An exonuclease safeguards cGAS immune signaling by degrading its 2'-5' phosphodiester intermediate

Cyclic oligonucleotide based anti-phage signaling systems (CBASS) are widespread in prokaryotes, providing innate immunity against phages1,2. CBASS systems typically use the signature cyclic GMP-AMP (cGAMP) synthase (cGAS)-like cyclase to synthesize second messengers in response to phage infection, which, in turn, activate effectors that induce abortive infection3-6. CBASS represents the ancestor of the cGAS-STING pathway that provides innate immunity for eukaryotes7,8. Due to the severe consequences triggered by the cGAMP signal, the activity of the cyclase must be tightly regulated within the cell, and for the first time, we discover a unique layer of regulation by thresholding the cGAMP synthesis. Many CBASS operons feature a predicted 3-5 exonuclease (Exo) of the RNase H fold9, but its role is unclear. In this study, we uncover the function of the Exo as a 2-5 phosphodiesterase. The Exo specifically degrades the intermediate 5-pppA[2-5]pG but not the final product cGAMP of cGAS, thereby setting a threshold of cGAMP synthesis. In prokaryotes, we showed that the Exo safeguards the CBASS immune signaling to prevent accident and excessive activation. Intriguingly, the Exo also degrades the reaction intermediate of human cGAS and inhibits human cGAS in vitro, suggesting a unified inhibitory mechanism of Exo across all species. Our results shed light on the molecular mechanisms underlying the regulation of the cGAS by Exo, highlighting its critical role in maintaining immune homeostasis and preventing autoimmunity, providing a candidate for developing novel cGAS antagonists.