title: Endonuclease EEPD1 Is a Gatekeeper for Repair of Stressed Replication Forks.
abstract: 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.
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202.
authors.forename: Hyun-Suk
authors.initials: HS
authors.lastname: Kim
authors.affiliation: the Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, Colorado 80523.
authors.forename: Jac A
authors.initials: JA
authors.lastname: Nickoloff
authors.affiliation: the Department of Medicine and the Cancer Center, University of Florida Health, Gainesville, Florida 32610, and.
authors.forename: Yuehan
authors.initials: Y
authors.lastname: Wu
authors.affiliation: the Department of Medicine and the Cancer Center, University of Florida Health, Gainesville, Florida 32610, and.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Williamson
authors.affiliation: the Department of Medicine and the Cancer Center, University of Florida Health, Gainesville, Florida 32610, and.
authors.forename: Gurjit Singh
authors.initials: GS
authors.lastname: Sidhu
authors.affiliation: the Department of Medicine and the Cancer Center, University of Florida Health, Gainesville, Florida 32610, and.
authors.forename: Brian L
authors.initials: BL
authors.lastname: Reinert
authors.affiliation: the Department of Medicine and the Cancer Center, University of Florida Health, Gainesville, Florida 32610, and.
authors.forename: Aruna S
authors.initials: AS
authors.lastname: Jaiswal
authors.affiliation: the Department of Medicine and the Cancer Center, University of Florida Health, Gainesville, Florida 32610, and.
authors.forename: Gayathri
authors.initials: G
authors.lastname: Srinivasan
authors.affiliation: the Department of Medicine and the Cancer Center, University of Florida Health, Gainesville, Florida 32610, and.
authors.forename: Bhavita
authors.initials: B
authors.lastname: Patel
authors.affiliation: the Department of Medicine and the Cancer Center, University of Florida Health, Gainesville, Florida 32610, and.
authors.forename: Kimi
authors.initials: K
authors.lastname: Kong
authors.affiliation: the Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, Texas 75390.
authors.forename: Sandeep
authors.initials: S
authors.lastname: Burma
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202, slee@iupui.edu.
authors.forename: Suk-Hee
authors.initials: SH
authors.lastname: Lee
authors.affiliation: the Department of Medicine and the Cancer Center, University of Florida Health, Gainesville, Florida 32610, and robert.hromas@medicine.ufl.edu.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Hromas
mesh_terms: D004260:DNA Repair; D004261:DNA Replication; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans
chemical_list: C000608105:EEPD1 protein, human; D004706:Endodeoxyribonucleases
keywords: DNA damage; DNA endonuclease; DNA repair; DNA replication; end resection; homologous recombination; nuclease; replication fork stress