Cyclic-G/AMP (cGAMP) synthase (cGAS) triggers host innate immune responses against cytosolic double-stranded (ds)DNA arising from genotoxic stress and pathogen invasion. The canonical activation mechanism of cGAS entails dsDNA-binding and dimerization. Here, we report an unexpected activation mechanism of cGAS in which Mn2+ activates monomeric cGAS without dsDNA. Importantly, the Mn2+-mediated activation positively couples with dsDNA-dependent activation in a concerted manner. Moreover, the positive coupling between Mn2+ and dsDNA length-dependent activation requires the cognate ATP/GTP substrate pair, while negative-cooperativity suppresses Mn2+ utilization by either ATP or GTP alone. Additionally, while Mn2+ accelerates the overall catalytic activity, dsDNA length-dependent dimerization specifically accelerates the cyclization of cGAMP. Together, we demonstrate how the intrinsic allostery of cGAS efficiently yet precisely tunes its activity.
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