Functional hierarchy of PCNA‐interacting motifs in DNA processing enzymes

Abstract

Numerous eukaryotic DNA processing enzymes, such as DNA polymerases and ligases, bind the processivity factor PCNA, which acts as a platform to recruit and regulate the binding of enzymes to their DNA substrate. Multiple PCNA‐interacting motifs (PIPs) are present in these enzymes, but their individual structural and functional role has been a matter of debate. Recent cryo‐EM reconstructions of high‐fidelity DNA polymerase Pol δ (Pol δ), translesion synthesis DNA polymerase κ (Pol κ) and Ligase 1 (Lig1) bound to a DNA substrate and PCNA demonstrate that the critical interaction with PCNA involves the internal PIP proximal to the catalytic domain. The ancillary PIPs, located in long disordered regions, are instead invisible in the reconstructions, and appear to function as flexible tethers when the enzymes fall off the DNA. In this review, we discuss the recent structural advancements and propose a functional hierarchy for the PIPs in Pol δ, Pol κ, and Lig1.

Canonical binding of Chaetomium thermophilum DNA polymerase δ/ζ subunit PolD3 and flap endonuclease Fen1 to PCNA

“…Instead, the PolD3 and PolD4 PIP motifs might facilitate recruitment of Pol δ to PCNA that has previously been loaded at the primer-template junction, before handing over the task of stabilizing Pol δ-PCNA interaction to PolD1 PIP-PCNA binding, or might ensure that should Pol δ disengage from the primer-template, it is retained in the vicinity of the DNA to allow efficient polymerase recycling. The PolD3 and PolD4 PIP motifs are located at the end of lengthy flexible regions that could, in either case, be employed in the "fly casting mechanism" (Shoemaker et al, 2000) to scan threedimensional space and latch onto PCNA prior to the Pol δ-PCNA complex being locked more securely into position via the PolD1 PIP-PCNA interaction seen in the cryo-EM structures (Hamdan and De Biasio, 2023). A similar mechanism has been proposed for DNA ligase I (Lig1)-PCNA interactions: a PIP motif at the N-terminal end of the flexible N-terminal region of Lig1 (PIP N-term ) tethers the protein to PCNA when the ligase is detached from DNA but once the ligase locates a nick, this interaction is disrupted and a second PIP motif (PIP DBD ), located near the centre of the Lig1 DNA binding domain, engages with PCNA instead (Blair et al, 2022).…”

“…This ring-shaped homotrimer encircles double-stranded DNA to act as a processivity factor for DNA polymerases and a landing pad for the assembly of various DNA processing factors such DNA ligase I, the flap endonuclease Fen1, and the clamp loader complex replication factor C (RF-C) which opens and closes the PCNA ring around dsDNA. Many of the proteins that interact with PCNA, including DNA polymerase δ, DNA ligase I, Fen1 and RF-C, do so via a common mechanism that involves a short linear interaction motif on the partner protein called a PCNA-interacting protein (PIP) motif (Warbrick, 1998;Hamdan and De Biasio, 2023). The best characterised PIP motifs have conserved sequence Qxxψxxθθ, where ψ and θ represent amino acids with hydrophobic and aromatic side chains, respectively, but other variations on this sequence (so-called non-canonical PIP motifs) have been identified Prestel et al, 2019) such as ψxxxθ (Gonzalez-Magana et al, 2019) and Qxxψxθ (Yang et al, 2023).…”