Figure 7. The N-terminus of USP1 is required for FANCD2-Ub deubiquitination when in complex with FANCI and DNA.
(A) Deubiquitination of di-monoubiquitinated FANCD2–FANCI complex with and without DNA by USP1FL–UAF1, USP1ΔN–UAF1, and USP1R22E–UAF1. A FANCD2–FANCI–ds DNA complex is monoubiquitinated by Ube2Tv4, FANCLΔELF, E1, and ubiquitin for 30 min and reactions arrested using apyrase. Ubiquitination reactions are treated with benzonase nuclease for 4 h on ice before being subject to USP1 for 30 min at room temperature. FANCD2 and FANCI are detected by Coomassie staining or Western blotting on the same nitrocellulose membrane and visualised using anti-FANCD2 or anti-V5 (FANCI). (B) Assay performed as in (A) except that FANCI with a K523R mutation is used. (C) Model of FANCD2-Ub deubiquitination by USP1–UAF1. USP1 targets FANCD2 via a direct interaction using its N-terminus. In addition to the N-terminus of USP1, FANCD2–Ub–FANCI complexes might be indirectly targeted by an interaction between UAF1 SLD and FANCI SUMO-like interacting motif (SLIM).
Source data are available online for this figure.
