Protein•DNA mesh assembly drives dsDNA-specific and duplex length-dependent activation of cGAS

Cyclic G/AMP synthase (cGAS) forms condensates on dysregulated double-stranded (ds) DNA to trigger inflammatory responses. Currently, how it specifically recognizes dsDNA and why activation depends on duplex length remain poorly understood. Using cryo-electron microscopy, biochemical assays, and single-molecule methods, we show that full-length cGAS assembles a protein•dsDNA mesh by reiteratively propagating dimers-of-dimers, driving an associative phase transition. Previously uncharacterized N- and C-terminal interactions, together with inter-dimer junction-loops, track the B-form groove and bend dsDNA to build an extensive mesh network spanning multiple duplexes. These interactions are critical for cross-stabilizing active cGAS, revealing why higher-order assembly on long duplexes maximizes signaling activity. Remarkably, cGAS can build the protein•dsDNA mesh on a single contiguous duplex by entangling its oligomerization platform, resulting in a mechanically resilient and kinetically stable signaling platform. Together, our findings establish higher-order mesh assembly of cGAS as the foundation for dsDNA selectivity, duplex-length-dependent activation, and condensate formation.