Extended Data Fig. 5: cGAS DNA-binding site B is required for cGAS tethering by the nucleosome.
From: Structural basis for sequestration and autoinhibition of cGAS by chromatin
a, SPR analysis of single-cycle-kinetics experiment with immobilized nucleosomes via biotinylated DNA and mouse cGAScat and cGAScat(R241E) mutant as analytes. Shown are injections of 1.1, 3.3, 9, 10, 30 and 90 nM mouse cGAScat and cGAScat(R241E). Data are representative of two biological replicates. b, SPR analysis with acidic patch mutant nucleosomes (H2A(E61A/E64A/D90A)) immobilized via biotinylated DNA and cGAScat as analyte. Shown are buffer injections, injections of 1.1, 3.3, 9, 10, 30 and 90 nM mouse cGAScat and the cGAScat background-corrected data. Mouse cGAScat has orders of magnitude lower affinity to acidic patch mutant nucleosomes than wild-type nucleosomes. Data are representative of two biological replicates. c, Human cGAS(R236E) mutant activity assay preincubated with dsDNA followed by titration of 0N0 nucleosome. Data are representative of two biological replicates. d, Human cGAS(R236E) mutant activity assay pre-incubated with 0N0 nucleosome followed by titration of dsDNA. Data are representative of two biological replicates. e, Mouse cGAS mutations tested affecting cGAS–nucleosome interactions were tested for DNA-dependent activation with plasmid DNA in the presence of 0N0 nucleosomes. cGAMP production was assayed by thin-layer chromatography. Data are representative of two biological replicates. f, Mouse cGAS mutations affecting cGAS–nucleosome interactions were tested for DNA-dependent activation with 147-bp nucleosomal DNA in the presence of 0N0 nucleosomes. Data are representative of two biological replicates. g, Mouse cGAS single and double mutations of tethering loop and DNA-binding site B were tested for cGAMP production in the presence of plasmid DNA alone or plasmid DNA and 0N0 nucleosome. Data are representative of two biological replicates. h, Mouse cGAS mutants R222E, K240E and R241E require plasmid DNA for activation. Data are representative of two biological replicates. i, Mouse cGAS mutants R222E, K240E and R241E were tested for DNA-dependent activation with plasmid DNA in the presence of 0N0 nucleosomes. Mutation of DNA-binding site A abolishes activation by plasmid DNA. Data are representative of two biological replicates. j, Human cGAS activity assay in the presence of dsDNA, followed by titration of 0N0 and 0N0 acidic patch mutant I (apI; H2A(E61A/E64A/D90A)). Data are representative of two biological replicates. k, Wild-type and R236E mutant cGAS activity assays with 40N40 nucleosomes (40-bp linker DNA on each side). Data are representative of two biological replicates. l, Fluorescence anisotropy analysis of human cGAScat human cGAScat site A mutant (K407E/K411E) binding to fluorescently labelled 20-bp dsDNA and in the presence of 0N0 nucleosomes. Data are representative of two biological replicates. m, Agarose gel of micrococcal nuclease (MNase)-digested synthetic 601 chromatin indicating a regular nucleosomal structure. Data are representative of two biological replicates.
