Extended Data Figure 10. BRCA1 requires similar loop region and H2A/H2B acidic patch for nucleosome ubiquitylation.

a, Alignment of the BRCA1/BARD1 and Ring1B/Bmi1 heterodimers using the RING domains of BRCA1 from the BRCA1/BARD1 NMR structure³⁵ (PDB ID 1JM7) and Ring1B. BRCA1 and BARD1 are shown in orange and purple, respectively; Cα atoms of essential arginine residues are indicated by spheres. b, Sequence alignment of Ring1B and BRCA1 shows conserved nucleosome interacting loop. c, BRCA1 Lys70Ala/Arg71Ala mutation eliminates E3 ligase activity of BRCA1/BARD1 RING heterodimer. Representative gel of one replicate of ubiquitylation assay using E1 Uba1, E2 UbcH5c, STR-His₁₀ ubiquitin, nucleosome core particles, and E3 BRCA1/BARD1 with the indicated mutants stained with Coomassie and scanned for fluorescent H2A. Nucleosome core particles in the experiment are doped with nucleosome core particles containing the H2A Thr10Cys mutant labeled with Oregon Green 488 maleimide. d, Quantitaion of mono- (H2Aub1, dark orange), di- (H2Aub2, orange) and tri-ubiquitylated H2A (H2Aub3, light orange) are shown as a fraction of total H2A. Means and standard deviations from three technical replicates are depicted. Ubiquitylation assay was repeated twice in our laboratory.