Wu Yuehan, Lee Suk-Hee, Williamson Elizabeth A, Reinert Brian L, Cho Ju Hwan, Xia Fen, Jaiswal Aruna Shanker, Srinivasan Gayathri, Patel Bhavita, Brantley Alexis, Zhou Daohong, Shao Lijian, Pathak Rupak, Hauer-Jensen Martin, Singh Sudha, Kong Kimi, Wu Xaiohua, Kim Hyun-Suk, Beissbarth Timothy, Gaedcke Jochen, Burma Sandeep, Nickoloff Jac A, Hromas Robert A
Department of Medicine and the Cancer Center, University of Florida Health, Gainesville, Florida, United States of America.
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.
PLoS Genet. 2015 Dec 18;11(12):e1005675. doi: 10.1371/journal.pgen.1005675. eCollection 2015 Dec.
Replication fork stalling and collapse is a major source of genome instability leading to neoplastic transformation or cell death. Such stressed replication forks can be conservatively repaired and restarted using homologous recombination (HR) or non-conservatively repaired using micro-homology mediated end joining (MMEJ). HR repair of stressed forks is initiated by 5' end resection near the fork junction, which permits 3' single strand invasion of a homologous template for fork restart. This 5' end resection also prevents classical non-homologous end-joining (cNHEJ), a competing pathway for DNA double-strand break (DSB) repair. Unopposed NHEJ can cause genome instability during replication stress by abnormally fusing free double strand ends that occur as unstable replication fork repair intermediates. We show here that the previously uncharacterized Exonuclease/Endonuclease/Phosphatase Domain-1 (EEPD1) protein is required for initiating repair and restart of stalled forks. EEPD1 is recruited to stalled forks, enhances 5' DNA end resection, and promotes restart of stalled forks. Interestingly, EEPD1 directs DSB repair away from cNHEJ, and also away from MMEJ, which requires limited end resection for initiation. EEPD1 is also required for proper ATR and CHK1 phosphorylation, and formation of gamma-H2AX, RAD51 and phospho-RPA32 foci. Consistent with a direct role in stalled replication fork cleavage, EEPD1 is a 5' overhang nuclease in an obligate complex with the end resection nuclease Exo1 and BLM. EEPD1 depletion causes nuclear and cytogenetic defects, which are made worse by replication stress. Depleting 53BP1, which slows cNHEJ, fully rescues the nuclear and cytogenetic abnormalities seen with EEPD1 depletion. These data demonstrate that genome stability during replication stress is maintained by EEPD1, which initiates HR and inhibits cNHEJ and MMEJ.
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