Dataset Information

The non-catalytic epsilon DNA polymerase subunit is an NPF motif recognition protein

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 epsilon (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 analysis 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 linking replication with other processes via broad NPF-motif recognition.

INSTRUMENT(S): timsTOF Pro 2

ORGANISM(S): Homo Sapiens

SUBMITTER: Markku Varjosalo Gergo Gogl

PROVIDER: MSV000097307 | MassIVE | Thu Mar 13 01:18:00 GMT 2025

SECONDARY ACCESSION(S): PXD061783

REPOSITORIES: MassIVE

ACCESS DATA

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Project description:BACKGROUND: In all living organisms, DNA replication is exquisitely regulated in a wide range of growth conditions to achieve timely and accurate genome duplication prior to cell division. Failures in this regulation cause DNA damage with potentially disastrous consequences for cell viability and human health, including cancer. To cope with these threats, cells tightly control replication initiation using well-known mechanisms. They also couple DNA synthesis to nutrient richness and growth rate through a poorly understood process thought to involve central carbon metabolism. One such process may involve the cross-species conserved pyruvate kinase (PykA) which catalyzes the last reaction of glycolysis. Here we have investigated the role of PykA in regulating DNA replication in the model system Bacillus subtilis.RESULTS: On analysing mutants of the catalytic (Cat) and C-terminal (PEPut) domains of B. subtilis PykA we found replication phenotypes in conditions where PykA is dispensable for growth. These phenotypes are independent from the effect of mutations on PykA catalytic activity and are not associated with significant changes in the metabolome. PEPut operates as a nutrient-dependent inhibitor of initiation while Cat acts as a stimulator of replication fork speed. Disruption of either PEPut or Cat replication function dramatically impacted the cell cycle and replication timing even in cells fully proficient in known replication control functions. In vitro, PykA modulates activities of enzymes essential for replication initiation and elongation via functional interactions. Additional experiments showed that PEPut regulates PykA activity and that Cat and PEPut determinants important for PykA catalytic activity regulation are also important for PykA-driven replication functions.CONCLUSIONS: We infer from our findings that PykA typifies a new family of cross-species replication control regulators that drive the metabolic control of replication through a mechanism involving regulatory determinants of PykA catalytic activity. As disruption of PykA replication functions causes dramatic replication defects, we suggest that dysfunctions in this new family of universal replication regulators may pave the path to genetic instability and carcinogenesis.

Dataset Information

The non-catalytic epsilon DNA polymerase subunit is an NPF motif recognition protein

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets