Mechanism of human Lig1 regulation by PCNA in Okazaki fragment sealing

During lagging strand synthesis, DNA Ligase 1 (Lig1) cooperates with the sliding clamp PCNA to seal the nicks between Okazaki fragments generated by Pol delta and Flap endonuclease 1 (FEN1). We present several cryo-EM structures combined with functional assays, showing that human Lig1 recruits PCNA to nicked DNA using two PCNA-interacting motifs (PIPs) located at its disordered N-terminus (PIPN-term) and DNA binding domain (PIPDBD). Once Lig1 and PCNA assemble as two-stack rings encircling DNA, PIPN-term is released from PCNA and only PIPDBD is required for ligation to facilitate the substrate handoff from FEN1. Consistently, we observed that PCNA forms a defined complex with FEN1 and nicked DNA, and it recruits Lig1 to an unoccupied monomer creating a toolbelt that drives the transfer of DNA to Lig1. Collectively, our results provide a structural model on how PCNA regulates FEN1 and Lig1 during Okazaki fragments maturation.

Automated summary

Using cryo-EM structures and functional assays, we have discovered the mechanism of action of DNA Ligase 1 (Lig1) and the sliding clamp PCNA in lagging strand synthesis. Lig1 recruits PCNA to nicked DNA using specific PCNA-interacting motifs, and facilitates substrate handoff from FEN1 for ligation. Additionally, PCNA forms a defined complex with FEN1 and nicked DNA, and recruits Lig1 for DNA transfer.