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. 2019 Aug 8;75(3):483–497.e9. doi: 10.1016/j.molcel.2019.06.002

Figure 2.

Figure 2

Integration of RAP80 and DUB Specificity of BRCA1-A

(A) The position of the UEV-N, RWD, and UEV-C domains of BRE and the VWA domain of MERIT40 (shown as cartoon) within the BRCA1-A complex (shown as surface). RAP80 is embedded into the complex through contacts with BRE, MERIT40, and ABRAXAS.

(B) BRCA1-A complex processes (Ub)4 faster than (Ub)3 and (Ub)2. K63-linked ubiquitin substrate (150 ng/lane) was incubated with 5 nM BRCA1-A on ice and analyzed by silver-stained SDS-PAGE. Under these conditions, (Ub)4 is cleaved within 5 min, whereas (Ub)2 and (Ub)3 are not cleaved within 7 h.

(C) Tetra-ubiquitin chains are pre-oriented on BRCA1-A complex during cleavage, as evident from comparison of the degradation of K63-linked (Ub)4 substrate labeled with TAMRA at the proximal and distal ubiquitin, respectively. Degradation products of 1 μM substrate incubated with 10 nM BRCA1-A at room temperature were resolved on an SDS-PAGE; TAMRA was subsequently visualized.