1. BRCA1-Independent Ubiquitination of FANCD2
Cassandra J. Vandenberg, Fanni Gergely, Chong Yi Ong, Paul Pace, Donna L. Mallery, Kevin Hiom, Ketan J. Patel 
Molecular Cell 
Volume 12, Issue 1, Pages (July 2003)
DOI: /S (03)

2. Figure 1 Monoubiquitination of FANCD2 in a Cell-free System
(A) FANCD2 is monoubiquitinated by the BRCA1/BARD1 complex in vitro. This reaction also requires E1 enzyme, UbcH5a as the E2 enzyme, and ATP as the energy source. Reactions (20 μl) were performed as described in Experimental Procedures using 125I-radiolabeled ubiquitin. Samples were analyzed by SDS-PAGE under reducing conditions. Ubiquitinated products were observed only when all components of the reaction were present (lane a). The high molecular weight polyubiquitinated BRCA1/BARD1 complex and the 160 kd monoubiquitinated FANCD2 products are labeled.
(B) Monoubiquitination of FANCD2 is specific because it does not occur on other purified FA proteins (FANCA, FANCC, or FANCE). To define a minimal domain of FANCD2 that acts as a substrate for ubiquitination, a truncated form of FANCD2 (ΔFANCD2) spanning K561 (residues 238–683) was examined as a substrate (lane c). A mutated derivative of this polypeptide ΔFANCD2 K561R was also ubiquitinated by BRCA1/BARD1 (lane d). Histone H2AX is known to be a substrate for BRCA1/BARD1 and was used as the positive control for this reaction (lane h).
(C) K561R mutation abolishes ubiquitination of full-length FANCD2 protein. Purified full-length FANCD2 K561R mutant protein (lane d) and another FA-associated mutant protein, FANCD2 K1236H (lane e), were examined as substrates for ubiquitination by BRCA1/BARD1 as described above.
(D) Mutation of the RING finger of BRCA1, but not BARD1, abolishes ubiquitination of FANCD2 in vitro. A naturally occurring mutation of the cysteine at position 61 to glycine is known to segregate in humans with breast cancer predisposition and disrupts BRCA1/BARD1 E3 activity. An equivalent RING mutation in BARD1 (BARD C83G) was also used.
Molecular Cell  , DOI: ( /S (03) )

3. Figure 2
siRNA Knockdown of BRCA1 Expression in HeLa Cells Reveals a Role for BRCA1 in the Localization of FANCD2 to Nuclear Foci Upon DNA Damage
(A) Immunofluorescence of control siRNA (CON) and BRCA1 siRNA. HeLa cells before (left) and after exposure to 13 Gy X-rays (right) are shown. The top panels were stained for FANCD2 (red), the middle panels were stained for BRCA1 (green), and the signals are merged in the bottom panels. Exposure of control cells to DNA damage leads to a marked accumulation of BRCA1 and FANCD2 into nuclear foci, while knockdown of BRCA1 expression ablates this response.
(B) Number of FANCD2 (green) and BRCA1 (red) foci in individual nuclei in control (top panels) and BRCA1 (bottom panels) siRNA-treated cells. The right-hand panels show cells irradiated with 13 Gy of ionizing radiation followed by 6 hr recovery. Blue circles represent nuclei that contain no BRCA1 or FANCD2 foci (these cells either express no detectable protein or show diffuse nuclear staining). The impact of BRCA1 knockdown is dramatic in that inducible FANCD2 foci do not form in these cells following DNA damage (compare right panel top and bottom). Sixty nuclei were analyzed for each graph.
(C) Bar chart representation of the average number of FANCD2 (green) and BRCA1 (red) foci per nuclei in control and BRCA1 siRNA-treated cells with (+ IRR) and without exposure to X rays.
(D) Western blotting of whole-cell lysates prepared from cells subjected to control (CON) and BRCA1 siRNA. Unirradiated and irradiated (IRR) samples were prepared 60 hr after the addition of the siRNAs. Irradiated cells were allowed to recover for 6 hr after exposure to 13 Gy X-rays, before being taken up in SDS loading buffer. Panels depict blots using antibodies against BRCA1 (top), FANCD2 (middle), and α-tubulin (as a loading control; bottom). BRCA1 siRNA results in greatly reduced BRCA1 protein, but does not affect the levels of FANCD2-L (monoubiquitinated form) or FANCD-S (unubiquitinated form).
Molecular Cell  , DOI: ( /S (03) )

4. Figure 3
The RING Finger Domains of Both BRCA1 and BARD1 Are Required for Repair of DNA Crosslinks but Are Not Essential for the Monoubiquitination of FANCD2
(A) Representative metaphase spreads of wild-type and RING mutant ΔBRCA1 and ΔBARD1 DT40 cells. Slides were prepared as described in Experimental Procedures. Isochromatid (open arrowheads) and chromatid (filled arrowheads) breaks are indicated.
(B) Chromosome breakage analysis of wild-type DT40, ΔBARD1, and ΔBRCA1 RING domain mutants. Cells were grown for 12 hr in the presence of mitomycin C (0.05 μg/ml), after which colcemid was added and the cells incubated for a further 4 hr. Metaphase spreads were prepared, stained, and coded as described in Experimental Procedures. The ablation of the RING fingers of either BRCA1 or BARD1 resulted in a marked increase in chromosome breakage.
(C) Survival after treatment with the DNA crosslinking agent cisplatin. Wild-type, ΔBRCA1, and ΔBARD1 DT40 cells were examined for their sensitivity to a range of different concentrations of cisplatin as described in Experimental Procedures. Survival was measured by spectrophotometry after treatment with MTS (PROMEGA).
Molecular Cell  , DOI: ( /S (03) )

5. Figure 4
ΔBRCA1 Mutant and ΔBARD1 Mutant Cell Lines Are Proficient for Ubiquitination of FANCD2 Protein
Western blot of cell lysates prepared from wild-type DT40, ΔBRCA1 mutant, and ΔBARD1 mutant for the presence of FANCD2 ubiquitination. Cells were either irradiated with 10 Gy of ionizing radiation (A) or treated with 0.02 μg/ml mitomycin C (B) and allowed to recover for the times indicated before being lysed, resolved by SDS-PAGE, and detected with polyclonal antiserum to full-length human FANCD2. The ubiquitinated (FANCD2-L) and unmodified (FANCD2-S) forms are indicated. In the control cells as well as both mutants, the induction of a large isoform of FANCD2 after DNA damage can be clearly seen.
Molecular Cell  , DOI: ( /S (03) )