BRCA1-BARD1 Complexes Are Required for p53 Ser-15 Phosphorylation and a G 1 /S Arrest following Ionizing Radiation-induced DNA Damage*

BRCA1 is a major player in the DNA damage response. This is evident from its loss, which causes cells to be-come sensitive to a wide variety of DNA damaging agents. The major BRCA1 binding partner, BARD1, is also implicated in the DNA damage response, and recent reports indicate that BRCA1 and BARD1 co-operate in this pathway. In this report, we utilized small interfering RNA to deplete BRCA1 and BARD1 to demonstrate that the BRCA1-BARD1 complex is required for ATM/ ATR (ataxia-telangiectasia-mutated/ATM and Rad3-re-lated)-mediated phosphorylation of p53 Ser-15 following IR- and UV radiation-induced DNA damage. In contrast, phosphorylation of a number of other ATM/ATR targets including H2AX, Chk2, Chk1, and c-jun does not depend on the presence of BRCA1-BARD1 complexes. Moreover, prior ATM/ATR-dependent phosphorylation of BRCA1 at Ser-1423 or Ser-1524 regulates the ability of ATM/ATR to phosphorylate p53 Ser-15 efficiently. Phosphorylation of p53 Ser-15 is necessary for an IR-induced G 1 /S arrest via transcriptional induction of the cyclin-dependent kinase inhibitor p21. Consistent with these data, re-pressing p53 Ser-15 phosphorylation by BRCA1-BARD1 depletion compromises p21 induction and the G 1 /S checkpoint arrest in response to IR but not UV radiation. These findings suggest that BRCA1-BARD1 complexes act as an adaptor to mediate ATM/ATR-directed phosphorylation of p53, influencing G 1 /S cell cycle pro- gression after DNA damage. J/m 2 UV light and were then incubated for 1 h. Cellular extracts were obtained and immunoblotted for p53 and Chk2 phosphorylation using phosphospecific antibodies against the indicated residues. The expression of Cdc25A was also assessed by immunoblotting. C , 293T cells were transfected and exposed to DNA-damaging agents as described in A . At 72 h post-transfection cells were immunostained for BRCA1 levels using an anti-BRCA1 antibody and for the phosphorylation status of p53 using the p53 Ser-15 phosphospecific antibody. Microscopy images demonstrate that p53 Ser-15 phosphorylation is repressed in cells lacking BRCA1 expression. D , 293T cells were transfected and treated as described in A and then immunostained with a H2AX phosphospecific antibody (H2AX Ser-139 ). In contrast to the effect of p53 Ser-15 phosphorylation, DNA damage-induced phosphorylation of H2AX Ser-139 was not affected by the loss of BRCA1 expression.

Damage to genetic material, which occurs as a consequence of exposure to environmental genotoxins or the byproducts of oxidative metabolism, represents a ubiquitous and persistent threat to genomic integrity. In eukaryotes and prokaryotes alike, the deleterious effects of genotoxic stress are countered by a robust DNA damage response pathway that monitors the genome for the presence of abnormal DNA structures, including single-stranded DNA, DNA double strand breaks, and chemically modified DNA bases. The DNA damage response can be reduced to detection, signal transduction, and effector phases, which are analogous to the signaling paradigms of growth factors and their cognate receptors. In mammals, the ATM 1 (ataxia-telangiectasia-mutated) and ATR (ATM and Rad3-related) protein kinases function as critical regulators of the cellular DNA damage response (1,2). ATM and ATR are Ser/Thr-Gln-directed protein kinases with overlapping substrate specificities that are activated in response to distinct, as well as partially overlapping, types of genotoxic stimuli (1,2). Despite their structural similarity and overlapping substrate specificities, ATM and ATR are functionally nonredundant protein kinases, and this is most convincingly demonstrated by comparing their respective gene knock-out phenotypes. In humans, inactivating mutations in ATM result in the cancer predisposition/neurodegeneration syndrome ataxia-telangiectasia (A-T) (3). Cells from A-T patients or ATM-nullizygous mice are exquisitely sensitive to ionizing radiation (IR) and other agents that induce double strand breaks and fail to activate the IR-induced G 1 /S or G 2 /M checkpoints. In addition, A-T cells exhibit radioresistant DNA synthesis, the failure to transiently down-regulate DNA replication in response to IR, which is indicative of an S-phase checkpoint defect. In contrast to ATM Ϫ/Ϫ mice, which are viable, ATR-deficient mice die early during embryogenesis, and conditional knock-out of ATR gene function in human cells leads to a loss of cellular viability (1). ATR mediates responses to a broad spectrum of genotoxic stimuli, including DNA replication inhibitors (e.g. hydroxyurea), UV radiation, IR, and agents such as cis-platinum that induce DNA interstrand cross-links.
