EEPD1 Rescues Stressed Replication Forks and Maintains Genome Stability by Promoting End Resection and Homologous Recombination Repair

Yuehan Wu, Suk-Hee Lee, Elizabeth A. Williamson, Brian L. Reinert, Ju Hwan Cho, Fen Xia, Aruna Shanker Jaiswal, Gayathri Srinivasan, Bhavita Patel, Alexis Brantley, Daohong Zhou, Lijian Shao, Rupak Pathak, Martin Hauer-Jensen, Sudha Singh, Kimi Kong, Xaiohua Wu, Hyun Suk Kim, Timothy Beissbarth, Jochen Gaedcke & 3 others Sandeep Burma, Jac A. Nickoloff, Robert A. Hromas

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Article numbere1005675
JournalPLoS Genetics
Volume11
Issue number12
DOIs
StatePublished - 2015

Fingerprint

Recombinational DNA Repair
Exonucleases
Genomic Instability
Endonucleases
homologous recombination
resection
Phosphoric Monoester Hydrolases
phosphatase
recombination
repair
genome
nucleases
homology
Homologous Recombination
cytogenetics
chromosome aberrations
DNA
cell death
phosphorylation
Protein Phosphatase 1

ASJC Scopus subject areas

  • Genetics
  • Molecular Biology
  • Ecology, Evolution, Behavior and Systematics
  • Cancer Research
  • Genetics(clinical)

Cite this

EEPD1 Rescues Stressed Replication Forks and Maintains Genome Stability by Promoting End Resection and Homologous Recombination Repair. / 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.

In: PLoS Genetics, Vol. 11, No. 12, e1005675, 2015.

Research output: Contribution to journalArticle

Wu, Y, Lee, S-H, Williamson, EA, Reinert, BL, Cho, JH, Xia, F, Jaiswal, AS, Srinivasan, G, Patel, B, Brantley, A, Zhou, D, Shao, L, Pathak, R, Hauer-Jensen, M, Singh, S, Kong, K, Wu, X, Kim, HS, Beissbarth, T, Gaedcke, J, Burma, S, Nickoloff, JA & Hromas, RA 2015, 'EEPD1 Rescues Stressed Replication Forks and Maintains Genome Stability by Promoting End Resection and Homologous Recombination Repair', PLoS Genetics, vol. 11, no. 12, e1005675. https://doi.org/10.1371/journal.pgen.1005675
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. / EEPD1 Rescues Stressed Replication Forks and Maintains Genome Stability by Promoting End Resection and Homologous Recombination Repair. In: PLoS Genetics. 2015 ; Vol. 11, No. 12.
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abstract = "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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AU - Wu, Yuehan

AU - Lee, Suk-Hee

AU - Williamson, Elizabeth A.

AU - Reinert, Brian L.

AU - Cho, Ju Hwan

AU - Xia, Fen

AU - Jaiswal, Aruna Shanker

AU - Srinivasan, Gayathri

AU - Patel, Bhavita

AU - Brantley, Alexis

AU - Zhou, Daohong

AU - Shao, Lijian

AU - Pathak, Rupak

AU - Hauer-Jensen, Martin

AU - Singh, Sudha

AU - Kong, Kimi

AU - Wu, Xaiohua

AU - Kim, Hyun Suk

AU - Beissbarth, Timothy

AU - Gaedcke, Jochen

AU - Burma, Sandeep

AU - Nickoloff, Jac A.

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N2 - 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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