BRCA1 copurifies with the holoenzyme complex. (a) HeLa whole-cell extract was chromatographed on Bio-Rex 70 matrix and step-eluted at 0.3 M, 0.6 M, and 1.5 M KOAc. Protein samples from each fraction were subjected to SDS/PAGE, and blots were probed with BRCA1, pol II, and cdk8 antibodies. (b) The Bio-Rex 70 0.6 M fraction was subjected to centrifugation through a 10–60% sucrose gradient in 0.2 M acetate salt and 0.1% Nonidet P-40. After centrifugation, samples were collected, analyzed for total protein content, and Western-blotted. Blots were probed for BRCA1, pol II large subunit, and cdk8. BRCA1 pool A includes fractions 10–15, and BRCA1 pool B includes fractions 1–3. (c) Sucrose sedimentation pool A was subjected to metal chelate chromatography. Fractions were eluted with a linear 5–130 mM gradient of imidazole. The indicated fractions were subjected to immunoblot analysis and probed with antibodies specific to BRCA1 (Top), pol II large subunit (Middle), and cdk8 (Bottom).

Differential purification of other transcription regulators. (a) The protein fractions from the Bio-Rex 70 chromatographic step were analyzed for the presence of YY1, TFII-I, the RelA subunit of NF-κB, RBP-Jκ, and hRad51. In each case, the indicated bands were the only significant bands of their type observed in the whole blot and migrated at the appropriate position relative to molecular weight markers. (b) Fractions from the sedimentation analysis of the 0.6 M Bio-Rex fraction were analyzed for the presence of YY1, TFII-I, and RBP-Jκ. The protein samples were analyzed for the presence of the RelA subunit of NF-κB and for hRad51, but they were not detected in these fractions.

BRCA1 coimmunopurifies with the holoenzyme. The 0.6 M column fraction from the Bio-Rex 70 column was immunoprecipitated with a panel of affinity-purified polyclonal or monoclonal antibodies indicated above each lane in a and c. The pol II antibody (23) is directed at the CTD of the large subunit and disrupts the SRB interaction with the core polymerase (24). In a, the immunoprecipitates were developed with anti-BRCA1 antibody (mAb MS110). In b, the hSRB7 immunoprecipitation was repeated but was competed with specific antigenic peptide (lane 2) or with control peptide (lane 3) and probed with mAb MS110. In c, proteins purified by the same immunoprecipitating antibodies as in a were probed with antibody to the large subunit of pol II, and the pol II polypeptide is indicated. In d, the anti-hSRB7 immunoprecipitation of pol II was performed in the absence and presence (lane 2) of antigenic peptide and the blot was probed with pol II antibody.

Immunoisolation of BRCA1 copurifies pol II and basal transcription factors. The Bio-Rex 70 0.6 M fraction (20 μg) was immunoprecipitated with the indicated antibodies, and transcription assays were performed on the washed beads. (a) Transcription assays contained each of the basal transcription factors except pol II. Antibodies to hSRB7 (lane 1), pol CTD (lane 2), adenovirus E1A protein (lane 3), BRCA1 carboxyl-terminal peptide (mAb SG11; lane 4), BRCA1 amino-terminal fragment (mAb MS13; lane 5), BRCA1 carboxyl-terminal fragment (mAb AP16; lane 6), simian virus 40 large tumor antigen (lane 7), and transcription factor E2F4 (lane 8) were used. (b) Transcription assays as in a were repeated with mAb SG11 (anti-BRCA1), and antibody binding was competed with 20 μg of antigenic peptide (lane 2) or 20 μg of control peptide (lane 3). (c) The basal factor composition of the proteins present in immunoprecipitates was assayed by supplying all basal factors except the indicated factor noted above each lane. The purified factors were analyzed (Top), and the film exposure time is the same time as in subsequent panels. When a band appears in a given lane, it means that the immunoprecipitate contains the basal activity identified above that lane. Polymerase was not separately added to transcription reactions from immunoprecipitates. Thus, for any transcription to occur, an immunoprecipitate must contain pol II as well as the indicated basal factor.

Deletion of 10 amino acid residues from the carboxyl terminus of BRCA1 results in reduced association with holoenzyme. The 293T cells were transfected with 10 μg (odd numbered lanes) or 20 μg (even numbered lanes) of plasmid expressing BRCA1 genes fused to the HA tag on the amino terminus. (a) Mutant BRCA1 (lanes 3 and 4) revealed a reduced association with the holoenzyme. Lysates prepared from transfected cells were immunoprecipitated with affinity-purified anti-hSRB7 antibody and subjected to SDS/PAGE, and the immunoblot was stained with anti-HA antibody. (b) Wild-type and mutant BRCA1 were equally well synthesized in transfected cells. Samples treated in parallel with the experiment in a were pulse-labeled with [³⁵S]methionine-containing medium. Lysates were prepared and immunoprecipitated with the anti-HA antibody and subjected to SDS/PAGE. (c) Steady-state levels of transfected protein were determined by subjecting lysates of the transfected cells used in (a) to SDS/PAGE and then immunoblotting and probing for the HA tag.