends Cell Biol. 14, 505–514 (2004).
Mao, Z. et al. SIRT6 promotes DNA repair under stress by activating PARP1. Science 332, 1443–1446 (2011).
Rogakou, E. P., Pilch, D. R., Orr, A. H., Ivanova, V. S. & Bonner, W. M. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J. Biol. Chem. 273, 5858–5868 (1998).
Stucki, M. et al. MDC1 directly binds phosphorylated histone H2AX to regulate cellular responses to DNA double-strand breaks. Cell 123, 1213–1226 (2005).
Landry, J. P., Fei, Y. & Zhu, X. Simultaneous measurement of 10,000 protein–ligand affinity constants using microarray-based kinetic constant assays. Assay Drug Dev. Technol. 10, 250–259 (2012).
Mao, Z., Seluanov, A., Jiang, Y. & Gorbunova, V. TRF2 is required for repair of nontelomeric DNA double-strand breaks by homologous recombination. Proc. Natl Acad. Sci. USA 104, 13068–13073 (2007).
Li, Z. et al. Impaired DNA double-strand break repair contributes to the age-associated rise of genomic instability in humans. Cell Death Differ. 23, 1765–1777 (2016).
Zhao, W. et al. Promotion of BRCA2-dependent homologous recombination by DSS1 via RPA targeting and DNA mimicry. Mol. Cell 59, 176–187 (2015).
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).
Escribano-Díaz, C. et al. A cell cycle-dependent regulatory circuit composed of 53BP1-RIF1 and BRCA1-CtIP controls DNA repair pathway choice. Mol. Cell 49, 872–883 (2013).
Caldecott, K. W.; KW. Mammalian single-strand break repair: mechanisms and links with chromatin. DNA Repair (Amst.) 6, 443–453 (2007).
Hu, Y. et al. PARP1-driven poly-ADP-ribosylation regulates BRCA1 function in homologous recombination-mediated DNA repair. Cancer Discov. 4, 1430–1447 (2014).
Rottenberg, S. et al. High sensitivity of BRCA1-deficient mammary tumors to the PARP inhibitor AZD2281 alone and in combination with platinum drugs. Proc. Natl Acad. Sci. USA 105, 17079–17084 (2008).
Steffen, J. D. et al. Targeting PARP-1 allosteric regulation offers therapeutic potential against cancer. Cancer Res. 74, 31–37 (2014).
Xie, S. et al. Timeless interacts with PARP-1 to promote homologous recombination repair. Mol. Cell 60, 163–176 (2015).
Young, L. M. et al. TIMELESS forms a complex with PARP1 distinct from its complex with TIPIN and plays a role in the DNA damage response. Cell Reports 13, 451–459 (2015).
Cerbinskaite, A., Mukhopadhyay, A., Plummer, E. R., Curtin, N. J. & Edmondson, R. J. Defective homologous recombination in human cancers. Cancer Treat. Rev. 38, 89–100 (2012).
Chernikova, S. B., Game, J. C. & Brown, J. M. Inhibiting homologous recombination for cancer therapy. Cancer Biol. Ther. 13, 61–68 (2012).
Hahn, W. C. et al. Creation of human tumour cells with defined genetic elements. Nature 400, 464–468 (1999).
Margolis, S. R., Wilson, S. C. & Vance, R. E. Evolutionary origins of cGAS–STING signaling. Trends Immunol. 38, 733–743 (2017).
Mao, Z., Bozzella, M., Seluanov, A. & Gorbunova, V. Comparison of nonhomologous end joining and homologous recombination in human cells. DNA Repair (Amst.) 7, 1765–1771 (2008).
Sanjana, N. E., Shalem, O. & Zhang, F. Improved vectors and genome-wide libraries for CRISPR screening. Nat. Methods 11, 783–784 (2014).
Huang, T. et al. G9A promotes tumor cell growth and invasion by silencing CASP1 in non-small-cell lung cancer cells. Cell Death Dis. 8, e2726 (2017).
