Novel BS complexes contain Topo IIIα, RPA, and MLH1. (A) A silver-stained gel showing the novel BS complexes isolated by immunoprecipitation with a BLM antibody from a HeLa nuclear extract. The polypeptides identified by mass spectrometry analysis are indicated by an asterisk. The polypeptides that remain to be identified (termed BLAPs) are also marked. The presence of immunoglobulins (immunoglobulin G [IgG] H and IgG L) are indicated. A control immunoprecipitation was included with the preimmune serum of the same rabbit. (B and C) Autoradiographs illustrating the immunoblotting analysis of load, flowthrough (FT), and eluted (Elute) fractions from the immunoaffnity purification by the BLM antibody. The antibodies utilized for immunoblotting are shown.

Five FA complementation group proteins are present in one complex with BLM, Topo IIIα, and RPA. (A) A silver-stained gel showing the polypeptides associated with endogenous FANCA (FAAPs) in comparison with BRAFT. Note that FANCA antibody was cross-linked to protein A-beads prior to immunoprecipitaion. The polypeptides identified by mass spectrometry analysis are marked with an asterisk. (B) Immunoblotting data showing the presence of multiple FA proteins in polypeptides isolated by using FANCA and BLM antibodies. As a positive control for FANCC and FANCF, whole-cell lysate from 293 cells transiently transfected with vectors expressing either epitope-tagged FANCC-HA or FANCF-Flag was used. HeLa nuclear extract was used as a positive control for BLM, FANCA, and FANCG. (C and D) A silver-stained gel (C) and Western blotting analysis (D) of the polypeptides associated with endogenous BLM, RPA70, Topo IIIα, and FANCA. The antibodies used for each immunoprecipitation are indicated on top of the figure. Mock immunoprecipitation with preimmune sera was used as the control. All immunoprecipitations were done with HeLa nuclear extract.

BLM and FANCA antibodies immunoprecipitate their associated complexes from wild-type but not their respective mutant cell lines. (A) Autoradiographs showing immunoblotting analysis of nuclear extracts prepared from wild-type (ManEBV), BLM^−/− (2036), and FANCA^−/− (VU388) human lymphoblastoid cell lines. After SDS-PAGE and Western transfer, the membrane was cut into two parts. The part corresponding to a molecular mass of >120 kDa was probed by using a mixture of BLM and FANCA antibodies (top panel). The other part was analyzed by using a mixture of Topo IIIα and FANCG antibodies (bottom panel). Note that FANCG was not detected in the FANCA-deficient cells. (B) Immunoblotting analysis of the polypeptides isolated by either BLM or FANCA antibodies from the three lymphoblastoid cell lines. The antibodies used for immunoprecipitation are indicated on the top, whereas those used for immunoblotting are listed on the right. (C) Immunoblotting analysis showing that the association between BLM and FA proteins remains intact in the presence of EtBr, a DNA-intercalating drug that can disassociate proteins from DNA. In this experiment, 50 μg of EtBr/ml was included in the immunoprecipitation mixture.

The BRAFT complex containing BLM and FA proteins is one of the three BLM complexes in the nuclear extract. (A) Immunoblotting analysis of different fractions from Superose-6 fractionation of HeLa nuclear extract. The fraction numbers are indicated on the top. The fractions corresponding to proteins of known molecular mass are denoted below the figure in kilodaltons. The void volume of the column is marked as “void.” The fractions in which BLM peaked are marked at the top as BCI, BCII, and BCIII (for BLM complexes I to III). The letters a, b, and c indicate the overlapping peak fractions between BLM and FANCA. A false-positive band derived from antibody cross-reactivity (which is also present in fractions from FANCA-deficient cells) is indicated by an asterisk. (B) Silver-stained gel showing the composition of three BLM complexes, which were immunoprecipitated with a BLM antibody (69D) from the three BLM peak fractions after Superose-6 fractionation (see panel A). The immunoprecipitations were carried out directly from each of the corresponding fractions (i.e., fractions 24, 30, and 36) without further dilution. BLM immunoprecipitate from total nuclear extract (NE) is shown for comparison. (C) Western blotting analysis showing the presence of multiple FANC proteins in the BRAFT complex. Fraction 24 of Superose-6, which corresponds to the 1.5- to 2-MDa fraction, was used as a positive control. (D) Immunoblotting of BLM immunoprecipitates (IPs) from three different Superose-6 fractions—a, b, and c—in which significant amounts of BLM and FANCA overlap. Notably, BLM coimmunoprecipitates with FANCA and FANCG only from fraction A, which corresponds to a molecular mass of 1.5 to 2 MDa (BRAFT).

The BLM complexes are present in smaller forms in an FA cell line. Immunoblotting analysis of different fractions from Superose-6 fractionation of the wild-type (ManEBV) and FANCA-deficient (VU388) lymphoblastoid cell lines. Notably, BLM peaks at fraction 21 in the FANCA-deficient cells, corresponding to a complex of ca. 1 MDa. This is smaller than the BLM from the wild-type cells, which peaks between fractions of 17 and 18, corresponding to a complex of 1.5 to 2 MDa (BRAFT). It was noted that BCII in this analysis is not that obvious, which may be due to its different levels in different cell lines.

Complexes isolated by BLM and FANCA antibodies possess a DNA-unwinding activity that requires the presence of BLM. (A-D) Autoradiographs showing the results of a helicase assay for complexes isolated by antibodies to BLM or FANCA. The partial DNA duplex substrate and the displaced single-strand oligonucleotide (Oligo) are illustrated on the left of the figure. Also analyzed were fractions isolated by control (mock) immunoprecipitation (Mock IP) with preimmune serum of the BLM antibody or normal rabbit IgG for the FANCA antibody. The cell lines and antibodies used for immunoprecipitations are indicated on the top. For complexes isolated from HeLa cells, 0 to 200 ng of the BLM-associated complexes or 0 to 500 ng of FANCA-associated complexes are used for the helicase assay (A and C). For lymphoblastoid cells, 0 to 50 ng of BLM-associated complexes and 0 to 100 ng of FANCA-associated complexes were analyzed (B and D). Maximum amounts of complexes were assayed in those isolated from BLM-deficient cells. To ensure that comparable amounts of BLM were present in complexes isolated from wild-type (WT) and FANCA-deficient cells, each immunoprecipitate was analyzed by immunoblotting with a BLM antibody, and the results are shown as a small figure at the bottom of panel B.

FA Proteins and BLM do not function in the linear signaling pathway through monoubiquitination. (A) Immunoblotting analysis to show that the monoubiquitination of FANCD2 is normal in BS cells. Nuclear extracts prepared from the indicated lymphoblast cells were probed with a FANCD2 antibody (FANCD2-L and FANCD2-S represent ubiquitinated and nonubiquitinated forms of this protein, respectively). (B) Immunoblotting analysis to show that monoubiquitination is not detectable for BLM, Topo IIIα, and RPA after HeLa cells are treated with MMC. As a control, monoubiquitination of FANCD2 is strongly induced under such conditions.