Project summary In metazoan, the presence of double-stranded (ds)DNA in the cytosol signals serious problems, which range from radiation damage to pathogen invasion. The innate immune system acts as the first line of defense against cytosolic dsDNA by initiating inflammatory signaling pathways. Cytosolic dsDNA sensing pathways are integral to host defense against numerous pathogens, and their malfunctions also result in various human maladies. Our research program is poised to resolve several long-standing mechanistic questions in understanding how cytosolic dsDNA sensing pathways are activated and regulated at the molecular level. We will also test new concepts as to how these sensors might be stigmatized as autoantigens, and explore the mechanical forces that drive and regulate their higher order assemblies. Our approaches include X-ray crystallography, rigorous biochemical measurements, cell-based assays, electron microscopy, and single molecule methods. Here, we will determine how cGAS coordinates different nucleotide substrates and metal co-factors at the active site to specifically generate 2'-5'/3'-5' linked cyclic G/AMP. We will also investigate how dimerization is allosterically coupled to activation. We will then determine how cGAS and ALR sensors selectively recognize and signal through dsDNA. Next, we will test the role of phase-separation/transition in the normal and aberrant activities of cytosolic dsDNA sensors. Finally, we will delineate mechanical forces and structural mechanisms that underpin the activation and regulation of cytosolic dsDNA sensors. The molecular insights resulting from the proposed studies will provide a foundation for developing new therapeutic strategies that target various human diseases caused by dysregulated cytosolic dsDNA sensing pathways.