The checkpoint functions of ATM and ATR are mediated, in part, by a pair of checkpoint effector kinases termed Chk1 and Chk2/Cds1 (4). Although structurally distinct, Chk1 and Chk2 are functionally related kinases that phosphorylate an overlapping pool of cellular substrates (5,6). Chk1 is phosphorylated on two Ser residues (Ser-317 and Ser-345) in an ATM-dependent manner following IR (7) and in an ATR-dependent manner following cellular exposure to hydroxyurea or UV light, two classic ATR stimuli. Chk2 is inducibly phosphorylated on multiple Ser/Thr residues, including a regulatory site at Thr-68, by ATM in response to double strand breaks. Substrates for Chk1 and Chk2 include members of the Cdc25 family of protein phosphatases, which are essen-tial for S-phase and G 2 /M-phase cell cycle transitions (1). Like ATM and ATR, Chk1 and Chk2 are nonredundant protein kinases that regulate distinct and partially overlapping cellular responses to genotoxic stimuli (8).
Although essential for transmission of DNA damage signals, the ATM/ATR and Chk1/Chk2 kinases are not sufficient for checkpoint signaling to occur. Genetic studies using budding and fission yeast have identified gene products that participate in the initiation and propagation of checkpoint signals (4,9). A critical element of the DNA damage-signaling network is the DNA replication factor C-related protein, Rad17. Studies in yeasts and human cells indicated that Rad17 interacts genetically and biochemically with a heterotrimeric complex of proteins consisting of Rad9, Rad1, and Hus1 (Rad9 complex). The Rad9 complex is structurally, and presumably functionally, related to the PCNA sliding clamp, leading to the hypothesis that hRad17-replication factor C functions as a clamp loader for the Rad9 complex at or near sites of DNA damage (10). Recently, Rad17 has been shown to be required for recruitment of Rad9 complex on damaged DNA and for ATR-dependent phosphorylation of Chk1 after IR (11).
Another protein with a putative adaptor/scaffolding function is the tumor suppressor, BRCA1. BRCA1 is a phosphoprotein that is mutated in ϳ50% of familial breast and ovarian cancers (12). BRCA1 is phosphorylated by ATM and ATR on multiple Ser/Thr residues in response to genotoxic stimuli (13,14), as well as by Chk2 (15). BRCA1 contains a BRCT motif that is found in a variety of DNA repair and checkpoint proteins (16), including budding yeast ScRad9p. ScRad9p mediates interaction between the ATM/ATR ortholog ScMec1p and its downstream target, the Chk2 ortholog ScRad53p (17). The adaptor function of Rad9 is dependent upon prior phosphorylation by ScMec1p. BRCA1 is an ATM/ATR target (14). Therefore, based on this analogy, it is conceivable that ATM/ATR-dependent phosphorylation of BRCA1 generates binding sites for BRCA1associated factors, which are ATM/ATR substrates. Consistent with this theory, BRCA1 in cells exists as a part of a complex known as BASC (BRCA1-associated genome surveillance complex), which contains many of the known ATM targets including NBS1, BLM, and SMC1 (18). BRCA1 is almost always found complexed with BARD1 in vivo. Based on these studies, we investigated whether BRCA1-BARD1 complexes are required for phosphorylation of ATM/ATR targets after exposure of cells to IR and UV radiation. The present study demonstrates that BRCA1-BARD1 dimers are required for efficient phosphorylation of p53 after exposure of cells to IR and UV radiation, respectively. Furthermore, ATM and ATR-dependent phosphorylation of BRCA1 is necessary for its adaptor function. Consequently, we show that the BRCA1-BARD1 complex is required for a G 1 /S arrest following IR but not UV radiation. These findings highlight the role of the BRCA1-BARD1 complex as an important mediator in the DNA damage response, affecting checkpoint function.

