Nat Commun. 2025 Dec 13. doi: 10.1038/s41467-025-67284-5
The non-catalytic DNA polymerase ε subunit is an NPF motif recognition protein
Salla Keskitalo#1, Boglarka Zambo#2,3, Dicle Malaymar Pinar1, Antti Tuhkala1, Kari Salokas1, Tanja Turunen1, Norbert Deutsch4, Norman Davey5, Zsuzsanna Dosztányi4, Markku Varjosalo6, Gergo Gogl7,8
Affiliations
1Institute of Biotechnology, Helsinki Institute of Life Science HiLIFE, University of Helsinki, Helsinki, Finland.
2Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), INSERM U1258/CNRS UMR 7104/Université de Strasbourg, Illkirch, France.
3Institut de Biologie Valrose – iBV, Université Côte d’Azur, CNRS UMR7277, Inserm, Nice, France.
4Department of Biochemistry, ELTE Eötvös Loránd University, Budapest, Hungary.
5Institute of Cancer Research, Chester Beatty Laboratories, London, UK.
6Institute of Biotechnology, Helsinki Institute of Life Science HiLIFE, University of Helsinki, Helsinki, Finland.
7Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), INSERM U1258/CNRS UMR 7104/Université de Strasbourg, Illkirch, France.
8Institut de Biologie Valrose – iBV, Université Côte d’Azur, CNRS UMR7277, Inserm, Nice, France.
#Contributed equally.
Abstract
Short linear motifs (SLiMs) in disordered protein regions direct numerous protein-protein interactions, yet most remain uncharacterized. The Asn-Pro-Phe (NPF) motif is a well-known EH-domain ligand implicated in endocytosis, but here we reveal that the non-catalytic subunit of human DNA polymerase ε (POLE2) also serves as a general NPF-motif receptor. Using a quantitative “native holdup” assay, we find that POLE2 selectively binds diverse NPF-containing peptides, including canonical EH-domain ligands (e.g., SYNJ1) and previously uncharacterized motifs. Biochemical measurements and mutational analyses show that NPF motifs interact with a shallow pocket near the POLE2 C-terminus, and AlphaFold predictions confirm key roles for Y513, E520, and S522 in motif coordination. Proteome-scale affinity screens identify NPF-containing nuclear proteins (e.g., WDHD1, DONSON, TTF2) that bind POLE2 with micromolar affinities, and their motif mutations abolish binding in cell extracts. Although POLE2 primarily tethers the catalytic POLE subunit to replication forks, these results indicate that it can also recruit various NPF-bearing partners involved in replication, DNA repair, and transcription regulation. Notably, NPF motifs optimized for EH-domain binding can still associate with POLE2, highlighting the inherent degeneracy of SLiM-mediated networks. Overall, these findings establish POLE2 as a central hub possibly linking replication with other processes via broad NPF-motif recognition.