DNA robustly stimulates FANCD2 monoubiquitylation in the complex with FANCI

Koichi Sato, Kazue Toda, Masamichi Ishiai, Minoru Takata, Hitoshi Kurumizaka

    Research output: Contribution to journalArticle

    49 Citations (Scopus)

    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.

    Original languageEnglish
    Pages (from-to)4553-4561
    Number of pages9
    JournalNucleic Acids Research
    Volume40
    Issue number10
    DOIs
    Publication statusPublished - 2012 May

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    DNA
    Fanconi Anemia
    Fanconi Anemia Complementation Group Proteins
    DNA Repair
    Catalytic Domain
    In Vitro Techniques

    ASJC Scopus subject areas

    • Genetics

    Cite this

    Sato, K., Toda, K., Ishiai, M., Takata, M., & Kurumizaka, H. (2012). DNA robustly stimulates FANCD2 monoubiquitylation in the complex with FANCI. Nucleic Acids Research, 40(10), 4553-4561. https://doi.org/10.1093/nar/gks053

    DNA robustly stimulates FANCD2 monoubiquitylation in the complex with FANCI. / Sato, Koichi; Toda, Kazue; Ishiai, Masamichi; Takata, Minoru; Kurumizaka, Hitoshi.

    In: Nucleic Acids Research, Vol. 40, No. 10, 05.2012, p. 4553-4561.

    Research output: Contribution to journalArticle

    Sato, K, Toda, K, Ishiai, M, Takata, M & Kurumizaka, H 2012, 'DNA robustly stimulates FANCD2 monoubiquitylation in the complex with FANCI', Nucleic Acids Research, vol. 40, no. 10, pp. 4553-4561. https://doi.org/10.1093/nar/gks053
    Sato, Koichi ; Toda, Kazue ; Ishiai, Masamichi ; Takata, Minoru ; Kurumizaka, Hitoshi. / DNA robustly stimulates FANCD2 monoubiquitylation in the complex with FANCI. In: Nucleic Acids Research. 2012 ; Vol. 40, No. 10. pp. 4553-4561.
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