Postdoct Associate The University of North Carolina at Chapel Hill Chapel Hill, North Carolina, United States
Disclosure(s):
Shuangshuang Yang, PhD: No relevant disclosure to display
Introduction/Rationale: 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.
