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DNA replication relies on precise coordination between proteins, including the sliding clamp proliferating cell nuclear antigen (PCNA), which encircles DNA to interact with key players in replication and repair. While biochemical studies have demonstrated interactions between PCNA and DNA polymerases δ and ε during DNA synthesis, the functional role of the Pol ε–PCNA interaction in vivo, particularly during leading strand synthesis, remains to be elucidated. To address this question, we employed AlphaFold to model how PCNA interact with four-subunit yeast Pol ε. Our models revealed two distinct points of interaction between Pol ε and PCNA: one at the P-domain and another at a PIP-box, a classical PCNA interaction motif. To validate these findings, we generated mutants that disrupted the Pol ε–PCNA interaction interface. Biochemical assays demonstrated that the PIP-box is critical for this interaction, with the P-domain serving as a secondary contact point. Notably, introducing these mutants into yeast, caused no phenotype in a wild-type background. However, when fewer origins are firing, resulting in longer stretches of leading strand synthesis before forks converge, strains expressing a Pol ε mutant lacking interaction with PCNA showed slower growth. These findings suggest that PCNA enhances the processivity of Pol ε both in vitro and in vivo.

DNA polymerase ε (Pol ε) participates in the synthesis of the leading strand during DNA replication in Saccharomyces cerevisiae. Pol ε comprises four subunits: the catalytic subunit, Pol2, and three accessory subunits, Dpb2, Dpb3 and Dpb4. DPB2 is an essential gene with unclear function. A genetic screen was performed in S. cerevisiae to isolate lethal mutations in DPB2. The dpb2-200 allele carried two mutations within the last 13 codons of the open reading frame, one of which resulted in a six amino acid truncation. This truncated Dpb2 subunit was co-expressed with Pol2, Dpb3 and Dpb4 in S. cerevisiae, but this Dpb2 variant did not co-purify with the other Pol ε subunits. This resulted in the purification of a Pol2/Dpb3/Dpb4 complex that possessed high specific activity and high processivity and holoenzyme assays with PCNA, RFC and RPA on a single-primed circular template did not reveal any defects in replication efficiency. In conclusion, the lack of Dpb2 did not appear to have a negative effect on Pol ε activity. Thus, the C-terminal motif of Dpb2 that we have identified may instead be required for Dpb2 to fulfill an essential structural role at the replication origin or at the replication fork.