title: Cell cycle stage-specific roles of Rad18 in tolerance and repair of oxidative DNA damage.
abstract: The E3 ubiquitin ligase Rad18 mediates tolerance of replication fork-stalling bulky DNA lesions, but whether Rad18 mediates tolerance of bulky DNA lesions acquired outside S-phase is unclear. Using synchronized cultures of primary human cells, we defined cell cycle stage-specific contributions of Rad18 to genome maintenance in response to ultraviolet C (UVC) and H(2)O(2)-induced DNA damage. UVC and H(2)O(2) treatments both induced Rad18-mediated proliferating cell nuclear antigen mono-ubiquitination during G(0), G(1) and S-phase. Rad18 was important for repressing H(2)O(2)-induced (but not ultraviolet-induced) double strand break (DSB) accumulation and ATM S1981 phosphorylation only during G(1), indicating a specific role for Rad18 in processing of oxidative DNA lesions outside S-phase. However, H(2)O(2)-induced DSB formation in Rad18-depleted G1 cells was not associated with increased genotoxin sensitivity, indicating that back-up DSB repair mechanisms compensate for Rad18 deficiency. Indeed, in DNA LigIV-deficient cells Rad18-depletion conferred H(2)O(2)-sensitivity, demonstrating functional redundancy between Rad18 and non-homologous end joining for tolerance of oxidative DNA damage acquired during G(1). In contrast with G(1)-synchronized cultures, S-phase cells were H(2)O(2)-sensitive following Rad18-depletion. We conclude that although Rad18 pathway activation by oxidative lesions is not restricted to S-phase, Rad18-mediated trans-lesion synthesis by Polη is dispensable for damage-tolerance in G(1) (because of back-up non-homologous end joining-mediated DSB repair), yet Rad18 is necessary for damage tolerance during S-phase.
authors.affiliation: Department of Pathology and Laboratory Medicine, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. yangyang@email.unc.edu
authors.forename: Yang
authors.initials: Y
authors.lastname: Yang
authors.forename: Michael
authors.initials: M
authors.lastname: Durando
authors.forename: Stephanie L
authors.initials: SL
authors.lastname: Smith-Roe
authors.forename: Chris
authors.initials: C
authors.lastname: Sproul
authors.forename: Alicia M
authors.initials: AM
authors.lastname: Greenwalt
authors.forename: William
authors.initials: W
authors.lastname: Kaufmann
authors.forename: Sehyun
authors.initials: S
authors.lastname: Oh
authors.forename: Eric A
authors.initials: EA
authors.lastname: Hendrickson
authors.forename: Cyrus
authors.initials: C
authors.lastname: Vaziri
mesh_terms: D002453:Cell Cycle / Q000235:genetics*; D002478:Cells, Cultured; D053903:DNA Breaks, Double-Stranded; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000502:physiology*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D016193:G1 Phase / Q000235:genetics; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D010084:Oxidation-Reduction; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D051716:Replication Protein A / Q000378:metabolism; D016196:S Phase / Q000235:genetics; D044767:Ubiquitin-Protein Ligases; D054875:Ubiquitination; D000099270:Y-Family DNA Polymerases
chemical_list: D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase; D006861:Hydrogen Peroxide; D018809:Proliferating Cell Nuclear Antigen; D051716:Replication Protein A; D044767:Ubiquitin-Protein Ligases; C485954:RAD18 protein, human; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases