Fig 2: DNA-flexibility controls DNA-potential to bind and activate cGAS.a DNA looping assay schematic and fluorescence images of single DNA in donor and acceptor channels after 1 M NaCl treatment for noted time. Scale bar = 10 μm. b Loop fraction and time of damaged ISDs. In this study, data are presented as mean values ± standard deviations (SDs), and error bars indicate SDs (Mean ± SD). n = 3 independent experiments. c Coomassie staining of purified human cGAS (hcGAS) protein. M: Marker; kD: kilodaltons. d Left: EMSA of hcGAS binding to noted ISD using HT-DNA as control. Right: EMSA bound fraction of 50 nM ISDs binding to serial dilutions of hcGAS protein. Mean ± SD. n = 3 independent experiments. e Representative images of hcGAS–DNA condensates after mixing 2 μM hcGAS with 2 μM ISD. Red: Cy3-labelled DNA; Green: Alexa Fluor- 488 (AF-488)-labelled hcGAS; BF: bright field. Scale bar = 20 μm. f Looping fraction and times of noted ISDs. Mean ± SD. n = 3 independent experiments. g Left: Representative images of hcGAS–DNA condensates after mixing 2 μM hcGAS with noted ISD. Red: Cy3-labelled DNA; Green: AF-488-labelled hcGAS; BF: bright field. Scale bar = 20 μm. Middle: cGAMP produced by mixing 100 nM cGAS with 100 nM ISD for 2 h. Right: Ifnb mRNA levels in the MEF cells transfected with 100 ng/mL ISD for 2 h. Mean ± SD. n = 6 biologically independent samples. h EMSA analyses of 50 nM ISD binding to hcGAS. Mean ± SD. n = 3 independent experiments. i Left: Representative images of cGAS–DNA condensate formation after mixing hcGAS with noted ISD. Red: Cy3-labelled DNA; Green: AF-488-labelled hcGAS; BF: bright field. Scale bar = 20 μm. Middle: cGAMP produced by mixing hcGAS and ISD. Right: Ifnb mRNA levels in the MEF cells transfected with ISD. Mean ± SD. n = 6 biologically independent samples. Unless specifically noted, P values were from unpaired two-tailed Student’s t test (***p < 0.001, **p < 0.01, *p < 0.05) in this study. Source data are provided as a Source Data file.

Fig 3: Flexsurveillance model suggests that low dose IR might activate cGAS-mediated AIS via repairable DNA.a Left: Schematic showing the time schedule of acute immune surveillance (AIS) and late immune surveillance (LIS). Right: Representative images of the nude mice at noted times after irradiation (IR) (ranging 150–600 mJ/cm2). b Representative erythema images of the mice pretreated with 150 μg/cm2 RU.521 for 1 h at noted times after IR (ranging 150–600 mJ/cm2). n = 6. c Skin immunofluorescence (IF) (upper) and hematoxylin-eosin (HE) (lower) staining images of the RU.521-pretreated nude mice at 4 h after 300 mJ/cm2 IR. Scale bar = 100 μm. Upper: Blue: Dapi; Green: Ifnb (anti-Ifnb antibody). Lower: Black dashed lines are used to evaluate the swell. d Skin IF images of the cGAS wild type (WT) and knock out (KO) mice at 4 h after 400 mJ/cm2 IR. Blue: Dapi; Green: Ifnb (anti-Ifnb antibody). Scale bar = 100 μm. e IF images of the nude mice treated with intraperitoneal injection of 100 mg/kg BrdU and IR. Intestinal cells that usually undergo rapid proliferation were used as a positive control for the BrdU staining. Skin: 0 and 8 h after the treatment. Intestine: 5 D after the treatment. Blue: Dapi; Red: BrdU. Scale bar = 100 μm. f Representative images of 5 μg/cm2 UCN-01 and/or niraparib-pretreated mice at the noted times after IR (ranging 150–600 mJ/cm2). g Enlarged erythema images of the mice in (f). h Regression times of erythema in the mice treated with 300 mJ/cm2 IR. P values were calculated by unpaired two-tailed Student’s t-test without adjustment for multiple comparisons. Mean ± SD. n = 6 mice. Source data are provided as a Source Data file.

Fig 4: DNA flexibility is associated with DNA potential to bind and activate cGAS.a Looping time of seq1–10 ISDs. Mean ± SD. n = 3 independent experiments. b Spearman analyses of the correlation between the ISD looping time and noted cGAS-binding or -activating potential. P and R values were from two-sided spearman analyses. c Pearson analyses of the correlation between the DNA looping time and noted cGAS-binding or -activating potential. P and R values were from two-sided Pearson analyses. Red dashed lines are the best fit lines from the linear regression. Mean ± SD. Looping time: n = 3 independent experiments; EMSA, MST, MARQTFQ cGAMP and Ifnb mRNA: n = 6 biologically independent samples. Source data are provided as a Source Data file.

Fig 5: Human R236 substitution relaxes the DNA-length specificity of cGAS.a EMSA analyses of 50 nM 20 bp ISDs binding the noted mouse mcGAS proteins. Mean ± SD. n = 3 independent experiments. b cGAMP produced by mixing 100 nM noted mcGAS and 100 nM 20 bp noted ISD for 2 h. Unless specifically noted, P values were from unpaired two-tailed Student’s t test without adjustment for multiple comparisons (***p < 0.001, **p < 0.01, *p < 0.05, N.S. = not statistically significant (p > 0.05)) in this study. Mean ± SD. n = 6 biologically independent samples. c Ifnb mRNA levels in the human THP-1 cells transfected with 100 ng/mL 20 bp noted ISDs for 2 h. Mean ± SD. n = 3 biologically independent samples. d Left: cGAMP produced by 100 nM human hcGAS in serial dilutions of noted ISD for 2 h. Right: cGAMP produced by serial dilutions of human hcGAS in 100 nM noted ISD for 2 h. Mean ± SD. n = 3 biologically independent samples. e EMSA analyses of 50 nM 20 bp ISDs binding to the noted human hcGAS proteins. Mean ± SD. n = 3 biologically independent samples. f cGAMP produced by mixing 100 nM noted human hcGAS and100 nM 20 bp noted ISD for 2 h. Mean ± SD. n = 6 biologically independent samples. g cGAMP produced by 100 nM noted mouse mcGAS (left) or human hcGAS (right) in the presence of 100 nM 28- or 20 bp ISD for 2 h. Mean ± SD. n = 6 biologically independent samples. h cGAMP produced by 100 nM noted mouse mcGAS (left) or human hcGAS (right) in the presence of 100 nM 44- or 20 bp ISD for 2 h. Mean ± SD. n = 6 biologically independent samples. i Sequence alignment to show that the human K185 and L195 residues are non-conserved between human and mouse. j cGAMP produced by 100 nM noted human hcGAS in the presence of 100 nM 44- or 20 bp ISD for 2 h. Mean ± SD. n = 6 biologically independent samples. Source data are provided as a Source Data file.