Cytosolic TOP3α facilitates mitochondrial DNA sensing by cGAS

Mitochondrial DNA (mtDNA) serves as a potent activator for cellular innate immune responses. Topoisomerase 3α (TOP3α), a type IA topoisomerase, is canonically localized to mitochondria and nuclei, but its enigmatic cytosolic fraction-observed over two decades ago-has remained functionally undefined. Here, we uncover a critical role for cytosolic TOP3α in amplifying mtDNA-triggered innate immunity. We observe that aberrant TOP3α expression causes mtDNA clustering and release via mPTP-VDAC, stimulating cGAS-mediated inflammatory responses. Cytosolic TOP3α facilitates the sensing of released mtDNA by cGAS and amplifies downstream innate immune signaling. Using an in vitro cell-free system, we reveal that TOP3α directly augments mtDNA interaction with cGAS, which in turn competes with TOP3α for mtDNA binding. A rare mutation of a highly conserved residue (G250D) of TOP3α impairs the assembly of TOP3α polypeptides into protein complexes and its binding to mtDNA. Furthermore, mutant TOP3α hinders cGAS-mtDNA interaction and compromises cGAS-driven immunity. Our findings reveal a function for cytosolic TOP3α as a regulator for cGAS-driven inflammation.