DHX9 resolves G-quadruplex condensation to prevent DNA double-strand breaks

Abstract DNA G-quadruplexes (G4s) structures are abundantly present in mammalian genomes and correlated with genome instability. However, the mechanism by which G4s are timely resolved remains unknown. Here, we report that DHX9 functions as a resolvase to unwind G4s globally in activated B cells. DHX9-deficient B cells show gross DNA double-strand breaks at the accumulated G4 sites, which are clustered together and form liquid condensates. We demonstrate that DHX9 also undergoes phase separation and fuses with G4 condensates for the productive unwinding of G4s in an ATP-dependent manner. Physiologically, G4-accumulation-induced DNA breaks can promote immunoglobulin class-switch recombination for producing high-affinity antibodies. Surprisingly, the DHX9Y1189C mutant identified in Hashimoto’s thyroiditis patients shows compromised phase separation and G4 unwinding abilities, causing elevated DNA damage and abnormal antibody production. Our findings suggest a DHX9-dependent G4 condensation-resolving mechanism to prevent DNA damage in mammalian cells. Disrupting this homeostasis may induce autoimmune disorders and lymphoid malignancies.