Extended Data Fig. 9: Effect of structure-guided mutations in cells.

a, Lines show FRAP recovery curves obtained after photo-bleaching WT cGAS-GFP or cGAS-GFP mutants inside the nucleus of cGAS KO HeLa cells. Data show mean ± SEM from 20-25 measurements. Graph is representative of n = 3 (left panel) or 4 (right panel) independent experiments. b–d, HeLa cGAS KO cells reconstituted with doxycycline-inducible WT cGAS or cGAS mutants were treated with doxycycline (1μg/ml) for 16h and 40h (b, c) or 40h (d), respectively. In b, cells were lysed and mRNA levels of IFI44, IFIT2 and CGAS were assessed. Data are presented as fold induction relative to non-treated WT cGAS and are mean ± s.d. of n = 5 independent experiments. Two-way ANOVA with post hoc Tukey multiple comparison test. In c, cells were lysed and STING and GAPDH levels were assessed by immunoblot. One representative experiment for n = 3 (16h) and n = 3 (40h) experiments with similar results is shown. In d, cells were stimulated with dsDNA (90mer) for 4h or left untreated (Ctrl.) and mRNA levels of IFI44 and IFIT2 were measured. Data show mean ± s.d. of n = 2 independent experiments. Individual data points represent biological replicates. For gel source data, see Supplementary Fig. 1. e, cGAS multiple sequence alignments showing the sequence conservation of residues involved in interactions with the acidic patch, nucleosome binding in cis, and nucleosome binding in trans. cGAS sequences from human, monkey, bovine, pig, mouse and rat corresponding to UniProt ID’s Q8N884, F7B8L6, E1BGN7, I3LM39, Q8C6L5 and A0A0G2JVC4 have been used in the alignment. The key residues involved in the interactions are highlighted in cyan. The consensus sequence and logo representation of the residues is shown below the sequence alignment. The alignment figure was created using Jalview⁵².