Defective FANCI Binding by a Fanconi Anemia-Related FANCD2 Mutant
Figure 1
FANCD2−/− DT40 cells expressing the chicken FANCD2 L234R mutant are defective in ICL repair.
(A) Schematic representation of human FANCD2. S1, HD1, S2, HD2, S3, and S4 denoted on the bar indicate the FANCD2 subdomains, solenoid 1, helical domain 1, solenoid 2, helical domain 2, solenoid 3, and solenoid 4, respectively. The FANCD2 C-terminal acidic region is colored red. The primary structure of the N-terminal regions of Homo sapiens, Mus musculus, Gallus gallus, Xenopus laevis, Danio rerio, Arabidopsis thaliana, and Drosophila melanogaster FANCD2 are aligned. The residues corresponding to human FANCD2 L231 are colored red. (B) ID complex monoubiquitination in cFANCD2−/− DT40 cells expressing the GFP-wild-type (WT) cFANCD2 or cFANCD2 L234R. Cells were treated with or without mitomycin C (MMC), and whole-cell extracts were analyzed by western blotting using anti-cFANCD2 (α-D2) and anti-cFANCI (α-I) antibodies. S-forms and L-forms indicate non-ubiquitinated and monoubiquitinated forms of cFANCD2 and cFANCI, respectively. Non-specific bands are marked by asterisks. (C) ID complex monoubiquitination in the chromatin fractions from cFANCD2−/− DT40 cells expressing GFP-WT cFANCD2 or cFANCD2 L234R. Cells were treated with or without MMC, and chromatin fractions were analyzed as in panel (B). (D) Cisplatin sensitivity assay of the cFANCD2−/− DT40 cells expressing H2B-GFP fusions with the WT cFANCD2, cFANCD2 K563R, or cFANCD2 L234R. The mean values are shown with s.d. from triplicate measurements.