Depletion of BACH1 and BRCA1 results in delayed G₁/S progression and accumulation of cells in G₁. (A) Flow chart depicting the sequence of siRNA transfections and S-phase analysis. (B) Western blot analysis of whole-cell lysate of HeLa cells treated with control, BACH1, or BRCA1 siRNA. Equal amounts of extract were loaded and probed with antibodies against BACH1 and BRCA1. Actin was used as a loading control. (C, D, and E) Cells transfected with control, BACH1, and BRCA1 siRNAs were synchronized at G₁/S and released into S phase, and the cell cycle progression was monitored at the indicated time points after release by flow cytometric analysis of the total DNA content. The percentage distribution of cells in the G₁, S, and G₂/M phases of the cell cycle in control and BACH1- and BRCA1-depleted cells, as determined by PI staining, is shown. Western blot analysis results of BACH1 and BRCA1 in the corresponding cells at different time points after release are shown under each panel. PCNA is shown as a loading control.

DNA-dependent ATPase activity of BACH1 is cell cycle regulated. (A) FLAG-BACH1 hydrolyzes ATP in a DNA-dependent manner. Equivalent amounts of purified WT BACH1 and K52R proteins were incubated with [γ-³²P]ATP in reaction mixtures containing 100 ng of double-stranded DNA (dsDNA) pcDNA3.1 and incubated at 30°C for 1 h. The reaction products were subjected to TLC on PEI-cellulose, and the inorganic phosphate (Pi) separated from ATP was analyzed by autoradiography of TLC plates (lanes 1 to 6). (B) Increasing amounts of BACH1 purified from FLAG-BACH1 cells at the S phase and G₁ phase of the cell cycle were subjected to immunoblot analysis with anti-BACH1 antibody. (C) BACH1 purified from the G₁ phase of the cell cycle has negligible ATPase activity. FLAG-BACH1 purified from nuclear extracts of cells synchronized in S and G₁ phases of the cell cycle was analyzed for ATPase activity as described for panel A. This is a representative of five independent experiments resulting in similar outcomes. (D) FLAG-BACH1 and FLAG-K52R cells were synchronized at G₁/S with aphidicolin and released into S phase, and at various times after release, BACH1 was immunoprecipitated from nuclear extracts with anti-FLAG antibody. The eluted fractions from each time point were assayed for ATPase activity as mentioned above.

Cells expressing mutations in BACH1 helicase display a delay in S-phase progression and increased sensitivity to ionizing radiation. (A) Nuclear extracts from FLAG-WT BACH1, FLAG-K52R (helicase mutant), and FLAG-S990A were immunoprecipitated with anti-FLAG antibody, and the fractions were analyzed by Western blotting with the antibodies shown to the right of the panel. (B) Control 293, FLAG-WT BACH1, FLAG-K52R, and FLAG-S990A cells were synchronized at G₁/S with aphidicolin and released into S phase, and the cell cycle progression was monitored at the indicated time points after release by flow cytometric analysis of the total DNA content. The percentage distribution of cells in G₁, S, and G₂/M phases of the cell cycle as determined by PI staining is shown. (C) Mutation in BACH1 (K52R) result in enhanced sensitivity to ionizing radiation compared to control 293 or WT BACH1 cells. (D) 53BP1 and γ-H2AX foci formation were studied in WT BACH1 and K52R cells. Nonirradiated 293 cells (negative control), irradiated (10 Gy) 293 cells (positive control), and WT BACH1 and K52R cells were stained by immunofluorescence with antibodies against 53BP1 and γ-H2AX as described in Materials and Methods. As a control, cells were stained with DAPI to mark the nuclear domain.

Purification of the BACH1-BRCA1 complex. (A) FLAG-BACH1, FLAG-BARD1, and FLAG-BRCC36 were immunoprecipitated from nuclear extracts of the corresponding cells with anti-FLAG-M2 agarose beads. The purified proteins were resolved on 4-12% Tris-glycine gels followed by Western blot analysis using the antibodies shown to the right of the panel. (B) Endogenous BACH1-BRCA1 complex was immunoprecipitated from HeLa nuclear extracts with polyclonal antibodies against BRCA1 and BACH1. The purified proteins were immunoblotted with antibodies against the proteins shown to the right of the panel. (C) Association of BACH1 and BRCA1 during cell cycle progression: the BACH1-BRCA1 interaction is cell cycle dependent. FLAG-BACH1 cells were synchronized with aphidicolin as described in Materials and Methods. The cells were released at the indicated times, followed by immunoprecipitation of nuclear extracts with anti-FLAG antibody. Immunoblot assays were carried out with the antibodies mentioned. Distribution of cells in the cell cycle as examined by flow cytometry is included for each time point. (D) Chromatin association of BRCA2, BRCA1, and BACH1 is cell cycle regulated. MCF7 cells were synchronized with aphidicolin and, at different time points after release, subjected to biochemical fractionation to isolate chromatin-bound proteins as described in Materials and Methods. Immunoblot assays of the chromatin-enriched fractions were carried out with the antibodies indicated. The cell cycle distribution at each time point is shown. Western blot analysis using the total lysate or the chromatin-bound and non-chromatin-bound amounts of BRCA2, BRCA1, BACH1, ORC2, and actin is shown on the right of the panel.

Progression of cells through S phase in control and BACH1- and BRCA1-depleted cells. Control (A) and BACH1-depleted (B) and BRCA1-depleted (C) cells after release from G₁/S arrest were labeled with BrdU at the indicated times and stained with anti-BrdU antibody followed by FITC-conjugated secondary antibody. The cells were stained with PI, and results were analyzed by two-color flow cytometry. Cell cycle populations are characterized as R2 (G₁ cells with 2N DNA content), R3 (S phase cells with variable DNA content) and R4 (G₂/M cells with 4N DNA content). The times after release from G₁/S arrest are indicated. Cells in R3 above the quadrant are BrdU positive and represent the percentage of replicating cells in the S phase of the cell cycle. Percentage incorporation of BrdU at each time point in control, BACH1, and BRCA1 siRNA-treated cells is shown, indicating DNA synthesis. Cells begin to enter S phase at 4 h after release from aphidicolin in the control population, while the BACH1- and BRCA1-depleted cells show a delay in entering S phase with considerable accumulation of cells in G₁. Loss of BACH1 and BRCA1 also led to a delay in exit from S to G₂ (compare results at 8 h and 9 h after release).

Dephosphorylation of BACH1 positively regulates G₁ ATPase activity. FLAG-BACH1 cells were synchronized at G₁/S with aphidicoiln and released into S phase, and at various times after release, cells were harvested followed by nuclear extract preparation and immunoprecipitation with M2-anti-FLAG antibody. The beads were treated with λ-protein phosphatase (+; lanes 4 to 6 and 10 to 12) or the heat-treated enzyme (−; lanes 1 to 3 and 7 to 9) and washed extensively, and the FLAG-eluted fractions from each time point were assayed for ATPase activity.