Year: 2025
Type: article
Abstract: 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.
Source: Nucleic Acids Research
DOI: 10.1093/nar/gkaf1339
Institutions: Umeå University, Lund University, Science for Life Laboratory
Language: English
Topic: DNA Repair Mechanisms
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $3,992
Awards: G1303920544, G1441096865, Fidelity of DNA replication – cancer associated substitutions in DNA polymerase epsilon, G2252777492, G2879713366, Interrogating bacteriophages to discover the universal secrets of host-virus and virus-virus warfare, G3321450376, G3631573628, Discovering the interplay between antibiotic and phage resistance in bacteria, G3983392107, G4066480865, National Microscopy Infrastructure, G516287535, G5181495137, G6265702644, G633570985, Discovering and exploiting the molecular basis of microbial warfare, G6625919268, G712881263, G7187285009, G7318745325, G796431723, G8436673617, NAISS, G8896341493
OpenAlex ID: W4417457361