MATERIALS AND METHODS
Cell Culture and Transfection-293T is a human embryonic kidney carcinoma cell line that has been transformed with the SV40 large T-antigen. U2OS is a human osteosarcoma cell line, and MCF-7 is a human breast carcinoma cell line. HeLa is a human cervical carcinoma cell line. HCC1937 is a human breast carcinoma cell line harboring a pathogenic hemizygous mutation in BRCA1 (19). These cells have been stably transfected with pcDNA vector, pcDNA-BRCA1wt, or pcDNA-BRCA1 S1423A/S1524A to generate three cell lines as described previously (20). C3ABR is a human lymphoblastoid cell line. All cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum and grown at 37°C in a humidified 5% CO 2 atmosphere. Cells were seeded onto 15-cm 2 dishes and transfected at 50 -60% confluence with 15 g of plasmid DNA by electroporation for immunoblotting. For immunofluorescence studies, a portion of the cells was taken from the above samples and cytospun onto slides prior to lysis. Alternatively, cells were seeded onto coverslips and transfected at 50 -60% confluence with 1.5 g of plasmid DNA with LipofectAMINE (Invitrogen) according to the manufacturer's instructions. For flow cytometry analysis, cells were seeded onto 6-cm 2 dishes and transfected at 50 -60% confluence with 5 g of plasmid DNA with LipofectAMINE according to the manufacturer's instructions. Cells were analyzed 72 h post-transfection. When required, cells were either irradiated with a 137 Cs ␥-ray source or exposed to UVC.
Plasmid Construction-The BRCA1 small interfering RNA (siRNA) construct was generated by annealing the following complementary oligonucleotides and inserted into the pSuper vector as a BglII Full-length wild-type YFP-tagged BRCA1 has been described previously (21). The QuikChange site-directed mutagenesis kit (Stratagene) was used according to the manufacturer's instructions to introduce alanine point mutations into the YFP-BRCA1 construct to disrupt the DNA damage-induced phosphorylation sites: Ser-1387, Ser-1423, and Ser-1524. To mutate residue serine 1387 to alanine, the following primers were used: forward, 5Ј-TCA GGG CTA TCC GCT CAG AGT GAC ATT-3Ј, and reverse, 5Ј-AAT GTC ACT CTG AGC GGA TAG CCC TGA-3Ј. To mutate residue serine 1423 to alanine, the following primers were used: forward, 5Ј-GAA CAG CAT GGG GCC CAG CCT TCT AAC-3Ј, and reverse, 5Ј-GTT AGA AGG CTG GGC CCC ATG CTG TTC-3Ј. To mutate residue serine 1524 to alanine, the following primers were used: forward, 5Ј-GA AAC TAC CCA GCT CAA GAG GAG CTC-3Ј, and reverse, 5Ј-GAG CTC CTC TTG AGC TGG GTA GTT TC-3Ј. The codons in boldface indicate the amino acids mutated to alanine. All plasmid mutations were confirmed by DNA sequencing.
Immunofluorescence Microscopy-Cells were fixed in 4% paraformaldehyde/PBS for 15 min at room temperature, washed three times in PBS, and then permeabilized in 0.2% Triton X-100/PBS. Cells were then washed three times in PBS and blocked in 3% bovine serum albumin/PBS for 45 min before the required primary antibody was applied. BRCA1 was detected with a monoclonal antibody Ab-1 (Oncogene Research) followed by incubation with fluorescein-labeled antimouse IgG (Zymed Laboratories Inc.. The phosphospecific antibodies p53 Ser-15 (Cell Signaling) and H2AX Ser-139 (Upstate Biotechnology) were used to detect the phosphorylation status of p53 and H2AX, respectively. Antibody bound to p53 Ser-15 or H2AX Ser-139 was detected with fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Molecular Probes). Cell nuclei were counterstained with the chromosome dye Hoechst 33285 (Sigma). After extensive washing, samples were mounted onto glass slides with a drop of Vectashield anti-fade mounting reagent (Vector Laboratories). Samples were analyzed with an epifluorescence microscope (Olympus). Images were captured digitally and processed with Adobe Photoshop.
