RPA hyperphosphorylation hinders the resolution of R-loops and G-quadruplex-associated R-loops during RAS-driven senescence.

Activation of RAS oncogenes in normal cells triggers a stable cell cycle arrest known as RAS-induced senescence (RIS), marked by persistent DNA damage and extensive epigenetic remodeling. Although bypassing RIS promotes tumorigenesis, the molecular mechanisms underlying this transition remain poorly defined. Here, we demonstrate that RIS cells accumulate high levels of R-loops-three-stranded DNA-RNA hybrids-that frequently co-localize with DNA G-quadruplexes formed on the non-template DNA strand, generating G-loop-like structures. RIS bypass is characterized by the resolution of these structures through the heterotrimeric RPA complex, which facilitates RNase H1-mediated R-loop processing. In pre-RIS and RIS cells, hyperphosphorylation of RPA32 disrupts the ability of RPA to enhance RNase H1 activity, thereby impairing its enzymatic processivity. Consequently, R-loops and G-loops remain unresolved, contributing to the accumulation of γH2AX. Remarkably, forced restoration of RPA-regulated RNase H1 activity in RAS-expressing cells reduces DNA damage and enables cell cycle re-entry, effectively bypassing senescence. These findings identify a regulatory axis involving RPA phosphorylation and RNase H1 activity that governs R-loop and G-loop resolution, acting as a critical genome maintenance mechanism during oncogene-induced stress.