title: EEPD1 Rescues Stressed Replication Forks and Maintains Genome Stability by Promoting End Resection and Homologous Recombination Repair.
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.
authors.affiliation: Department of Medicine and the Cancer Center, University of Florida Health, Gainesville, Florida, United States of America.
authors.forename: Yuehan
authors.initials: Y
authors.lastname: Wu
authors.affiliation: Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.
authors.forename: Suk-Hee
authors.initials: SH
authors.lastname: Lee
authors.affiliation: Department of Medicine and the Cancer Center, University of Florida Health, Gainesville, Florida, United States of America.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Williamson
authors.affiliation: Department of Medicine and the Cancer Center, University of Florida Health, Gainesville, Florida, United States of America.
authors.forename: Brian L
authors.initials: BL
authors.lastname: Reinert
authors.affiliation: Department of Radiation Oncology, Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, United States of America.
authors.forename: Ju Hwan
authors.initials: JH
authors.lastname: Cho
authors.affiliation: Department of Radiation Oncology, Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, United States of America.
authors.forename: Fen
authors.initials: F
authors.lastname: Xia
authors.affiliation: Department of Medicine and the Cancer Center, University of Florida Health, Gainesville, Florida, United States of America.
authors.forename: Aruna Shanker
authors.initials: AS
authors.lastname: Jaiswal
authors.affiliation: Department of Medicine and the Cancer Center, University of Florida Health, Gainesville, Florida, United States of America.
authors.forename: Gayathri
authors.initials: G
authors.lastname: Srinivasan
authors.affiliation: Department of Medicine and the Cancer Center, University of Florida Health, Gainesville, Florida, United States of America.
authors.forename: Bhavita
authors.initials: B
authors.lastname: Patel
authors.affiliation: Department of Medicine and the Cancer Center, University of Florida Health, Gainesville, Florida, United States of America.
authors.forename: Alexis
authors.initials: A
authors.lastname: Brantley
authors.affiliation: Department of Pharmaceutical Sciences, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America.
authors.forename: Daohong
authors.initials: D
authors.lastname: Zhou
authors.affiliation: Department of Pharmaceutical Sciences, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America.
authors.forename: Lijian
authors.initials: L
authors.lastname: Shao
authors.affiliation: Department of Pharmaceutical Sciences, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America.
authors.forename: Rupak
authors.initials: R
authors.lastname: Pathak
authors.affiliation: Department of Pharmaceutical Sciences, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America.
authors.forename: Martin
authors.initials: M
authors.lastname: Hauer-Jensen
authors.affiliation: Department of Medicine and the Cancer Center, University of Florida Health, Gainesville, Florida, United States of America.
authors.forename: Sudha
authors.initials: S
authors.lastname: Singh
authors.affiliation: Department of Craniofacial Regeneration, College of Dental Medicine, Columbia University, New York, New York, United States of America.
authors.forename: Kimi
authors.initials: K
authors.lastname: Kong
authors.affiliation: Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, California, United States of America.
authors.forename: Xaiohua
authors.initials: X
authors.lastname: Wu
authors.affiliation: Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.
authors.forename: Hyun-Suk
authors.initials: HS
authors.lastname: Kim
authors.affiliation: Department of Medical Statistics, and General, Visceral, and Pediatric Surgery, University Medical Center Göttingen, Göttingen, Germany.
authors.forename: Timothy
authors.initials: T
authors.lastname: Beissbarth
authors.affiliation: Department of Medical Statistics, and General, Visceral, and Pediatric Surgery, University Medical Center Göttingen, Göttingen, Germany.
authors.forename: Jochen
authors.initials: J
authors.lastname: Gaedcke
authors.affiliation: Department of Radiation Oncology, University of Texas Southwestern, Dallas, Texas, United States of America.
authors.forename: Sandeep
authors.initials: S
authors.lastname: Burma
authors.affiliation: Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, Colorado, United States of America.
authors.forename: Jac A
authors.initials: JA
authors.lastname: Nickoloff
authors.affiliation: Department of Medicine and the Cancer Center, University of Florida Health, Gainesville, Florida, United States of America.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Hromas
mesh_terms: D053903:DNA Breaks, Double-Stranded; D004249:DNA Damage / Q000235:genetics; D059766:DNA End-Joining Repair / Q000235:genetics; D004265:DNA Helicases / Q000235:genetics*; D004706:Endodeoxyribonucleases / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics; D005786:Gene Expression Regulation; D042822:Genomic Instability; D057809:HEK293 Cells; D006657:Histones / Q000235:genetics; D059765:Homologous Recombination / Q000235:genetics*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics*; D059767:Recombinational DNA Repair / Q000235:genetics*; D000071857:Tumor Suppressor p53-Binding Protein 1
chemical_list: D029968:Escherichia coli Proteins; D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; C498301:TP53BP1 protein, human; D000071857:Tumor Suppressor p53-Binding Protein 1; C000608105:EEPD1 protein, human; D004706:Endodeoxyribonucleases; C071059:Holliday junction DNA helicase, E coli; D004265:DNA Helicases