Human REV1 interacts with DHX36 to promote replication and tolerance of G-quadruplex DNA.

G-quadruplex DNA is a barrier to replication, but how the replisome couples G4 bypass with fork progression is not well-defined. Here, we establish that REV1 is central to coordination of G4 resolution, replication fidelity, and tolerance of G4 stabilization. REV1 loss switched fork elongation to a PrimPol-driven mechanism and resulted in defective ssDNA gap suppression in cells treated with pyridostatin (PDS). Mutagenic G4 replication on the leading strand was more impacted by REV1 loss than lagging strand bypass, but only lagging strand mutagenesis was sensitive to PDS. REV1 deficiency increased nuclear G4 signal, amplified ATM/ATR signaling, and sensitized cells to G4-stabilizing agents. We discovered that the REV1 C-terminal domain interacts with the G4 helicase DHX36 to restrain PrimPol activity. The REV1-DHX36 interaction is direct and requires a newly defined REV1-interacting region at the C-terminus of DHX36. Prolonged G4 stabilization uncoupled REV1 and DHX36, with the G4 helicase accumulating at a site distal from REV1 and the DNA synthesis machinery. Our findings establish a two-tiered mechanism for REV1 action that coordinates helicase-dependent G4 unwinding with suppression of ssDNA gaps in response to G4 stabilization.