RNA transcripts suppress G-quadruplex structures through G-loop formation

The eukaryotic genome contains numerous guanine-rich sequences that regulate DNA replication and transcription by folding into non-canonical structures called G-quadruplexes (G4s)1. Compromised G4 resolution causes genomic instability and diseases linked to cancer susceptibility and aging2. However, how G4s are resolved is poorly understood. G4 structures are found in genomic regions containing DNA:RNA hybrids, also referred to as R-loops3, 4. When an R-loop and G4 structure form exactly opposite to each other, a G-loop structure is formed. G-loops have been observed directly in prokaryotes5 but their function is unknown. Using the Xenopus egg extract system, we show that G-loop structures act in suppressing mutagenic G4 structures thereby preventing genomic instability. Mechanistically, the RAD51 recombinase binds to the G4-opposing strand and promotes hybridization of RNA transcripts supplied by the hnRNPA1 ribonucleoprotein complex independently of ongoing transcription. The resulting G-loop then induces FANCI-FANCD2 monoubiquitination that triggers site-specific incision of the hybrid strand by the SLX4-XPF-ERCC1 complex. G-loop incision requires prior G4 unwinding by the DHX36 and FANCJ helicases and allows immediate DNA synthesis past the G4 motif. This resynthesis step impacts local chromatin states. This work establishes a G-loop-dependent mechanism that prevents mutagenic consequences of both G4 and R-loop structures.