RNA transcripts regulate G-quadruplex landscapes through G-loop formation

G-quadruplexes (G4s) are epigenetic DNA structures that regulate transcription but also threaten genome stability when dysregulated. It remains unknown how G4 dynamics is organized in a cell-specific manner. Here, we report that RNA transcripts govern G4 landscapes through homology-directed invasion opposite the G4 strand, forming 'G-loops'. G-loops are disassembled via DHX36-FANCJ-mediated G4 unwinding and subsequent nucleolytic incision by XPF-ERCC1, leading to hybrid strand renewal through DNA synthesis. G-loop disassembly requires stochiometric invading RNAs, by contrast, excess of which stabilizes G-loops against disassembly. Defects in G-loop disassembly accumulate G-loops predominantly in gene regulatory regions, leading to transcriptional dysregulation, replication stress, and DNA damage throughout the cell cycle. These findings establish nuclear RNAs as pivotal G-loop regulators, linking the transcriptome to gene regulation and genome maintenance. Overall design: Wild-type, DHX36KO, FANCJKO or DHX36/FANCJKO were subjected to scEdU-seq as previously described by van den Berg 2024