Robert, X. & Gouet, P. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 42, W320–W324 (2014).
Capes-Davies, A. et al. Match criteria for human cell line authentication: where do we draw the line? Int. J. Cancer 132, 2510–2519 (2013).
Acknowledgements
This project was supported by grants from the Chinese National Program on Key Basic Research Project (2017YFA0505900 to B.G. and 2015CB964800 to Z.M.) and the National Natural Science Foundation of China (81770006 and 81370108 to H.Liu, 91542111 and 31030028 to B.G., and 81622019 and 31570813 to Z.M.). H.Liu is sponsored by the Shanghai Pujiang Program (16PJ1408600) and by the Shanghai Medical and Health Services Outstanding Youth Talent Program (2017YQ078).
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Contributions
H.Liu, Z.M. and B.G. conceived the project, designed the experiments and wrote the manuscript. H.Liu, H.Z. and X.W. performed most of the experiments and analysed the data. D.M., J.Wu and L.W. generated cGAS knockout cells and performed the co-IP experiments. Y.J. performed the animal experiments. Y.F. and C.Z. performed the label-free biomolecular interaction assays. R.T. performed the laser microirradiation experiments. P.J. conducted the mass spectrometry analyses. Q.Y. performed the multiple sequence alignment. F.Z. carried out analyses of gene expression and Kaplan–Meier survival plots. R.Z., S.L., H.Liang, Z.L., H.Y., J.C., P.W., T.T., W.P., Z.H., Z.X., X.H., J.Wang, H.Li, Y.Z., F.L. and D.Y. assisted with experiments and provided technical help. G.P., A.D., S.H.E.K. and C.C. provided comments and assisted with manuscript preparation.
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Extended data figures and tables
Extended Data Fig. 1 cGAS translocates to the nucleus in non-apoptotic cells with intact nuclear membranes.
a–e, Immunofluorescence results depicting localization of endogenous cGAS (anti-cGAS) in HCA2-TERT cells, an TERT-immortalized human fibroblast line. Cells were untreated (NT) (a) or exposed to etoposide (100 μg ml−1) (b), camptothecin (1 μM) (c) or H2O2 (10 mM) (d) for the indicated times. Data are representative of n = 3 independent experiments. Quantitative data are shown in e. At least 100 cells were counted in each experiment. Data are mean ± s.e.m. of n = 3 independent experiments. f–i, Results of immunofluorescence analysis showing localization of overexpressed HA–cGAS in human HCA2-TERT fibroblasts that were untreated (NT) (f) or exposed to camptothecin (1 μM) (g) or H2O2 (10 mM) (h) for the indicated times. Data are representative of n = 3 independent experiments. Quantitative data are shown in i. At least 100 transfected cells were counted in each experiment. Data are mean ± s.e.m. of n = 3 independent experiments. j–n, Representative immunofluorescence analysis showing the localization of HA–cGAS (anti-HA, green) in transfected primary human skin fibroblasts from one healthy donor that were untreated (NT) (j) or exposed to etoposide (100 μg ml−1) (k), camptothecin (1 μM) (l) or H2O2 (10 mM) (m) for the indicated times. Data represent n = 2 independent experiments. The quantification of nuclear translocation of cGAS in primary skin fibroblasts from two donors in response to indicated DNA-damaging agents are shown in n. At least 100 transfected cells were counted in each experiment. o, Representative FACS results depicting apoptosis of human fibroblast HCA2-TERT cells treated with etoposide (100 μg ml−1), camptothecin (1 μM), H2O2 (10 mM) or carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (10 μM) for the indicated times. Data represent n = 3 independent experiments. Scale bar, 5 μm.
