title: DNA robustly stimulates FANCD2 monoubiquitylation in the complex with FANCI.
abstract: FANCI and FANCD2 form a complex, and play essential roles in the repair of interstrand DNA crosslinks (ICLs) by the Fanconi anemia (FA) pathway. FANCD2 is monoubiquitylated by the FA core complex, composed of 10 FA proteins including FANCL as the catalytic E3 subunit. FANCD2 monoubiquitylation can be reconstituted with purified minimal components, such as FANCI, E1, UBE2T (E2) and FANCL (E3) in vitro; however, its efficiency is quite low as compared to the in vivo monoubiquitylation of FANCD2. In this study, we found that various forms of DNA, such as single-stranded, double-stranded and branched DNA, robustly stimulated the FANCD2 monoubiquitylation in vitro up to a level comparable to its in vivo monoubiquitylation. This stimulation of the FANCD2 monoubiquitylation strictly required FANCI, suggesting that FANCD2 monoubiquitylation may occur in the FANCI-FANCD2 complex. A FANCI mutant that was defective in DNA binding was also significantly defective in FANCD2 monoubiquitylation in vitro. In the presence of 5' flapped DNA, a DNA substrate mimicking the arrested replication fork, about 70% of the input FANCD2 was monoubiquitylated, while less than 1% FANCD2 monoubiquitylation was observed in the absence of the DNA. Therefore, DNA may be the unidentified factor required for proper FANCD2 monoubiquitylation.
authors.affiliation: Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan.
authors.forename: Koichi
authors.initials: K
authors.lastname: Sato
authors.forename: Kazue
authors.initials: K
authors.lastname: Toda
authors.forename: Masamichi
authors.initials: M
authors.lastname: Ishiai
authors.forename: Minoru
authors.initials: M
authors.lastname: Takata
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Kurumizaka
mesh_terms: D000818:Animals; D004247:DNA / Q000378:metabolism*; D052236:Fanconi Anemia Complementation Group D2 Protein / Q000378:metabolism*; D051856:Fanconi Anemia Complementation Group Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D054875:Ubiquitination
chemical_list: D052236:Fanconi Anemia Complementation Group D2 Protein; D051856:Fanconi Anemia Complementation Group Proteins; D004247:DNA