title: A novel role for non-ubiquitinated FANCD2 in response to hydroxyurea-induced DNA damage.
abstract: Fanconi anemia (FA) is a genetic disease of bone marrow failure, cancer susceptibility, and sensitivity to DNA crosslinking agents. FANCD2, the central protein of the FA pathway, is monoubiquitinated upon DNA damage, such as crosslinkers and replication blockers such as hydroxyurea (HU). Even though FA cells demonstrate unequivocal sensitivity to crosslinkers, such as mitomycin C (MMC), we find that they are largely resistant to HU, except for cells absent for expression of FANCD2. FANCD2, RAD51 and RAD18 form a complex, which is enhanced upon HU exposure. Surprisingly, although FANCD2 is required for this enhanced interaction, its monoubiquitination is not. Similarly, non-ubiquitinated FANCD2 can still support proliferation cell nuclear antigen (PCNA) monoubiquitination. RAD51, but not BRCA2, is also required for PCNA monoubiquitination in response to HU, suggesting that this function is independent of homologous recombination (HR). We further show that translesion (TLS) polymerase PolH chromatin localization is decreased in FANCD2 deficient cells, FANCD2 siRNA knockdown cells and RAD51 siRNA knockdown cells, and PolH knockdown results in HU sensitivity only. Our data suggest that FANCD2 and RAD51 have an important role in PCNA monoubiquitination and TLS in a FANCD2 monoubiquitination and HR-independent manner in response to HU. This effect is not observed with MMC treatment, suggesting a non-canonical function for the FA pathway in response to different types of DNA damage.
authors.affiliation: Department of Pediatrics, Section of Hematology/Oncology, Yale Cancer Center, Yale School of Medicine, New Haven, CT, USA.|Department of Pathology, Section of Hematology/Oncology, Yale Cancer Center, Yale School of Medicine, New Haven, CT, USA.
authors.forename: X
authors.initials: X
authors.lastname: Chen
authors.affiliation: Department of Pediatrics, Section of Hematology/Oncology, Yale Cancer Center, Yale School of Medicine, New Haven, CT, USA.|Department of Pathology, Section of Hematology/Oncology, Yale Cancer Center, Yale School of Medicine, New Haven, CT, USA.
authors.forename: L
authors.initials: L
authors.lastname: Bosques
authors.affiliation: Department of Molecular, Cellular, and Developmental Biology, Yale Cancer Center, Yale School of Medicine, New Haven, CT, USA.
authors.forename: P
authors.initials: P
authors.lastname: Sung
authors.affiliation: Department of Pediatrics, Section of Hematology/Oncology, Yale Cancer Center, Yale School of Medicine, New Haven, CT, USA.|Department of Pathology, Section of Hematology/Oncology, Yale Cancer Center, Yale School of Medicine, New Haven, CT, USA.
authors.forename: G M
authors.initials: GM
authors.lastname: Kupfer
mesh_terms: D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D005199:Fanconi Anemia / Q000188:drug therapy* / Q000235:genetics* / Q000378:metabolism; D052236:Fanconi Anemia Complementation Group D2 Protein / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006801:Humans; D006918:Hydroxyurea / Q000009:adverse effects / Q000494:pharmacology*; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D051135:Rad51 Recombinase / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases; D054875:Ubiquitination; D000099270:Y-Family DNA Polymerases
chemical_list: D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase; D052236:Fanconi Anemia Complementation Group D2 Protein; D006918:Hydroxyurea; D018809:Proliferating Cell Nuclear Antigen; D051135:Rad51 Recombinase; D044767:Ubiquitin-Protein Ligases; C499905:FANCD2 protein, human; C485954:RAD18 protein, human; C495153:RAD51 protein, human; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases