Structural and biological bases of cGAS bending bubble DNA to form a hyperactive dimer 2310028

Introduction The abnormal presence of DNA in the cytosol signals diverse intracellular crises. Cyclic GMP-AMP synthase (cGAS) is a predominant pattern-recognition receptor for double-stranded DNA (dsDNA), forming 2:2 cGAS-DNA dimers that further oligomerize into phase-separated condensate to initiate interferon responses. However, immunostimulatory DNA is rarely a perfectly contiguous duplex; instead, it frequently harbors additional conformations and local structures. How cGAS senses and responds to such nonlinear or non-contiguous dsDNAs remains poorly appreciated. Methods To address this, we created dsDNA containing unpaired regions, termed bubble DNA (Bu-DNA), and examined macrophage responses to Bu-DNA across multiple DNA topologies. To elucidate the underlying mechanism, we determined cryo-EM structures and performed biochemical, biophysical and single-molecule FRET (sm-FRET) analyses. Results Here, we demonstrate that Bu-DNA engages cGAS through a distinct binding configuration, eliciting enhanced signaling and cytokine production. Hyperactivation is observed by Bu-DNA embedded in linear DNA, circular DNA, plasmids and mitochondrial DNA. Bu-DNA binds cGAS with significantly higher affinity than fully complementary dsDNA, yet suppresses higher-order condensation. We further reveal that cGAS forms a 2cGAS:1DNA complex by bending Bu-DNA into a V-shape using the unpaired region as a hinge, thereby limiting its oligomeric state. Conclusion Taken together, these findings uncover a novel mode of cGAS activation and highlight the versatility of host defense strategies, in which cGAS recognizes structural features as a “pattern within a pattern” to fine-tune inflammatory responses. Funding Source NIH R01AI029564, R35CA232109, R01AI158314 (J.P-Y.T) and R35GM145363 (J.