Figure 5. Characterization of c[G(2′,5′) pA(3′,5′)p] Formation by cGAS.

Generation of c[G(2′,5′)pA(3′,5′)p] and linear products and intermediates were assayed by thin-layer chromatography (TLC) using purified recombinant truncated (A, amino acids 147–507, used in crystallization studies) and full-length cGAS (B–D, amino acids 1–507). Long- and short-dashed lines indicate the origin and solvent fronts, respectively.

(A) A 45 nt dsDNA was incubated with truncated cGAS in reaction buffer containing indicated divalent cation (or EDTA) and α³²p-ATP and -GTP. Chemically synthesized cGAMP containing both 3′,5′ linkages was cospotted in every sample, and its migration, visualized by UV, is indicated (dashed outlines).

(B) cGAS was incubated with single (ss)-stranded or double (ds)-stranded DNA, RNA, DNA/RNA duplex, or 8-oxoguanine (8-O-G)-modified DNA of similar sequence, and c[G(2′,5′)pA(3′,5′)p] formation was monitored using α³²p-ATP.

(C) Mono- and diphosphorylated adenosine and guanosine were used as substrates to determine the order of c[G(2′,5′)pA(3′,5′)p] formation. Slow-migrating 2′,5′-linked species was observed when cGAS and dsDNA is incubated with α³²p-ATP and GMP (5′-pGpA) or GDP (5′-ppGpA).

(D) dsDNA-dependent cGAMP reaction intermediates were visualized by using 2′ or 3′ dATP and dGTP. Slow-migrating intermediate species, corresponding to pppGpA (lane 1) or pppGpdA (lanes 2 and 3), are seen by changing TLC mobile-phase composition. Intermediate species were confirmed using γ³²p-GTP.

See also Figures S5 and S6 and Table S6.