DHX30 is a replication stress-responsive R-loop helicase that safeguards genome stability
R-loops are three-stranded nucleic acid structures formed primarily during transcription that can obstruct replication fork progression, leading to replication stress and genome instability. Although RNA helicases are increasingly implicated in R-loop metabolism, how individual helicases safeguard genome integrity remains incompletely understood. Here we identify DHX30, a neurodevelopmental disorder-associated DExH-box RNA helicase, as a replication stress-responsive regulator of R-loops. We show that DHX30 localizes to the nucleus in addition to its known cytoplasmic and mitochondrial pools, and that its nuclear abundance in-creases following aphidicolin or camptothecin treatment. DHX30 deficiency results in RNA:DNA hybrid accumulation in human cells and zebrafish, R-loop-dependent DNA double-strand breaks, micronucleus formation, reduced DNA synthesis, and altered expression of immediate-early stress genes under basal conditions and following replication perturbation. Biochemically, DHX30 binds branched nucleic acid structures and directly unwinds R-loop substrates containing a 3' single-stranded RNA overhang in an ATP- and polarity-dependent manner. Proteomic analyses place DHX30 within a nuclear network of RNA- and R-loop-associated factors, including the RNA helicase DHX9. These findings establish DHX30 as a conserved R-loop-resolving helicase that protects from R-loop-induced replication perturbations, contributes to transcriptional homeostasis, and maintains genome stability, thereby providing a mechanistic framework for understanding DHX30-associated neurodevelopmental disorders.