Structural basis of nick translation in human DNA replication

Abstract

Nick translation during Okazaki fragment maturation requires iterative coordination of DNA polymerase delta (Pol {delta}), 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 {delta}-PCNA, Pol {delta}-PCNA-FEN1 and FEN1-PCNA on flap DNA, capturing four states of the nick translation cycle. Pol {delta} 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 {delta}-PCNA-FEN1 toolbelt, FEN1 is pre-positioned opposite Pol {delta} on PCNA to receive the substrate. Nucleotide removal triggers DNA handoff while both enzymes remain clamp-bound, followed by Pol {delta} 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.