Mechanism of human Lig1 regulation by PCNA in Okazaki fragment sealing

Abstract During lagging strand synthesis, DNA Ligase 1 (Lig1) cooperates with the sliding clamp PCNA to seal the nicks between Okazaki fragments generated by Flap endonuclease 1 (FEN1), but the structural basis of this mechanism is unknown. We present cryo-EM structures of human Lig1 bound to nicked DNA and PCNA in absence and presence of FEN1. Lig1 and PCNA form a two-ring complex encircling the DNA substrate. The ligase DNA binding domain (DBD) dynamically tethers Lig1 to one PCNA protomer, leaving one protomer fully accessible for FEN1 engagement. Ligation assays argue that Lig1 binding to PCNA is critical for substrate handoff from FEN1 preassembled with nicked DNA and PCNA. Consistently, cryo-EM data show that when Lig1 and FEN1 are simultaneously bound to one PCNA ring, the nicked DNA is trapped within the ligase active site and poised for the end-joining reaction. Thus, PCNA functions as a bivalent platform for the synchronous coordination of Lig1 and FEN1 in Okazaki fragment sealing.