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Replication is not as continuous as once thought, with DNA damage frequently stalling replication forks. Aberrant repair of stressed replication forks can result in cell death or genome instability and resulting transformation to malignancy. Stressed replication forks are most commonly repaired via homologous recombination (HR), which begins with 5' end resection, mediated by exonuclease complexes, one of which contains Exo1. However, Exo1 requires free 5'-DNA ends upon which to act, and these are not commonly present in non-reversed stalled replication forks. To generate a free 5' end, stalled replication forks must therefore be cleaved. Although several candidate endonucleases have been implicated in cleavage of stalled replication forks to permit end resection, the identity of such an endonuclease remains elusive. Here we show that the 5'-endonuclease EEPD1 cleaves replication forks at the junction between the lagging parental strand and the unreplicated DNA parental double strands. This cleavage creates the structure that Exo1 requires for 5' end resection and HR initiation. We observed that EEPD1 and Exo1 interact constitutively, and Exo1 repairs stalled replication forks poorly without EEPD1. Thus, EEPD1 performs a gatekeeper function for replication fork repair by mediating the fork cleavage that permits initiation of HR-mediated repair and restart of stressed forks., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

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. [ABSTRACT FROM AUTHOR]

Chromosome translocations are caused by inappropriate religation of two DNA double-strand breaks (DSBs) in heterologous chromosomes. These DSBs can be generated by endogenous or exogenous sources. Endogenous sources of DSBs leading to translocations include inappropriate recombination activating gene (RAG) or activation-induced deaminase (AID) activity during immune receptor maturation. Endogenous DSBs can also occur at noncanonical DNA structures or at collapsed replication forks. Exogenous sources of DSBs leading to translocations include ionizing radiation (IR) and cancer chemotherapy. Spatial proximity of the heterologous chromosomes is also important for translocations. While three distinct pathways for DNA DSB repair exist, mounting evidence supports alternative nonhomologous end joining (aNHEJ) as the predominant pathway through which the majority of translocations occur. Initiated by poly (ADP-ribose) polymerase 1 (PARP1), aNHEJ is utilized less frequently in DNA DSB repair than other forms of DSB repair. We recently found that PARP1 is essential for chromosomal translocations to occur and that small molecule PARP1 inhibitors, already in clinical use, can inhibit translocations generated by IR or topoisomerase II inhibition. These data confirm the central role of PARP1 in aNHEJ-mediated chromosomal translocations and raise the possibility of using clinically available PARP1 inhibitors in patients who are at high risk for secondary oncogenic chromosomal translocations. [ABSTRACT FROM AUTHOR]