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 (EXOI) 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 EXOI 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 gamma-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 EXOI 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. (C) 2019 Elsevier Inc. All rights reserved.