Multifactorial contributions to an acute DNA damage response by BRCA1/BARD1-containing complexes

Abstract

The BRCA1 gene product and its stoichiometric binding partner, BARD1, play a vital role in the cellular response to DNA damage. However, how they acquire specific biochemical functions after DNA damage is poorly understood. Following exposure to genotoxic stress, DNA damage-specific interactions were observed between BRCA1/BARD1 and the DNA damage-response proteins, TopBP1 and Mre11/Rad50/NBS1. Two distinct DNA damage-dependent super complexes emerged; their activation was dependent, in part, on the actions of specific checkpoint kinases, and each super complex contributed to a distinctive aspect of the DNA damage response. The results support a new, multifactorial model that describes how genotoxic stress enables BRCA1 to execute a diverse set of DNA damage-response functions.

Figure 1.

BRCA1 is required for DSB localization of BARD1, Rad51, and BRCA2. (A–D) Laser microirradiation was performed on HCC1937 cells reconstituted with empty vector (top panels) or wild-type BRCA1 (bottom panels). Cells were fixed ∼30 min after laser treatment, and laser stripe localization for BARD1 (A), Rad51 (B), BRCA2 (C), and NBS1 (D) assessed by dual color immunofluorescence with specific Ab to each protein (Rhodamine-Red) and Ab to H2AX-phosphate (FITC-Green).

Figure 2.

Damage-induced BRCA1 interactions with TopBP1 and the M/R/N complex. (A) Silver-stained gel demonstrating purified complexes after single Flag (Flag) and consecutive Flag and HA purification steps (Flag/HA) for eBARD1 (B) and mock (M) transduced HeLa S3 cell lines. The BRCA1 and BARD1 bands are denoted in bold black type. Selected interacting partners that had been reported previously are indicated in nonbold black type. Novel interacting proteins are indicated with a red asterisk (*). (B) Complexes were purified 3 h after 0 (–) or 10 Gy (+) by serial Flag and HA-immunoaffinity chromatography/elution (Flag/HA). BRCA1 bands from undamaged cells and post-IR phosphorylated BRCA1 (BRCA1-P) are indicated. Damage-specific bands are indicated by red asterisks (*). A new band for NBS1 was not detected due to its comigration with BARD1. (C) Immunoblot detection of IR-dependent TopBP1 and NBS1 association with Flag/HA-purified BARD1-containing complexes. Damage-induced binding proteins are indicated in red. (D) Flag/HA-purified eBARD1 complexes were isolated from HeLa S3 cells following mock treatment, IR, or a thymidine block. Damage-activated binding events are indicated in red. (E) IF for H2AX-P and TopBP1 in microirradiated HCC1937 cells reconstituted with vector (top panels) or wild-type BRCA1 (bottom panels).

Figure 3.

PIKK and CHK2 kinases are required for damage-induced BARD1–BRCA1 protein-binding events. (A) ATM-null lymphoblasts (–/–) (GM 03189D) and ATM wild-type lymphoblasts (+/+) (GM 03323A) were treated with DMSO (control) or the DNA-PK inhibitor, LY294002 (100 μM), as indicated. Endogenous BRCA1 was immunoprecipitated, and immunoblots were performed as noted. (B) DNA-PK-deficient glioblastoma cells (M059J) were treated with DMSO, or the indicated doses of caffeine and wortmannin and immunoprecipitated 1 h after 10 Gy. Immunoblots for BRCA1, phosphorylated BRCA1 at Ser 1423, and TopBP1 were performed as indicated. (C) HCT115 (CHK2-deficient) cells, expressing eBARD1, were reconstituted with either wild-type HA-tagged CHK2 or a vector control and were immunoprecipitated with Flag Ab 2 h after exposure to 10 Gy. Immunoblotting was subsequently performed as indicated.

Figure 4.