Bromodeoxyuridine (BrdUrd) Incorporation Assay-Cells were mock irradiated or exposed to 6 Gy of IR or 50 J/m 2 UVC. After 16 h, cells were pulsed by 20 mM BrdUrd for 30 min and then harvested by washing with PBS. Cells were fixed in 70% ethanol at ؊20°C for at least 1 h. After fixation, the cells were washed with 1% fetal calf serum, PBS and then treated with 2 M HCl containing 0.5% Triton X-100 for 30 min at room temperature. The acid was neutralized by resuspending the cell pellet in 0.1 M sodium tetraborate (pH 8.5) and incubating at room temperature for 5 min. Following centrifugation, the cell pellet was resuspended in 100 l of wash/stain buffer (1% fetal calf serum, 0.5% Tween 20, PBS) and then 10 l of anti-BrdUrd-FITC antibody (BD Biosciences). Cells were incubated in the dark for 30 min and then washed twice in wash/stain buffer before being stained with 25 g of propidium iodide/ml and 0.1 mg of RNase A/ml in PBS. Cellular fluorescence was measured by a BD Biosciences FACS Calibur flow cytometer. The data were analyzed using CellQuest software.

BRCA1 and BARD1 Require Heterodimerization to Maintain
Stability-BRCA1 predominantly is found complexed in vivo with its major binding partner, BARD1 (22). These proteins co-localize in nuclear foci during S-phase (23) and following exposure to DNA-damaging agents relocate to sites of DNA repair (24). Furthermore, they co-fractionate in various DNA repair-associated nuclear complexes (25), implicating the BRCA1-BARD1 heterodimer in the DNA damage response. Previous studies have demonstrated that the stability of BRCA1 is dependent on the expression of BARD1 and vice versa (26,27). Therefore, we sought to confirm these data by using siRNA to specifically inhibit the expression of either BRCA1 or BARD1 in cells. 293T cells were transfected with siRNAs targeting BRCA1 and GFP. Immunoblotting results show that only BRCA1 siRNA specifically decreased BRCA1 expression with or without prior exposure of cells to IR (10 Gy) or UV (50 J/m 2 ). In contrast, GFP siRNA had no apparent effect (Fig. 1A). Similar results were obtained by immunostaining (Fig.  2C). Consistent with published data, BARD1 levels were reduced in BRCA1 siRNA transfected cells compared with control transfected cells (Fig. 1A, GFP siRNA). The same phenomenon applied to BARD1 siRNA, i.e. 293T cells transfected with BARD1 siRNA not only specifically reduced BARD1 expression but also repressed BRCA1 expression, whereas GFP siRNA had no effect on either protein (Fig. 1B). These findings indicate that BRCA1-BARD1 complex formation is essential for mutual stability.
ATM is the primary signal transducer in response to DNA double strand breaks caused by exposure to IR. A recent paper illustrated that ATM is activated by autophosphorylation on Ser-1981 in response to IR (28). Therefore, to confirm that BRCA1-BARD1 complexes do not disrupt ATM activity directly or indirectly, we immunoblotted GFP, BRCA1, and BARD1 siRNA cellular extracts with a Ser-1981 phosphospecific antibody as a marker of ATM activity in cells. As expected, ATM Ser-1981 phosphorylation was markedly increased following exposure to IR, but not UV radiation, in control transfected cells (Fig. 1, GFP siRNA). Importantly, phosphorylation of ATM Ser-1981 was not decreased by the depletion of BRCA1 and BARD1 in cells transfected with siRNAs targeting these proteins (see Fig. 1). In contrast, IR-induced phosphorylation of ATM at Ser-1981 was slightly increased in BRCA1 and BARD1 siRNA-transfected cells relative to control cells (GFP siRNA), indicating that BRCA1-BARD1 complexes may set a threshold for ATM autophosphorylation in response to IR. These findings indicate that BRCA1-BARD1 complexes are not required for the activation of ATM.
