Proximity labeling reveals OTUD3 as a DNA-binding deubiquitinase of cGAS.

Cyclic GMP-AMP synthase (cGAS), as the major DNA sensor, initiates DNA-stimulated innate immune responses and is essential for a healthy immune system. Although some regulators of cGAS have been reported, it still remains largely unclear how cGAS is precisely and dynamically regulated and how many potential regulators govern cGAS. Here we carry out proximity labeling of cGAS with TurboID in cells and identify a number of potential cGAS-interacting or -adjacent proteins. Deubiquitinase OTUD3, one candidate identified in cytosolic cGAS-DNA complex, is further validated to not only stabilize cGAS but also enhance cGAS enzymatic activity, which eventually promotes anti-DNA virus immune response. We show that OTUD3 can directly bind DNA and is recruited to the cytosolic DNA complex, increasing its association with cGAS. Our findings reveal OTUD3 as a versatile cGAS regulator and find one more layer of regulatory mechanism in DNA-stimulated innate immune responses.