Figure 4. The BRCA1^5382insC mutation likely alters interactions with RNAP II and BARD1.

(a) Close-up view of the BRCT density with respect to the RPB1 subunit of RNAP II that is disordered beyond P1455 (black arrow), where the C-terminus emanates. Theoretical molecular models of the BRCT domain for wild type BRCA1 (gray; pdbcode, 1KOH) (b) compared to the mutated BRCA1^5382insC (red) (c) revealed the hydrophobic binding pocket (gray rectangle) is disrupted in the mutated BRCT domain. This significant disruption in the peptide-binding site suggests that native substrates may not interact with BRCA1^5382insC in the same manner as with wild type BRCA1. (d) Western blot analysis indicates that RNAP II (RPB1 subunit) interacts with BRCA1^5382insC in co-IP experiments, and the RNAP II core is similarly ubiquitinated by K63-specific moieties in cell lines expressing both mutated (Mut) and wild type (WT) BRCA1. The large subunit of the RNAP II core (RPB1) migrates at ~260 kDa. BRCA1 migrates at ~220 kDa. (e) The BRCA1^5382insC protein contained in nuclear extracts showed some interaction with BARD1 in comparison to negative control IPs performed using species-specific mouse normal (nMo) IgG antibodies (top panel). Wild type (WT) BRCA1 shows a strong interaction with BARD1 in Ni-NTA eluted fractions used as input material for microchip-capture experiments (bottom panel). BARD1 migrates at ~87 kDa. IN (input material); DEP (unbound); IP (immunoprecipated protein); IB (immunoblot).