Extended Data Fig. 2 DNA damage induces nuclear translocation of cGAS in lung cancer cells.
a–e, Immunofluorescence assay results showing localization of transfected HA–cGAS (anti-HA, green) in A549 cells that were untreated (a) or treated with etoposide (100 μg ml−1) (b), camptothecin (1 μM) (c) or H2O2 (10 mM) (d) for the indicated times. Data represent n = 3 independent experiments. Quantitative data are depicted in e. At least 100 transfected cells were counted in each experiment. Data are expressed as mean ± s.e.m. Scale bar, 5 μm. f, g, Immunofluorescence assay results showing localization of transfected HA–cGAS (anti-HA, green) in PC-9 cells that were untreated (NT) or treated with indicated concentrations of etoposide, camptothecin or H2O2 for the indicated times (f). Data represent n = 3 independent experiments. Quantitative data were depicted in g. At least 100 transfected cells were counted in each experiment. Data are expressed as mean ± s.e.m. Scale bar, 5 μm. h, Immunoblot results showing cytoplasmic and nuclear fractions of A549 cells transfected with pcDNA3.1-HA (HA) or pcDNA3.1-HA–cGAS (HA–cGAS) for 48 h and then treated with etoposide (100 μg ml−1) for the indicated times. Data are representative of n = 2 independent experiments. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 3 cGAS translocates to the nucleus in response to DNA damage independently of its DNA-binding ability or enzymatic activity.
a, Immunofluorescence assay results showing localization of transfected HA–cGAS and corresponding mutants (anti-HA, green) in PC-9 cells exposed to etoposide (100 μg ml−1) for 4 h. Data represent n = 3 independent experiments. Quantitative data are depicted in b. At least 100 transfected cells were counted in each experiment. Data are expressed as mean ± s.e.m. of n = 3 independent experiments. One-way ANOVA was used for statistical analysis. NS, not significant.
Extended Data Fig. 4 BLK-mediated phosphorylation of cGAS at Y215 regulates the nuclear translocation of cGAS in response to DNA damage.
a, Alignment of primary sequences of H. sapiens cGAS and its homologues in 22 species: H. sapiens (Hsa), Pan troglodytes (Ptr), Pan paniscus (Pps), Gorilla gorilla (Ggo), Pongo abelii (Pon), Macaca mulatta (Mmu), Ailuropoda melanoleuca (Aml), Canis familiaris (Cfa), Felis catus (Fca), Sus scrofa (Ssc), Bos taurus (Bta), Mus musculus (Mms), Rattus norvegicus (Rno), Monodelphis domestica (Mdo), Sarcophilus harrisii (Shr), Meleagris gallopavo (Mgp), Gallus gallus (Gga), Taeniopygia guttata (Tgu), Anolis carolinensis (Acs), Xenopus tropicalis (Xtr), Oryzias latipes (Ola) and Danio rerio (Dre). Consensus sequences (similarity score, >0.7) are listed at the bottom of the column; conserved amino acids are highlighted in yellow, and absolutely conserved amino acids are highlighted in red. The filled triangle indicates the conserved Y215. The primary sequence numbers of H. sapiens cGAS are labelled at the top of the line. b–e, Immunofluorescence assay results showing the localization of HA–cGAS and HA–cGAS(Y215E) (anti-HA, green) in PC-9 cells exposed to camptothecin (1 μM) for 4 h (b) or to H2O2 (10 mM) for 30 min (d). Data represent n = 3 independent experiments. Quantitative data for b and d are depicted in c and e, respectively. Data are expressed as mean ± s.e.m. of n = 3 independent experiments. f, Immunoblot results of anti-HA immunoprecipitates from HEK293T cells transfected with HA–cGAS or the HA–cGAS(Y215A) mutant in the absence or presence of pervanadate for 30 min, followed by cell collection. Data represent n = 3 independent experiments. g, Immunoblot of lysates from PC-9 cells that had been stimulated with camptothecin (1 μM) for the indicated times. Data represent n = 3 independent experiments. h, i, Results of immunofluorescence analysis showing localization of HA–cGAS (anti-HA, green) in PC-9 HA–cGAS cells transfected with control shRNA (sh-Ctrl) or with shRNA targeting BLK (sh-BLK) and then exposed to camptothecin (1 μM) for 4 h (h). Nuclei were stained with DAPI (blue). Data represent n = 3 independent experiments. Quantitative data are shown in i. At least 100 transfected cells were counted in each experiment. Data are expressed as mean ± s.e.m. of n = 3 independent experiments. j, Immunoblot results of lysates from PC-9 HA–cGAS cells that had been transfected with either control shRNA or with shRNA targeting BLK and then exposed to camptothecin (1 μM) for 4 h. Data represent 3 independent experiments. Student’s t tests (unpaired and two-tailed) were used for statistical analysis (c, e, i). Scale bar, 5 μm. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 5 cGAS interacts with importin-α and translocates to the nucleus in a manner dependent on importin-α.
