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

Abstract

SUMMARY 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.

Ribonuclease H2 mutations induce a cGAS/STING‐dependent innate immune response

“…Finally, the fact that both dsDNA (Sun et al , 2013; Gao et al , 2013) and RNA:DNA hybrids (Mankan et al , 2014) can bind and activate the cytoplasmic nucleic acid sensor cGAS prompted us to consider the origin of the cGAS ligand present in RNase H2‐deficient cells. Abrogated RNase H2 function could give rise to cytosolic accumulation of RNA:DNA heteroduplexes as a consequence of reduced RNA:DNA degradation of structures such as R‐loops or active retroelements/endogenous retroviruses (Chon et al , 2013; Rigby et al , 2014; Moelling & Broecker, 2015).…”

TREX1 Deficiency Triggers Cell-Autonomous Immunity in a cGAS-Dependent Manner

“…Cyclic GMP-AMP synthase (cGAS) has been identified as a cytosolic DNA receptor responsible for the induction of an antiviral and proinflammatory gene expression profile (2)(3)(4). Upon dsDNA binding, cGAS catalyzes the production of the second messenger molecule cGAMP(29-59), a unique cyclic dinucleotide molecule containing one 29-59 phosphodiester linkage in addition to a canonical 39-59 linkage (5)(6)(7)(8). Cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) subsequently stimulates the endoplasmic reticulum localized protein STING to induce cytokine expression through activation of the transcription factors IRF3 and NF-kB via the protein kinase TBK1 (9).…”

Building unique bonds to fight misplaced DNA

“…Taking the bacterial synthesis as a model and based on the fact that chemically synthesized cGAMP with the 3′-5′-phosphodiester linkage stimulates STING-dependent type I IFN production in mammalian cells [3], one would assume that cGASderived cGAMP likely contains the same phosphodiester linkage. However, in an outstanding paper published by Cell, Gao et al [7] challenged this view. Combining structural, chemical, biochemical and biological techniques, they definitely establish that cGAMP contains a 2′-5′ linkage, position this second messenger as the first 2′-5′ linkage-containing metazoan second messenger ever described, and distinguish it from the bacterial cyclic dinucleotides.…”