Project summary Excessive DNA either from pathogen or host activates the cytosolic DNA sensor, cyclic GMP-AMP synthase (cGAS) that produces the second messenger, cyclic GMP-AMP (cGAMP). cGAMP triggers a signal cascade leading to type I interferon (IFN) expression. It has been thought that cGAS is sequestered in the cytosol and cannot access host DNA normally restricted in cellular compartments, such as the nucleus. The recent discovery of nuclear cGAS has dramatically changed this model. Structural studies showed cGAS binds to the H2A-H2B heterodimer of the nucleosome and the binding immobilizes cGAS on the chromatin; thus, cGAS cannot access the nearby DNA. However, the micronuclei caused by DNA damage are immunostimulatory, and the chromatin fragments in micronuclei activate cGAS. Currently, we don’t know whether cGAS is still immobilized on the nucleosomes in the micronuclei and how cGAS is activated by the interaction with chromatin upon DNA damage. Endogenous defects in the DNA damage response (DDR) induce autoinflammatory diseases, such as Ataxia-Telangiectasia (A-T). Thus, there is a critical need to elucidate the mechanisms controlling cGAS activation upon DNA damage. Our objective is to define the role of K63-linked ubiquitination in DNA damage-induced cGAS activation and elucidate the underlying mechanisms by which K63-linked polyubiquitin regulates cGAS activity. Our preliminary data found that cGAS was subject to K63-linked ubiquitination upon DNA damage and inhibition K63-linked ubiquitination abrogated DNA damage-induced cGAS signaling pathway and interferon-stimulated gene expression. Based on the existing literature and our preliminary data, we hypothesize that K63-linked polyubiquitin facilitates cGAS activation in the micronuclei. We will examine two aims: First, we will determine the role of K63-linked ubiquitination in cGAS activation. Second, we will determine the mechanisms by which K63-linked polyubiquitin facilitates DNA damage-induced cGAS activation. The chromatin fragments in the micronuclei are a strong immunostimulatory agent for cGAS, but how cGAS overcome the histone sequestration and competes with other DNA binding proteins to access the DNA has yet to be determined. This exploratory R21 application proposes that K63-linked ubiquitination facilitates cGAS activation upon DNA damage. Overall, this study elucidates the regulatory mechanisms of nuclear cGAS activation and provides foundations for developing novel therapeutic strategies for infectious and autoimmune diseases.