a, Diagram showing the location and sequence of two predicted nuclear localization sequences of cGAS. b, Alignment of primary sequences of H. sapiens cGAS and its homologues in 22 species were performed as indicated in Extended Data Fig. 4a. The conserved NLS2 sequences are shown in the rectangular box. c, Immunoblot results of cell lysates and anti-Flag immunoprecipitates from HEK293T cells that had been transfected with HA–cGAS and the indicated Flag–KPNA and then exposed to etoposide (100 μg ml−1) for 4 h. Data represent n = 3 independent experiments. d, Immunoblot findings of cell lysates or anti-Flag immunoprecipitates from HEK293T cells that had been transfected with Flag–KPNA2 and HA–cGAS or the cGAS nuclear localization sequence deletion mutants (HA–cGAS(ΔNLS1) and HA–cGAS(ΔNLS2)) and exposed to etoposide (100 μg ml−1) for 4 h. Data represent n = 2 independent experiments. e–j, Immunofluorescence findings showing the localization of transfected HA–cGAS or HA–cGAS(ΔNLS2) (anti-HA, green) in PC-9 cells treated with etoposide (100 μg ml−1) (e) or camptothecin (1 μM) (g) for 4 h or with H2O2 (10 mM) for 30 min (i). Nuclei were stained with DAPI (blue). Data represent n = 3 independent experiments. Quantitative data for e, g, i are shown in f, h, j, respectively, and are expressed as mean ± s.e.m. of n = 3 independent experiments. k–p, Representative immunofluorescence of HA–cGAS (anti-HA, green) in PC-9 HA–cGAS cells treated with etoposide (100 μg ml−1) (k) or camptothecin (1 μM) (m) for 4 h or with H2O2 (10 mM) for 30 min (o) in the presence of dimethyl sulfoxide (DMSO; mock treatment) or importazole (1 μM). Nuclei were stained with DAPI (blue). Data represent n = 3 independent experiments. Quantitative data for k, m, o are shown in l, n, p, respectively, and are expressed as mean ± s.e.m. of n = 3 independent experiments. At least 100 transfected cells were counted in each experiment. Student’s t tests (unpaired and two-tailed) were used for statistical analysis (f, h, j, l, n, p). Scale bar, 5 μm. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 6 cGAS interacts with γH2AX directly.
