Cyclic [G(2′,5′)pA(3′,5′)p] Is the Metazoan Second Messenger Produced by DNA-Activated Cyclic GMP-AMP Synthase

• Published in: Cell Vol. 153; no. 5; pp. 1094 - 1107
• Main Authors: Gao, Pu, Ascano, Manuel, Wu, Yang, Barchet, Winfried, Gaffney, Barbara L., Zillinger, Thomas, Serganov, Artem A., Liu, Yizhou, Jones, Roger A., Hartmann, Gunther, Tuschl, Thomas, Patel, Dinshaw J.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 23-05-2013
• Subjects: 2',5'-Oligoadenylate Synthetase - chemistry; adenosine triphosphate; Adenosine Triphosphate - metabolism; Amino Acid Sequence; Animalia; Animals; Crystallography, X-Ray; Dinucleoside Phosphates - metabolism; DNA - chemistry; DNA - metabolism; guanosine triphosphate; Guanosine Triphosphate - metabolism; Humans; interferons; Mice; Models, Chemical; Models, Molecular; Molecular Sequence Data; mutants; Nucleotides, Cyclic - metabolism; Nucleotidyltransferases - chemistry; Nucleotidyltransferases - metabolism; Second Messenger Systems; second messengers; Sequence Alignment
• ISSN: 0092-8674; 1097-4172
• DOI: 10.1016/j.cell.2013.04.046

Recent studies identified cyclic GMP-AMP (cGAMP) as a metazoan second messenger triggering an interferon response. cGAMP is generated from GTP and ATP by cytoplasmic dsDNA sensor cGAMP synthase (cGAS). We combined structural, chemical, biochemical, and cellular assays to demonstrate that this second messenger contains G(2′,5′)pA and A(3′,5′)pG phosphodiester linkages, designated c[G(2′,5′)pA(3′,5′)p]. We show that, upon dsDNA binding, cGAS is activated through conformational transitions, resulting in formation of a catalytically competent and accessible nucleotide-binding pocket for generation of c[G(2′,5′)pA(3′,5′)p]. We demonstrate that cyclization occurs in a stepwise manner through initial generation of 5′-pppG(2′,5′)pA prior to cyclization to c[G(2′,5′)pA(3′,5′)p], with the latter positioned precisely in the catalytic pocket. Mutants of cGAS dsDNA-binding or catalytic pocket residues exhibit reduced or abrogated activity. Our studies have identified c[G(2′,5′)pA(3′,5′)p] as a founding member of a family of metazoan 2′,5′-containing cyclic heterodinucleotide second messengers distinct from bacterial 3′,5′ cyclic dinucleotides.
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•A pronounced change in cGAS on DNA binding generates an accessible catalytic pocket•Structural/biochemical studies of cGAS-DNA with substrate, intermediate, and product•cGAS contains a single active site for stepwise formation of GpA and ApG linkages•HPLC/NMR of synthesized standards validate cGAS product as c[G(2′,5′)pA(3′,5′)p]
cGAS is a cytosolic DNA sensor that generates cyclic GMP-AMP as a second messenger to trigger the interferon response. Structural and biochemical studies of cGAS show that cGMP-AMP contains A(3′,5′)pG, but also G(2′,5′)pA, phosphodiester linkages. The latter may potentially represent a new family of cellular second messengers.