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. | Nature

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.

From: Nuclear cGAS suppresses DNA repair and promotes tumorigenesis

Extended Data Fig. 7

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. gl, 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.

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