title: The Fanconi anemia pathway is required for the DNA replication stress response and for the regulation of common fragile site stability.
abstract: Fanconi anemia (FA) is a rare multi-genic, autosomal and X-linked recessive disorder characterized by hematological abnormalities, developmental defects and increased cancer susceptibility. Patient-derived FA cells display heightened sensitivity to DNA cross-linking agents such as mitomycin C (MMC). In response to DNA damaging agents, and during S-phase of the cell cycle, the FA pathway is activated via the mono-ubiquitination of FANCD2 (FANCD2-Ub), signaling its translocation to discrete nuclear foci, where it co-localizes with the central DNA repair proteins BRCA1 and RAD51. However, the exact function of activated FANCD2-Ub remains unclear. Here, we have characterized the role of the FA pathway in response to DNA replicative stress by aphidicolin (APH) and hydroxyurea (HU). The FA pathway is strongly activated in response to both agents. In addition, using patient-derived FA cell lines and siRNA targeting FANCD2, we demonstrate a functional requirement for the FA pathway in response to low doses of APH: a replicative stress treatment known to result in chromosome breakage at common fragile sites. Both the total number of chromosome gaps and breaks and breaks at the specific common fragile sites FRA3B and FRA16D were significantly elevated in the absence of an intact FA pathway. Furthermore, we demonstrate that APH activates the mono-ubiquitination of both FANCD2 and PCNA and the phosphorylation of RPA2, signaling processive DNA replication arrest. Following APH treatment, FANCD2-Ub co-localizes with PCNA (early) and RPA2 (late) in discrete nuclear foci. Our results demonstrate an integral role for the FA pathway in the DNA replication stress response.
authors.affiliation: Department of Human Genetics, University of Michigan, Ann Arbor, MI 48109, USA. niallg@umich.edu
authors.forename: Niall G
authors.initials: NG
authors.lastname: Howlett
authors.forename: Toshiyasu
authors.initials: T
authors.lastname: Taniguchi
authors.forename: Sandra G
authors.initials: SG
authors.lastname: Durkin
authors.forename: Alan D
authors.initials: AD
authors.lastname: D'Andrea
authors.forename: Thomas W
authors.initials: TW
authors.lastname: Glover
mesh_terms: D016590:Aphidicolin / Q000494:pharmacology; D043171:Chromosomal Instability / Q000187:drug effects; D043283:Chromosome Fragile Sites / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D004261:DNA Replication / Q000187:drug effects / Q000502:physiology*; D005199:Fanconi Anemia / Q000378:metabolism*; D052236:Fanconi Anemia Complementation Group D2 Protein; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D009687:Nuclear Proteins / Q000235:genetics; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D013997:Time Factors; D025801:Ubiquitin / Q000378:metabolism
chemical_list: C499905:FANCD2 protein, human; D052236:Fanconi Anemia Complementation Group D2 Protein; D009687:Nuclear Proteins; D019384:Nucleic Acid Synthesis Inhibitors; D018809:Proliferating Cell Nuclear Antigen; D034741:RNA, Small Interfering; D025801:Ubiquitin; D016590:Aphidicolin; D006918:Hydroxyurea