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Fanconi anaemia (FA) is a cancer predisposition syndrome characterized by cellular sensitivity to DNA interstrand crosslinkers. The molecular defect in FA is an impaired DNA repair pathway. The critical event in activating this pathway is monoubiquitination of FANCD2. In vivo, a multisubunit FA core complex catalyzes this step, but its mechanism is unclear. Here, we report purification of a native avian FA core complex and biochemical reconstitution of FANCD2 monoubiquitination. This demonstrates that the catalytic FANCL E3 ligase subunit must be embedded within the complex for maximal activity and site specificity. We genetically and biochemically define a minimal subcomplex comprising just three proteins (FANCB, FANCL, and FAAP100) that functions as the monoubiquitination module. Residual FANCD2 monoubiquitination activity is retained in cells defective for other FA core complex subunits. This work describes the in vitro reconstitution and characterization of this multisubunit monoubiquitin E3 ligase, providing key insight into the conserved FA DNA repair pathway.

Original publication

DOI

10.1016/j.molcel.2014.05.001

Type

Journal article

Journal

Mol Cell

Publication Date

05/06/2014

Volume

54

Pages

858 - 869

Keywords

Animals, Avian Proteins, Cell Line, Chickens, Fanconi Anemia, Fanconi Anemia Complementation Group D2 Protein, Fanconi Anemia Complementation Group L Protein, Fanconi Anemia Complementation Group Proteins, Humans, Ubiquitin-Specific Proteases, Ubiquitination