a, b, Immunoblot of cell lysates and anti-H2AX (a) or anti-γH2AX (b) immunoprecipitates from PC-9 cells treated with etoposide (100 μg ml−1) for the indicated times. Data represent n = 3 independent experiments. c, Immunoblot of cell lysates or anti-HA immunoprecipitates from HEK293T cells transfected with Flag–cGAS and HA–H2AX in the absence or presence of etoposide (100 μg ml−1) for 4 h or H2O2 (10 mM) for 30 min. Data represent n = 3 independent experiments. d, e, Immunoblot of cell lysates or anti-Flag immunoprecipitates from HEK293T cells that had been transfected with Flag–H2AX and the indicated HA–cGAS truncated mutants in the presence of etoposide (100 μg ml−1) for 4 h. Data represent n = 3 independent experiments. f, Immunoblot of cell lysates or anti-Flag immunoprecipitates from HEK293T cells transfected with Flag–cGAS, HA–H2AX, or HA–H2AX(S139A). Data represent n = 3 independent experiments. g, Coomassie blue staining of purified GST–cGAS protein. Data represent n = 2 independent experiments. h, Binding curves of surface-immobilized nonphosphorylated peptide with GST-labelled cGAS at concentrations of 368 nM, 184 nM and 92 nM, and control GST protein at a concentration of 92 nM. Vertical lines mark the starts of association and dissociation phases of binding events. Data represent n = 4 independent experiments. i, Immunoblot of cell lysates of U2OS cells that had been stably transfected with either control shRNA or shRNA targeting H2AX (sh-H2AX). Data represent n = 2 independent experiments. j, k, Representative images of U2OS cells that had been stably transfected with either control shRNA or shRNA targeting H2AX, showing the recruitment of GFP–cGAS to sites of DNA damage induced by laser microirradiation. Data represent n = 3 independent experiments. Quantification data are shown in k and represent the mean ± s.d. of n = 17 (sh-Ctrl) and n = 22 (sh-H2AX) cells, respectively, from 3 independent experiments. Two-way ANOVA was used for statistical analysis. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 7 cGAS inhibits repair mediated by homologous recombination in a manner that is independent of IFNβ, without altering the cell cycle or DNA replication.
a, Schematic of reporter constructs prepared for the analysis of efficiency of DNA DSB repair by homologous recombination and NHEJ. Both reporter cassettes were constructed on GFP–Pem1. There are two non-functional copies of the GFP-Pem1 gene in the homologous recombination construct. The first copy of GFP–Pem1 contains a 22-nucleotide deletion and an insertion of 2 inverted I-SceI recognition sites. The second copy lacks the ATG start codon and the second GFP exon. After the induction of a DSB with I-SceI, only gene conversion can restore the functional GFP gene. The NHEJ cassette contains a copy of the GFP gene in which a Pem1 intron is interrupted by an adenoviral exon (Ad2). The I-SceI endonuclease recognition sites flank the adenoviral exon. The GFP gene is restored when a DSB induced by I-SceI digestion is successfully repaired by NHEJ. SD, splice donor; SA, splice acceptor. b, c, Effect of cGAS overexpression on the efficiency of homologous recombination (b) and NHEJ (c) in primary cells, including primary skin fibroblast from two donors. Primary cells were transfected with linearized homologous recombination constuct (b) or NHEJ construct (c) and pDsRed2-N1 together with pcDNA3.1-HA (HA) or HA–cGAS, respectively, and analysis of the efficiency of homologous recombination or NHEJ was performed. Data represent mean ± s.e.m. of n = 3 independent experiments. Student’s t tests (unpaired and two-tailed) were used for statistical analysis. d, Immunoblot of lysate of parental HCA2-H15c cells and cGAS knockout cells (cGAS KO) in which cGAS was deleted from the genome by CRISPR–Cas9 editing. Data represent n = 2 independent experiments. e, Quantitative PCR detection of the relative expression of IFNβ transcripts in HCA2-H15c cells at different time points after transfection with the pcDNA3.1-HA plasmid (HA) or the pcDNA3.1-HA–cGAS plasmid (HA–cGAS) with or without the I-SceI expression