Figure 4.

The FA Core Complex Has Higher Activity and Specificity than Isolated FANCL

(A) Titration of the FA core complex in a 2-fold dilution series (lanes 2–5) is compared to isolated FANCL (lanes 8 and 9) in the monoubiquitination assay after 90 min at 30°C monitored by western blotting. See Figure S2A for analysis after an additional 12 hr at room temperature. The bottom panel is a silver-stained gel showing the relative amounts of isolated FANCL (1 μM) used for the reactions in lanes 8 and 9 and FANCL in the FA core complex (∼200 nM) at the highest concentration in lanes 5–7. The full gel is shown in Figure S2B.

(B) Time course of FANCD2 monoubiquitination by the FA core complex (top) and a molar equivalent amount of recombinant FANCL (bottom). Anti-His western blots for detecting His-tagged FANCI and FANCD2 are shown. Anti-HA western blots of the same samples to detect HA-ubiquitin are shown in Figure S2C.

(C) The amounts of FANCD2-Ub in the reactions shown in (B) were quantified and are plotted as a percentage of total FANCD2 (ubiquitinated FANCD2/total FANCD2).

(D) Monoubiquitination of WT and nonubiquitinatable (lysine to arginine, KR) mutants of FANCI and FANCD2 by ∼100 nM core complex in the presence of DNA over 3 hr at 30°C.

See also Figures S2 and S3.