title: 5-Aza-2'-deoxycytidine causes replication lesions that require Fanconi anemia-dependent homologous recombination for repair.
abstract: 5-Aza-2'-deoxycytidine (5-azadC) is a DNA methyltransferase (DNMT) inhibitor increasingly used in treatments of hematological diseases and works by being incorporated into DNA and trapping DNMT. It is unclear what DNA lesions are caused by 5-azadC and if such are substrates for DNA repair. Here, we identify that 5-azadC induces DNA damage as measured by γ-H2AX and 53BP1 foci. Furthermore, 5-azadC induces radial chromosomes and chromatid breaks that depend on active replication, which altogether suggest that trapped DNMT collapses oncoming replication forks into double-strand breaks. We demonstrate that RAD51-mediated homologous recombination (HR) is activated to repair 5-azadC collapsed replication forks. Fanconi anemia (FA) is a rare autosomal recessive disorder, and deaths are often associated with leukemia. Here, we show that FANCG-deficient cells fail to trigger HR-mediated repair of 5-azadC-induced lesions, leading to accumulation of chromatid breaks and inter-chromosomal radial fusions as well as hypersensitivity to the cytotoxic effects of 5-azadC. These data demonstrate that the FA pathway is important to protect from 5-azadC-induced toxicity. Altogether, our data demonstrate that cytotoxicity of the epigenetic drug 5-azadC can, at least in part, be explained by collapsed replication forks requiring FA-mediated HR for repair.
authors.affiliation: Department of Cell Biology, Cell Culture and Radiobiology Research Group, University of Seville, 41012 Seville, Spain. morta2@us.es
authors.forename: Manuel Luís
authors.initials: ML
authors.lastname: Orta
authors.forename: José Manuel
authors.initials: JM
authors.lastname: Calderón-Montaño
authors.forename: Inmaculada
authors.initials: I
authors.lastname: Domínguez
authors.forename: Nuria
authors.initials: N
authors.lastname: Pastor
authors.forename: Estefanía
authors.initials: E
authors.lastname: Burgos-Morón
authors.forename: Miguel
authors.initials: M
authors.lastname: López-Lázaro
authors.forename: Felipe
authors.initials: F
authors.lastname: Cortés
authors.forename: Santiago
authors.initials: S
authors.lastname: Mateos
authors.forename: Thomas
authors.initials: T
authors.lastname: Helleday
mesh_terms: D000818:Animals; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000633:toxicity; D002460:Cell Line; D002842:Chromatids / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D053960:DNA Breaks; D004261:DNA Replication / Q000187:drug effects*; D051747:DNA-Activated Protein Kinase / Q000037:antagonists & inhibitors; D000077209:Decitabine; D004791:Enzyme Inhibitors / Q000633:toxicity*; D052241:Fanconi Anemia Complementation Group G Protein / Q000502:physiology*; D007976:Leupeptins / Q000494:pharmacology; D061988:Proteasome Inhibitors / Q000494:pharmacology; D059767:Recombinational DNA Repair
chemical_list: D004791:Enzyme Inhibitors; D052241:Fanconi Anemia Complementation Group G Protein; D007976:Leupeptins; D061988:Proteasome Inhibitors; D000077209:Decitabine; D051747:DNA-Activated Protein Kinase; D001374:Azacitidine; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde