Fig. 3. Substrate specificity of the BRCA1/BARD1 complex. A 0.1 µg aliquot of BRCA1/BARD1 was incubated with 1 µg of the indicated substrate as described in Materials and methods. Lysozyme (lane g) was denatured previously, by heating to 100°C for 15 min in a waterbath. ¹²⁵I-Labelled products were analysed by SDS–PAGE and visualized with a Molecular Dynamics Typhoon PhosphorImager.

Fig. 6. BAP1 does not deubiquitylate BRCA1/BARD1 complex. (A) Recombinant BAP1 has C-terminal ubiquitin hydrolase activity. Recombinant BAP1 and the mutated protein BAP1(C91S) were incubated with ubiquitin-AMC as described in Materials and methods. Ubiquitin hydrolysis was measured by fluorescence using a Perkin Elmer Luminescence Spectrometer with excitation wavelength of 380 nm and emission wavelength of 460 nm. Fluorescence is plotted as arbitrary units (A.U.) (B) The ¹²⁵I-polyubiquitylated form of BRCA1/BARD1 was isolated using nickel-NTA resin, washed in buffer and incubated with 100 nM BAP1 protein or 100 nM isopeptidase T as described in Materials and methods. Products were analysed by SDS–PAGE and deubiquitylation determined by the release of [¹²⁵I]ubiquitin.

Fig. 2. The RING domains of BRCA1 and BARD1 are necessary but not sufficient for E3 activity. (A) Sequences outside the minimal interacting RING domains of BRCA1 and BARD1 are required for ubiquitin ligase activity. Ubiquitin ligase activity of two RING domain complexes, BRCA1^1–109/BARD1^26–119 and BRCA1^1–250/BARD1^14–186, was investigated. Reactions were incubated for 1.5 h at 37°C as described in Materials and methods. Ubiquitylated H2A product (Ub-H2A) and ubiquitylated BRCA1 and BARD1 products (Ub-BRCA1^1–250 and Ub-BARD1^14–186) are indicated. (B) Mutation of a key metal-binding residue in BRCA1 but not BARD1 abolishes the E3 activity of BRCA1/BARD1. Mutant complexes BRCA1(C61G)/BARD1 and BRCA1/BARD1(C83G) were examined for E3 activity as described in Figure 1. Each reaction contained 0.1 µg of BRCA1/BARD1 complex. Ubiquitylated H2A (Ub-H2A) product is indicated.

Fig. 4. Auto-ubiquitylation of BRCA1/BARD1 complex involves addition of multiple polyubiquitin chains. (A) BRCA1/BARD1-dependent ubiquitylation of H2A requires all the key reaction components. Reactions were carried out as described in Materials and methods, but omitting the indicated component from the reaction. The complete reaction is shown in lane a. Ubiquitylated BRCA1/BARD1 com plex (Ub-BRCA1/BARD1) and ubiquitylated H2A are indicated. (B) Ubiquitylation of the BRCA1/BARD1 complex in the presence of unmodified ubiquitin (lanes a–d) or methyl ubiquitin (lanes e–h). Reactions were carried out as outlined in Materials and methods using 0, 0.1, 0.3 or 1 µg (lanes a and e, b and f, c and g, and d and h) of ubiquitin or methyl ubiquitin as indicated. Reactions were performed for 60 min. Products were analysed by SDS–PAGE on a 4–20% Tris–glycine gel. Ubiquitylated H2A (Ub-H2A) and auto-ubiquitylated BRCA1/BARD1 products (Ub-BRCA1/BARD1) are indicated.

Fig. 5. Ubiquitylation of BRCA1/BARD1 complex potentiates its E3 ligase activity. (A) A 0.025 µg aliquot of BRCA1/BARD1 was pre-incubated at 37°C with the indicated amounts of ubiquitin or methyl ubiquitin in conditions permissible for auto-ubiquitylation (see Materials and methods). After 2 h, 1 µg of H2A substrate, excess [¹²⁵I]ubiquitin and ATP were added to each reaction, followed by a further 20 min incubation at 37°C. Reactions were stopped by the addition of SDS loading dye and boiling for 2 min (+pre-incubation). In control reactions, BRCA1/BARD1 was omitted from the pre-incubation step and added instead with H2A and [¹²⁵I]ubiquitin (–pre-incubation). Products were analysed by SDS–PAGE and visualized using a PhosphorImager. The ubiquitylated H2A and BRCA1/BARD1 products are indicated. (B) Ubiquitylation of H2A by polyubiquitylated and non-polyubiquitylated BRCA1/BARD1 complex. Polyubiquitylated and non-ubiquitylated forms of the BRCA1/BARD1 complex were isolated as described in Materials and methods. E3 activity was examined in fresh reactions as described in Figure 1, and analysed by SDS–PAGE. Ubiquitylated BRCA1/BARD1 complexes and ubiquitylated H2A are indicated.

Fig. 1. BRCA1/BARD1 has E3 monoubiquitin ligase activity in vitro. (A) A Coomassie-stained gel of the full-length BRCA1/BARD1 complex. His-tagged BRCA1 and BARD1 proteins were co-expressed and co-purified as a complex from Sf9 insect cells as described in Materials and methods. A 20 µl aliquot of the purified complex was analysed by SDS–PAGE using a 6% Tris–glycine gel and visualized by staining with Simply Blue Safe Stain (Invitrogen). Molecular weight markers (M) and the BRCA1 and BARD1 proteins are indicated. (B) BRCA1/BARD1 complex exhibits ubiquitin ligase activity in the presence of the E2 enzyme, UbcH5a. Ubiquitin E3 ligase activity was investigated by incubating histone H2A substrate with BRCA1/BARD1 complex in the presence of E1 enzyme and a variety of E2 enzymes (as indicated). Reactions were carried out with a 30–60 min pre-incubation step as described in Materials and methods. ¹²⁵I-Labelled products were analysed by SDS–PAGE and visualized using a PhosphorImager. [¹²⁵I]ubiquitin substrate and ¹²⁵I-ubiquitylated H2A products (Ub-H2A) are indicated.