title: The E3 ubiquitin ligase RAD18 regulates ubiquitylation and chromatin loading of FANCD2 and FANCI.
abstract: Fanconi anemia (FA) is a rare genetic disorder characterized by bone marrow failure, congenital abnormalities, and an increased risk for cancer and leukemia. Components of the FA-BRCA pathway are thought to function in the repair of DNA interstrand cross-links. Central to this pathway is the monoubiquitylation and chromatin localization of 2 FA proteins, FA complementation group D2 (FANCD2) and FANCI. In the present study, we show that RAD18 binds FANCD2 and is required for efficient monoubiquitylation and chromatin localization of both FANCD2 and FANCI. Human RAD18-knockout cells display increased sensitivity to mitomycin C and a delay in FANCD2 foci formation compared with their wild-type counterparts. In addition, RAD18-knockout cells display a unique lack of FANCD2 and FANCI localization to chromatin in exponentially growing cells. FANCD2 ubiquitylation is normal in cells containing a ubiquitylation-resistant form of proliferating cell nuclear antigen, and chromatin loading of FA core complex proteins appears normal in RAD18-knockout cells. Mutation of the RING domain of RAD18 ablates the interaction with and chromatin loading of FANCD2. These data suggest a key role for the E3 ligase activity of RAD18 in the recruitment of FANCD2 and FANCI to chromatin and the events leading to their ubiquitylation during S phase.
authors.affiliation: Department of Pathology, Yale University School of Medicine, New Haven, CT, USA.
authors.forename: Stacy A
authors.initials: SA
authors.lastname: Williams
authors.forename: Simonne
authors.initials: S
authors.lastname: Longerich
authors.forename: Patrick
authors.initials: P
authors.lastname: Sung
authors.forename: Cyrus
authors.initials: C
authors.lastname: Vaziri
authors.forename: Gary M
authors.initials: GM
authors.lastname: Kupfer
mesh_terms: D015153:Blotting, Western; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism; D004249:DNA Damage / Q000502:physiology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D052236:Fanconi Anemia Complementation Group D2 Protein / Q000378:metabolism*; D051856:Fanconi Anemia Complementation Group Proteins / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D055786:Gene Knockout Techniques; D006801:Humans; D047468:Immunoprecipitation; D034741:RNA, Small Interfering; D016196:S Phase / Q000502:physiology; D014162:Transfection; D044767:Ubiquitin-Protein Ligases; D054875:Ubiquitination
chemical_list: D002843:Chromatin; D004268:DNA-Binding Proteins; C499905:FANCD2 protein, human; C519921:FANCI protein, human; D052236:Fanconi Anemia Complementation Group D2 Protein; D051856:Fanconi Anemia Complementation Group Proteins; C485954:RAD18 protein, human; D034741:RNA, Small Interfering; D044767:Ubiquitin-Protein Ligases