Structural basis of nick translation in human DNA replication

Nick translation during Okazaki fragment maturation requires iterative coordination of DNA polymerase delta (Pol δ), which displaces the downstream primer, and flap endonuclease FEN1, which cleaves the resulting flap, on the sliding clamp PCNA. The structural basis of this coordination is unknown. We present cryo-EM structures of human Pol δ–PCNA, Pol δ–PCNA–FEN1 and FEN1–PCNA on flap DNA, capturing four states of the nick translation cycle. Pol δ strand displacement emerges from structural elements intrinsic to the B-family fold rather than dedicated separation machinery, with a conserved palm loop acting as separation wedge and PCNA engagement required for melting of the downstream duplex. In the Pol δ–PCNA–FEN1 toolbelt, FEN1 is pre-positioned opposite Pol δ on PCNA to receive the substrate. Nucleotide removal triggers DNA handoff while both enzymes remain clamp-bound, followed by Pol δ dissociation. A post-handoff structure reveals stable DNA retention by FEN1–PCNA after flap cleavage, explaining the slow nick translation kinetics and the obligate role of Ligase 1 in sealing.