vector. Data represent mean ± s.e.m. of n = 3 independent experiments. Student’s t tests (unpaired and two-tailed) were used for statistical analysis. f, Effect of exogenous IFNβ on NHEJ efficiency. Values represent the ratios of the quantity of GFP+ cells (corresponding to successful repair events) to the DsRed+ transfection controls. Data represent the mean ± s.e.m. of n = 3 independent experiments. One-way ANOVA was used for statistical analysis. g–l, Representative immunofluorescence of RPA2 foci at multiple time points in HCA2-H15c cells that had been transfected with pcDNA3.1-HA control (HA) or HA–cGAS, followed by ionizing irradiation (IR) (dose of 8 Gy) (g) or exposure to etoposide (100 μg ml−1) (i) or camptothecin (1 μM) (k) for 4 h. Data represent n = 6 independent experiments. The percentage of RPA2-positive cells (with >5 nuclear foci per cell) was quantified at the indicated time points in h, j, l and expressed as mean ± s.e.m. of n = 6 independent experiments. Student’s t tests (unpaired and two-tailed) were used for statistical analysis. m, Representative comet assay showing the tail moment of parental and cGAS knockout HCA2-H15c cells under alkaline conditions. Data represent n = 3 independent experiments. n, Quantification of the tail moment of parental and cGAS knockout HCA2-H15c cells under alkaline conditions. Data are expressed as mean ± s.d. of the tail moment of n = 87 (parental) and n = 75 (cGAS knockout) cells from 3 independent experiments. The Mann–Whitney U-test was used for statistical analysis. o, Immunoblot of cell lysates of skin fibroblast cells isolated from the tail of wild-type (WT) and cGAS knockout mouse. Data represent n = 2 independent experiments. p, Representative results of the comet assay showing the tail moment of skin fibroblast cells isolated from the tail of wild-type and cGAS knockout mouse under alkaline conditions. q, Quantification of the tail moment of wild-type and cGAS knockout skin fibroblast cells under alkaline conditions. Data are expressed as mean ± s.d. of the tail moment of n = 299 (wild type) and n = 346 (cGAS knockout) cells from 3 independent experiments. The Mann–Whitney U-test was used for statistical analysis. r, Representative FACS results depicting the cell-cycle distribution in HCA2-H15c cells that had been transfected with pcDNA3.1-HA (HA) or pcDNA3.1-HA–cGAS (HA–cGAS). Data are representative of n = 3 independent experiments. s, t, Representative FACS results showing the DNA content with EdU (s). Quantitative data are shown in t. Data represent the mean ± s.e.m. of n = 3 independent experiments. Student’s t tests (unpaired and two-tailed) were used for statistical analysis. NS, not significant. Scale bar, 5 μm. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 8 cGAS inhibits repair mediated by homologous recombination independently of its DNA-binding ability or enzymatic activity.
a–g, Human fibroblast HCA2-H15c cells containing chromosomally integrated reporter cassettes (a, c, d, g) or the corresponding cGAS knockout cells (b, e, f) were co-transfected with I-SceI endonuclease (to induce DSBs), pcDNA3.1-HA (HA) (control), HA–cGAS and corresponding mutants, and DsRedN-1 plasmid (transfection control) and assessed for efficiency of homologous recombination. h, HCA2-H15c cells were treated with either DMSO (control) or the importin-β inhibitor importazole (10 μM), followed by analysis of the efficiency of homologous recombination. Values in a–h represent the ratio of the quantity of GFP+ cells (corresponding to successful repair events) to the DsRed+ transfection controls. Data represent the mean ± s.e.m. of n = 3 independent experiments. Student’s t tests (unpaired and two-tailed) were used for statistical analysis. NS, not significant. i, Quantitative PCR analysis of BLK expression in the homologous-recombination-repair reporter fibroblast HCA2-H15c cells, transfected with control shRNA or shRNA targeting BLK. Data represent the mean ± s.e.m. of n = 3 independent experiments.
Extended Data Fig. 9 cGAS interacts with PARP1 via PAR and prevents formation of the PARP1–Timeless complex.