BRCA1-BARD1 Complexes Are Required for ATM/ATR-dependent Phosphorylation of p53 Ser-15 -Previous work from our laboratory and those of others (14,29) has shown that although ATM and ATR phosphorylate the same set of targets in vitro, functionally they are nonredundant kinases in vivo. Rapid IR-induced phosphorylation of downstream targets are catalyzed by ATM in vivo, whereas ATR mediates rapid UVCinduced phosphorylation events. BRCA1 has been shown to bind many ATM/ATR targets as a component of BASC (BRCA1-associated genome surveillance complex) (18). To address whether the BRCA1-BARD1 heterodimer is required for the phosphorylation of ATM/ATR targets, we used siRNA to specifically inhibit the expression of BRCA1 and BARD1 in cells. By immunoblotting we examined the phosphorylation status of p53 (anti-p53 Ser-15 ), Chk2 (anti-Chk2 Thr-68 ), and c-jun (anti-c-Jun Ser-63 ) in BRCA1, BARD1, and GFP siRNA-transfected cells before and after exposure to IR (10 Gy) and UV radiation (50 J/m 2 ). In cells transfected with GFP siRNA, phosphorylation of p53 and c-jun increased following exposure to IR and UV radiation, whereas phosphorylation of Chk2 only increased after IR ( Fig. 2A). Interestingly, the IR-and UV-induced increase in p53 phosphorylation was significantly less in BRCA1 and BARD1 siRNA-transfected cells after DNA damage (Fig. 2, A and B). Similar results were obtained by immunostaining (Fig. 2C). In contrast, Chk2 and c-jun phosphorylation was not affected by the loss of BRCA1 and BARD1 expression (Fig. 2, A and B). The effect of Chk1 phosphorylation (anti-Chk1 Ser-317 ) was also assessed in GFP and BRCA1 siRNA-transfected cells in the absence and presence of DNAdamaging agents. As expected, Chk1 Ser-317 phosphorylation increased following exposure to UV radiation in control transfected cells (Fig. 2A, GFP siRNA). Interestingly, Chk1 Ser-317 phosphorylation was not affected in cells depleted of BRCA1 ( Fig. 2A). It should be noted that publications from our laboratory and others (7,40) have shown that phosphorylation of 293T cells were transfected with siRNAs targeting GFP, BRCA1 (A), or BARD1 (B). Cells were mock irradiated or exposed to 10 Gy IR or 50 J/m 2 UV light. At 1 h post-irradiation, cellular extracts were prepared, and BRCA1 and BARD1 levels were analyzed by immunoblotting. Blots demonstrate that siRNA targeting of BRCA1 not only depletes BRCA1 expression but also BARD1 expression and vice versa. Furthermore, cellular extracts were immunoblotted with an ATM Ser-1981 phosphospecific antibody. Immunoreactivity with this antibody is indicative of ATM kinase activity (28). Blots indicate that ATM kinase function is not affected by loss of BRCA1 and BARD1. Chk1 Ser-317 is increased following IR in a number of lymphoblastoid cell lines in an ATM-dependent manner; however, we did not observe an increase in Chk1 Ser-317 phosphorylation in 293T cells following IR ( Fig. 2A). In contrast, as a positive control, we demonstrate that Chk1 Ser-317 phosphorylation is markedly increased 1 h post-IR in C3ABR lymphoblastoid cells ( Fig. 2A). A recent publication (30) has demonstrated that SV40 transformation can interfere with Chk1 phosphorylation, which would provide an explanation as to why we did not observe an increase in this phosphorylation event in 293T cells. Cdc25A is a downstream target of the Chk1 and Chk2 kinases. Both kinases can mediate the phosphorylation of Cdc25A, stimulating its degradation and resulting in activation of the Sphase checkpoint (31). Therefore, Cdc25A expression was analyzed in GFP, BRCA1, and BARD1 siRNA-transfected cells. As expected, Cdc25A expression decreased in GFP siRNA-transfected cells following DNA damage compared with untreated cells. Consistent with our findings on Chk1/Chk2 phosphorylation, Cdc25A degradation was not affected by the loss of BRCA1 and BARD1 expression (Fig. 2, A and B). We conclude that BRCA1-BARD1 dimers are required for ATM/ATR-dependent phosphorylation of p53 Ser-15 in response to DNA dam-age but are dispensable for phosphorylation of various other ATM/ATR targets.
BRCA1 Is Not Required for DNA Damage-induced H2AX Phosphorylation-One of the first proteins phosphorylated by ATM after DNA double strand breaks (32) or by ATR after replication stress (33) is the histone variant H2AX. Phosphorylated H2AX forms nuclear foci at the sites of double strand breaks and is thought to be important for increasing the concentration of several repair factors to the sites of DNA damage (24) such as MDC1 (34), the MRN complex (Mre11-Rad50-Nbs1), Rad51, and BRCA1 (35,36). Therefore, to confirm that BRCA1 acts downstream of H2AX, the phosphorylation status of H2AX was examined by immunostaining GFP and BRCA1 siRNA-transfected cells 1 h after exposure to IR or UV radiation with a phosphospecific antibody (anti-H2AX Ser-139 ). Phosphorylation of H2AX was observed in control cells (GFP siRNAtransfected) following IR and UV radiation (see Fig. 2D). As predicted, H2AX Ser-139 phosphorylation after DNA damage was not affected by the loss of BRCA1 expression (see Fig. 2D). These findings indicate that H2AX phosphorylation is not dependent on BRCA1 and suggest that BRCA1 acts downstream of H2AX in the DNA damage response.