BARD1–BRCA1 damage-induced complexes are biochemically distinct. (A) eBARD1/BRCA1-containing complexes were purified from irradiated HeLa S3 nuclear extracts by Flag IP and elution with Flag peptide. Eluted material was then separated by centrifugation through a 10%–40% glycerol gradient, and alternate fractions (renumbered 1, 2, 3 etc.) were probed by Western blot with Abs to the proteins listed. (B) Flag-eluted material (input) was immunoprecipitated with either mouse IgG (negative control) or a mouse monoclonal Ab specific for Rad50 or for BACH1. Immunoprecipitated material was then immunoblotted with the indicated Abs. (C) HeLa cells were stably transduced with a retrovirus encoding an shRNA for Luciferase (Luc), for BACH1, or for CtIP. (Right) After 5 Gy, BRCA1 IP and Western blotting for BRCA1 and TopBP1 was performed. (Left) Standardized quantities of control whole-cell extract (WCE) were probed for BRCA1, BACH1, CtIP, and TopBP1. (D) One hour after 10 Gy IR, BRCA1 was immunoprecipitated from HCC1937 cells that had been reconstituted with vector or wild-type BRCA1. Western blotting for coimmunoprecipitated proteins was performed. (E) Myc-tagged wild-type and BACH1 mutant alleles were transfected into 293 cells and immunoprecipitated with anti-myc Ab 2 h after 10 Gy IR. Western blotting was performed for TopBP1 and Myc-tagged BACH1 (eBACH1).

Figure 5.

BRCA1 super complexes control distinct cell cycle checkpoints after DNA damage. (A) HeLa cells were transfected with siRNA to BRCA1, BACH1, or CtIP, as indicated; and the G2/M checkpoint assay was performed. (B) U2OS cells were transfected with siRNA as indicated, and the radiation-resistant DNA synthesis (RDS) assay was performed. DNA synthesis after 10 Gy is plotted graphically as a percentage of DNA synthesis in unirradiated cells. (C, top) Cell cycle profile of adherent HeLa cells expressing shRNA to luciferase, or either of two different shRNAs targeting BACH1. (Bottom) Quantitation of S phase in HeLa S3 cells targeted by shRNAs to Luciferase (Luc), BACH1, or CtIP. FACS analysis was performed 18 h after exposure of the relevant cells to 1 μg/mL aphidicolin (+). Control cells were mock treated (–). The arrowheads in each FACS profile correspond to 2N and 4N cells, respectively. (D) Western blotting for phospho-CHK1 was performed 1 h after IR on BACH1, CtIP, and BRCA1-depleted cells using phosphospecific Ab to CHK1 (CHK1 pS317).

Figure 6.

BRCA1–BACH1 control Cdc45 association with sites of replication initiation after DNA damage. (A) ChIP was performed with Ab to BRCA1, BACH1, or CtIP, and precipitated genomic DNA was PCR amplified for β-globin replication origin core sequences. (B) ChIP was performed for eBARD1 with Flag Ab, followed by Flag peptide elution. ReChIP on eluted material was performed with the indicated Abs as in A. (C) ChIP was performed as indicated in A, but in this experiment, it was undertaken both before and after 10 Gy. (D) ChIP was performed for BRCA1 and BACH1 in the absence or presence of 5 mM caffeine. (E) ChIP was performed with rabbit IgG (negative control) or rabbit polyclonal antibodies to Cdc45 before and after IR in HeLa cells stably tranduced with retrovirus encoding shRNA for Luciferase (Luc) or BACH1. PCR for the β-globin origin of replication was performed on input genomic DNA (input) and ChIP samples.

Figure 7.

Model for BRCA1 checkpoint function. Model proposing that prior to DNA damage, BRCA1-containing complexes (dotted circles) fail to interact efficiently with TopBP1 and the M/R/N. Upon genotoxic stress, specific kinase signaling pathways enable BRCA1 super-complex assembly (colored circles). DSB recruitment of proteins that constitutively interact with BRCA1 (i.e., BARD1, BACH1, BRCA2, Rad51) is largely BRCA1 dependent, while damage-inducible BRCA1-associated proteins (M/R/N and TopBP1) access DNA damage sites in a BRCA1-independent manner. Each BRCA1–BARD1 super complex is responsible for executing distinct elements of BRCA1-dependent damage response activity.