a, Coomassie-blue-stained SDS-polyacrylamide gel image showing the anti-HA immunoprecipitates of HEK293T cells stably transfected with pcDNA3.1-HA (HA) or HA–cGAS (HA–cGAS) and treated with H2O2 (10 mM) for 30 min. M, pre-stained protein markers. Distinct protein bands (indicated as 1 and 2) in the anti-HA immunoprecipitates of HEK293T cells that had been stably transfected with HA–cGAS were identified by mass spectrometry. Data represent n = 2 independent experiments. b, Results of mass spectrometry analysis of bands 1 and 2 in the anti-HA immunoprecipitates of HEK293T cells that had been stably transfected with HA–cGAS. c, Immunoblot of cell lysates or anti-Flag immunoprecipitates from HEK293T cells that were transfected with Flag–PARP1 and HA–cGAS in the presence or absence of DNase I. Data represent n = 3 independent experiments. d, Immunoblot of cell lysates or anti-Flag immunoprecipitates from HEK293T cells that were transfected with Flag–cGAS and HA–PARP1 in the absence or presence of the PARP1 inhibitor, olaparib (20 μM). Data represent n = 3 independent experiments. e, Immunoblot results of cell lysates or anti-Flag immunoprecipitates from HEK293T cells that had been transfected with Flag–cGAS, HA–PARP1 or its enzyme-inactivated mutant HA–PARP1(E988K). Data represent n = 3 independent experiments. f–h, Immunoblot of cell lysates or anti-Flag immunoprecipitates from HEK293T cells that had been transfected with Flag–PARP1, Myc–Timeless and HA–cGAS and then exposed to etoposide (100 μg ml−1) (f) or camptothecin (1 μM) (g) for 4 h, or ionizing irradiation (8 Gy) (h). Data represent n = 3 independent experiments. i, j, Representative images of U2OS cells showing the recruitment of GFP–PARP1 to sites of DNA damage induced by laser microirradiation. Cells had been transfected with pcDNA3.1-HA control (HA) or HA–cGAS. Quantification data are shown in j and represent the mean ± s.d. of n = 19 cells (both HA and HA–cGAS) from 3 independent experiments. Two-way ANOVA was used for statistical analysis. NS, not significant. k–m, Representative immunofluorescence of HA–cGAS (anti-HA, green) in PC-9 HA–cGAS cells treated with etoposide (100 μg ml−1) (k) or camptothecin (1 μM) (l) for 4 h in the presence of DMSO (mock treatment) or olaparib (20 μM). Nuclei were stained with DAPI (blue). Data represent n = 3 independent experiments. Quantitative data are shown in m. At least 100 transfected cells were counted in each experiment. Data are expressed as mean ± s.e.m. of n = 3 independent experiments (m). Student’s t tests (unpaired and two-tailed) were used for statistical analysis. Scale bar, 5 μm. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 10 cGAS promotes tumorigenesis.
a, Results of the MTT assay showing the proliferation of PC-9 cells that had been stably transfected with pcDNA3.1-HA (HA) or HA–cGAS (HA–cGAS). Data are expressed as mean ± s.e.m. of n = 3 independent experiments. Student’s t tests (unpaired and two-tailed) were used for statistical analysis. *P < 0.05; ***P < 0.001. b, Colonogenic assay results depicting the colony-forming ability of PC-9 cells that were stably transfected with pcDNA3.1-HA (HA) or HA–cGAS (HA–cGAS). Data represent the mean ± s.e.m. of n = 6 independent experiments. The Mann–Whitney U-test was used for statistical analysis. c, The survival fraction of control shRNA LLC and Cgas shRNA LLC cells was determined by the MTT assay 6 days after exposure to etoposide (100 μg ml−1) or camptothecin (1 μM) for 4 h or H2O2 (10 mM) for 30 min. Data are expressed as mean ± s.e.m. of n = 3 independent experiments. Student’s t tests (unpaired and two-tailed) were used for statistical analysis. d, The survival fraction of PC-9 cells that had been stably transfected with pcDNA3.1-HA (HA) or HA–cGAS was determined by the MTT assay 6 days after exposure to etoposide (100 μg ml−1) or camptothecin (1 μM) for 4 h or H2O2 (10 mM) for 30 min. Data are expressed as mean ± s.e.m. of n = 3 independent experiments. Student’s t tests (unpaired and two-tailed) were used for statistical analysis. e–f, Soft agar assays of anchorage-independent colony formation of human skin fibroblast from two different