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Evidence for a functional interaction between yeast Pol ε and PCNA in vivo

Singh, Noopur ; Odai, Roni LU ; Persson, Ulf LU ; Bylund, Göran O. LU ; Obi, Ikenna ; Sabouri, Nasim ; Atkinson, Gemma C. LU orcid and Johansson, Erik LU (2025) In Nucleic Acids Research 53(22).
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... (More)

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.

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organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Nucleic Acids Research
volume
53
issue
22
article number
gkaf1339
publisher
Oxford University Press
external identifiers
  • scopus:105025062180
  • pmid:41404803
ISSN
0305-1048
DOI
10.1093/nar/gkaf1339
language
English
LU publication?
yes
additional info
Publisher Copyright: © The Author(s) 2025. Published by Oxford University Press.
id
c8e253ad-3961-4c93-b52c-488a5a681c37
date added to LUP
2026-02-11 15:16:11
date last changed
2026-02-12 15:06:51
@article{c8e253ad-3961-4c93-b52c-488a5a681c37,
  abstract     = {{<p>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.</p>}},
  author       = {{Singh, Noopur and Odai, Roni and Persson, Ulf and Bylund, Göran O. and Obi, Ikenna and Sabouri, Nasim and Atkinson, Gemma C. and Johansson, Erik}},
  issn         = {{0305-1048}},
  language     = {{eng}},
  month        = {{12}},
  number       = {{22}},
  publisher    = {{Oxford University Press}},
  series       = {{Nucleic Acids Research}},
  title        = {{Evidence for a functional interaction between yeast Pol ε and PCNA in vivo}},
  url          = {{http://dx.doi.org/10.1093/nar/gkaf1339}},
  doi          = {{10.1093/nar/gkaf1339}},
  volume       = {{53}},
  year         = {{2025}},
}