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Updated: Jan 25, 2026
Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
Published on: May 3, 2018
The human Exonuclease-1 interactome and phosphorylation sites
Wassim Eid1, Daniel Hess2, Christiane König1
1Institute of Molecular Cancer Research, University of Zurich, Winterthurerstrasse 190, CH-8057, Zurich, Switzerland.
Abstract:
Error-free repair of DNA double-strand breaks is orchestrated by homologous recombination (HR) pathways and requires the concerted action of several factors. Among these, Exonulcease-1 (EXO1) and DNA2/BLM execute extensive resection of DNA ends to produce 3'-overhangs, which are key intermediates for downstream steps of HR. To help shedding light on regulatory aspects of DNA repair pathways in which EXO1 participates, we set out to identify proteins interacting with EXO1. Affinity purification of EXO1 followed by Orbitrap mass spectrometry led to the identification of novel partners that are involved in RNA processing or that are the causative agents of rare X-linked disorders. Depletion of a selected subset of EXO1 interacting proteins led to reduction of the DNA damage response. Among those, we examined the RRP5-homologue and NF-kappa-B-interacting protein PDCD11/ALG-4, which has roles in apoptosis and is a putative driver gene in cutaneous T-cell lymphoma. We provide evidence that depletion of PDCD11 decreased the formation of γ-H2AX foci and the phosphorylation of DNA damage response (DDR) signaling intermediates in response to camptothecin or bleomycin, resulting in increased cellular resistance to DNA damage. Furthermore, extensive coverage of EXO1 sequence by mass spectrometry allowed conducting an in-depth analysis of its phosphorylation sites, with the identification of 26 residues that are differentially modified in untreated conditions or upon induction of DNA damage.
Insights
Researchers identified novel proteins interacting with Exonuclease-1 (EXO1), a key factor in DNA repair. Depleting PDCD11, an EXO1 partner, reduced DNA damage response and increased cellular resistance, highlighting its role in DNA repair pathways.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- DNA double-strand breaks are critical lesions repaired by homologous recombination (HR).
- Exonuclease-1 (EXO1) and DNA2/BLM are essential for DNA end resection, generating 3'-overhangs crucial for HR.
- Understanding the regulation of EXO1 function is vital for comprehending DNA repair fidelity.
Purpose of the Study:
- To identify novel protein interactors of Exonuclease-1 (EXO1).
- To investigate the role of EXO1-interacting proteins in DNA damage response (DDR).
- To analyze the phosphorylation status of EXO1 under various conditions.
Main Methods:
- Affinity purification of EXO1 followed by Orbitrap mass spectrometry.
- Depletion of identified interacting proteins using RNA interference.
- Assessment of DNA damage response markers (e.g., γ-H2AX foci) and cellular resistance.
- Mass spectrometry-based phosphoproteomic analysis of EXO1.
Main Results:
- Identification of novel EXO1 interacting partners involved in RNA processing and X-linked disorders.
- Depletion of specific EXO1 interactors, including PDCD11/ALG-4, diminished the DNA damage response.
- PDCD11 depletion decreased γ-H2AX foci and DDR signaling, conferring increased cellular resistance to DNA damage.
- Identification of 26 differentially phosphorylated EXO1 residues under basal and DNA-damaged conditions.
Conclusions:
- PDCD11/ALG-4 is a novel regulator of DNA damage response pathways involving EXO1.
- EXO1 phosphorylation sites are dynamically regulated during DNA repair.
- These findings provide insights into the complex regulatory network governing homologous recombination and DNA repair.
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