donors that were transfected with vectors encoding SV40 large tumour antigen (LT), HRAS V12 (Ras) and TERT together with pcDNA3.1-HA (HA) or HA–cGAS followed by plating in soft agar. Colonies were photographed after 5 weeks of growth at 200× original magnification (e). To assess anchorage-independent growth, 105 cells were plated in 0.4% Noble agar and colonies were counted 5 weeks after seeding. Quantification data are shown in f and represent the mean ± s.e.m. of n = 3 independent experiments. The Mann–Whitney U-test was used for statistical analysis. g, Representative results of the comet assay depicting the tail moment of PC-9 cells that had been stably transfected with pcDNA3.1-HA (HA) or HA–cGAS under alkaline conditions. h, Quantification of the tail moment of PC-9 cells that had been stably transfected with pcDNA3.1-HA (HA) or HA–cGAS under alkaline conditions. Data represent mean ± s.d. of the tail moment of n = 143 (HA) and n = 127 (HA–cGAS) cells from 3 independent experiments. The Mann–Whitney U-test was used for statistical analysis. i, Schematic of the role of cGAS in control of the DNA-damage response. In response to DNA damage, cGAS translocates to the nucleus and is recruited to DSBs where it interacts with PARP1 via PAR and prevents the formation of the PARP1–Timeless complex. This interferes with the process of homologous recombination. Nuclear cGAS suppresses DNA repair and promotes tumorigenesis. j, The relative expression of cGAS in normal and tumour (T) stages from specimens of patients with lung adenocarcinoma (LUAD). Data were obtained from the TCGA database. log2(fold-changes) and P values of cGAS expression are 0.47 (P = 0.12, T1 versus normal), 0.73 (P = 0.01, T2 versus normal), 0.76 (P = 0.005, T3 versus normal) and 0.93 (P = 0.003, T4 versus normal). NS, not significant. k, The relative expression of cGAS in normal and T stages from specimens of patients with lung squamous carcinoma (LUSC). Data were obtained from the TCGA database. log2(fold-changes) and P values of cGAS expression are 0.56 (P = 0.06, T1 versus normal), 0.78 (P = 0.006, T2 versus normal), 0.94 (P = 0.0003, T3 versus normal) and 0.79 (P = 0.004, T4 versus normal). NS, not significant. l, m, Kaplan–Meier curve analysis of the overall survival probabilities of patients with lung adenocarcinoma or lung squamous carcinoma based on the expression levels of BLK and KPNA2. Patients with lung adenocarcinoma were assigned to 1 of 4 groups based on low or high levels of gene expression.
Supplementary information
Supplementary Data
This file contains Supplementary Figure 1: Source data of western blot scans.
Supplementary Table
This file contains Supplementary Table 1: Primers for site-directed mutagenesis.
Supplementary Table
This file contains Supplementary Table 2: Screening results showing the effect of knockdown of indicted protein tyrosine kinases on cGAS nuclear translocation. Nuclear translocation of GFP-cGAS was monitored in PC-9 cells transfected with shRNAs targeting 89 protein tyrosine kinases. Transfection of cells with shRNA targeting B-lymphoid tyrosine kinase (BLK) produced >1.5 fold increase in cGAS nuclear translocation.
Supplementary Table
This file contains Supplementary Table 3: Antibodies and dilutions used.
Video 1
Recruitment of cGAS to DNA damage sites. Laser microirradiation showed that GFP-cGAS was recruited to DNA lesions. Data represent n=21 cells from 3 independent experiments.
Video 2
Knocking down H2AX significantly inhibited the recruitment of cGAS to DNA damage sites. Recruitment of cGAS to DNA damage sites in U2OS cells stably transfected with scrambled shRNA (sh-Ctrl) and shRNA targeting H2AX (sh-H2AX). Data represent n=17 sh-Ctrl U2OS cells and n=22 sh-H2AX U2OS cells from 3 independent experiments, respectively.
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Liu, H., Zhang, H., Wu, X. et al. Nuclear cGAS suppresses DNA repair and promotes tumorigenesis. Nature 563, 131–136 (2018). https://doi.org/10.1038/s41586-018-0629-6
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DOI: https://doi.org/10.1038/s41586-018-0629-6
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