RNA transcripts regulate G-quadruplex landscapes through G-loop formation (RNA-Seq)

G-quadruplexes (G4s) are prevalent DNA structures that regulate transcription but also threaten genome stability. How G4 dynamics is controlled remains poorly understood. Here, we report that RNA transcripts govern G4 landscapes through coordinated ‘G-loop’ assembly and disassembly. G-loop assembly involves activation of the ATM and ATR kinases followed by homology-directed invasion of RNA opposite the G4 strand mediated by BRCA2 and RAD51. Disassembly of the G-loop resolves the G4 structure via DHX36-FANCJ-mediated G4 unwinding that triggers nucleolytic incision and subsequent hybrid strand renewal by DNA synthesis. Inhibition of G-loop disassembly causes global G4 and R-loop accumulation, leading to transcriptome dysregulation, replication stress, and genome instability. These findings establish an intricate G-loop assembly-disassembly mechanism that controls G4 landscapes and is essential for cellular homeostasis and survival.