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 Pol δ 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.

Functional hierarchy of PCNA‐interacting motifs in DNA processing enzymes

“…[ 67,68 ] A cryo‐EM structure of human Lig1 bound to a non‐ligatable nicked DNA and PCNA has been recently determined, and corresponds to the second step of the ligation reaction, right before the joining of the nick ends (Figure 3B). [ 69 ] The ligase occupies the top of the PCNA ring, with the DNA running across the ligase and through the PCNA hole, an architecture similar to that described in an archaeal ligase complex. [ 70 ] The Lig1−DNA structure closely resembles that reported in the absence of PCNA.…”

“…(B) Cryo‐EM model of Lig1 bound to PCNA and nicked DNA. [ 69 ] The inset shows the model details at the Lig1−PCNA interface. Lig1 PIP DBD is shown as red sticks, PCNA as a blue surface.…”

“…In this process PCNA forms two dynamic toolbelts, a Pol δ−PCNA−FEN1 toolbelt that mediates the handoff of the flap substrate from Pol δ to FEN1 and, upon FEN1 cleavage of the flap substrate, a Lig1−PCNA−FEN1 toolbelt that transfers the nicked DNA from FEN1 to Lig1 for its ligation. [30,69,74] The structures of these toolbelts show how Pol δ and Lig1 binding to their substrates results in the adjacent free PIP pockets on PCNA to be less accessible and the one across from them to be more exposed (Figure 3E). The PIPs of Pol δ, Lig1 and FEN1 interacting with PCNA in the toolbelts are all located in flexible loops which confer considerable dynamics to the bound enzymes.…”

“…Strikingly, the entire Lig1 N-terminus, including PIP N-term , is invisible in the cryo-EM map, suggesting that PIP N-term does not mediate an interaction with PCNA once the ternary complex has formed (Figure 3B). However, the off-DNA interaction between Lig1 and PCNA mediated by PIP N-term is strong (K d ∼ 40 nM), [69] presumably due to the basic residues flanking the PIP. [17,63] In addition, Förster Resonance Energy transfer (FRET) studies using Lig1 variants with one or both PIPs inactivated demonstrate that PIP N-term is important to recruit PCNA on the DNA, and cryo-EM data show that PIP N-term deletion partly compromises the formation of the ternary complex.…”

“…[17,63] In addition, Förster Resonance Energy transfer (FRET) studies using Lig1 variants with one or both PIPs inactivated demonstrate that PIP N-term is important to recruit PCNA on the DNA, and cryo-EM data show that PIP N-term deletion partly compromises the formation of the ternary complex. [69] Furthermore, a peptide spanning the PIP N-term of the yeast homolog of Lig1 (CDC9) has been previously co-crystallized with PCNA [71] (Figure 3C). Therefore, PIP N-term , with its long capture radius and high affinity, appears important for the initial recruitment of Lig1 to PCNA, while the PIP in the DBD stabilizes the complex with DNA once the three components (Lig1, PCNA and DNA) have acquired the correct relative configuration.…”