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EEPD1 (endonuclease/exonuclease/phosphatase family domain-containing 1) is an uncharacterized human protein that we found to be transcriptionally increased upon induction of DNA double strand breaks (DSBs). A549 cells with EEPD1 repressed by siRNA arrested at the G1/S and G2/M transitions and had markedly increased sensitivity to various DSB-inducing agents, such as camptothecin, VP-16, hydroxyurea (HU), and ionizing irradiation (IR). There are two major types of DSB repair, non-homologous end-joining (NHEJ) and homologous recombination (HR), which is essential for replication fork repair. Using the EJ5 NHEJ reporter system, EEPD1 repression resulted in a 2-fold increase in NHEJ repair. However, formation of 53BP1 foci was unaffected in these cells, indicating that the initial step towards NHEJ was intact. However, in the HT256 HR reporter system, EEPD1 repression produced a 5-fold decrease in HR repair, indicating that EEPD1 mediates HR and inhibits NHEJ, placing it at the decision point for DSB repair. Immunofluorescence assays of HR components were performed in the presence of HU. EEPD1 repression significantly decreased γ-H2Ax, RAD51, and RPA32 foci formation, so placing EEPD1 above these HR components. The initial activity that commits a DSB to HR repair and away from NHEJ is 5′ end resection. This generates the 3′ ss DNA that results in ATR/Chk1 activation as well as RPA32 and γ-H2Ax foci. To assess this, a ss BRDU end resection assay was done after IR and there was a 4-fold decrease in 5′ end resection when EEPD1 was repressed. Consistently, there was also a decrease in ATR and Chk1 phosphorylation. Co-immunoprecipitation studies found that EEPD1 interacts with Ku80 and NBS1. In vitro assays using purified recombinant EEPD1 protein found its 5′ endonuclease activity was distinct from both Exo1 and DNA2, the two major mediators of 5′ end resection. This activity was, however, repressed by the Ku complex, indicating antagonism between EEPD1 and Ku. Cells with decreased EEPD1 expression showed severe nuclear anomalies, such as micronuclei, nucleoplasmic bridges that may have been caused by unopposed NHEJ when the functional HR was not available. Metaphase analysis in these cells also showed a significant increase in chromosomal aberrations, especially after IR and HU. These data suggest a novel model of collapsed replication fork repair pathway choice, where the free DS end binds the MRN complex, and NBS1 recruits EEPD1, inhibiting NHEJ and promoting HR via initiation of 5′ end resection. This is supported by the finding that EEPD1 is required for RPA32/ATRIP activation of ATR/Chk1, and all subsequent steps in repairing collapsed replication forks. Without such repair, the collapsed forks are end-joined to create chromosome fusions, which generate the nucleoplasmic bridging during mitosis. Lung and endometrial cancers have significant rates of EEPD1 mutation, further indicating that EEPD1 might be a novel tumor suppressor. Citation Format: Yuehan Wu, Suk-Hee Lee, Elizabeth A. Williamson, Brian L. Reinert, Gayathri Srinivasan, Sudha Singh, Aruna-Shanker Jaiswal, Silvia Tornaletti, Alexis C. Brantly, Robert A. Hromas. The 5′ endonuclease EEPD1 maintains genomic stability by mediating DNA repair pathway choice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2427. doi:10.1158/1538-7445.AM2014-2427