FIG. 2. BRCA1-BARD1 complexes mediate ATM/ATR-dependent phosphorylation of p53 Ser-15 following DNA damage. A, cellular extracts were obtained from 293T cells transfected with siRNAs targeting either GFP or BRCA1 in the absence or presence of irradiation at 1 h after exposure to 10 Gy IR or 50 J/m 2 UV light. The level of Cdc25A was analyzed by Western blotting. The phosphorylation of the indicated residues within the ATM/ATR substrates p53, Chk2, Chk1, and c-jun was also examined using phosphospecific antibodies. Chk1 phosphorylation was also assessed in C3ABR lymphoblastoid cells with (ϩ) and without (Ϫ) prior exposure to ionizing radiation (10 Gy). B, 293T cells were transfected with either GFP or BARD1 siRNAs. At 72 h post-transfection, cells were treated with 10 Gy IR or 50 J/m 2 UV light and were then incubated for 1 h. Cellular extracts were obtained and immunoblotted for p53 and Chk2 phosphorylation using phosphospecific antibodies against the indicated residues. The expression of Cdc25A was also assessed by immunoblotting. C, 293T cells were transfected and exposed to DNA-damaging agents as described in A. At 72 h post-transfection cells were immunostained for BRCA1 levels using an anti-BRCA1 antibody and for the phosphorylation status of p53 using the p53 Ser-15 phosphospecific antibody. Microscopy images demonstrate that p53 Ser-15 phosphorylation is repressed in cells lacking BRCA1 expression. D, 293T cells were transfected and treated as described in A and then immunostained with a H2AX phosphospecific antibody (H2AX Ser-139 ). In contrast to the effect of p53 Ser-15 phosphorylation, DNA damage-induced phosphorylation of H2AX Ser-139 was not affected by the loss of BRCA1 expression.
ATM/ATR-directed Phosphorylation of BRCA1 Is Required for p53  Phosphorylation-We have previously reported that BRCA1 is rapidly phosphorylated by ATM and ATR after exposure to IR and UV radiation, respectively. Specifically, residues Ser-1423 and Ser-1524 are phosphorylated in response to IR and UV radiation, whereas Ser-1387 is phosphorylated only following IR (14). By using phosphospecific antibodies, phosphorylated BRCA1 has been detected in DNA damage-induced nuclear foci (14). These foci are thought to be sites of DNA damage/repair (37) and contain many proteins, some of which are ATM/ATR substrates (18). Therefore, site-directed mutagenesis was employed to abolish phosphorylation of YFP-BRCA1 at Ser-1387, Ser-1423, and Ser-1524 by changing these residues to alanine. To confirm that these mutations disrupted YFP-BRCA1 phosphorylation, wildtype YFP-BRCA1, YFP-BRCA1 S1423A/S1524A , and YFP-BRCA1 S1387A/S1423A/S1524A were transfected into 293T cells, and their phosphorylation status was observed by immunoblotting 1 h after IR with affinity purified BRCA1 phosphospecific antibodies against residues Ser-1387, Ser-1423, and Ser-1524. As expected, all three BRCA1 phosphospecific antibodies recognized wild-type YFP-BRCA1 but not YFP-BRCA1 S1387A/S1423A/S1524A (Fig. 3A). In addition, the double mutant YFP-BRCA1 S1423A/S1524A was detected only by the anti-BRCA1 Ser-1387 antibody. To determine whether phosphorylation affects the ability of BRCA1 to form DNA damage-induced foci, these YFP-BRCA1 mutants were transfected into COS-7 cells, synchronized in S-phase, and then visualized by immunofluorescence in the absence of IR and at 1 or 4 h post-IR (6 Gy). S-phase wild-type YFP-BRCA1 nuclear foci were observed in untreated cells, which dispersed following IR and then reappeared 4 h post-IR (Fig. 3B) as reported previously (38). Interestingly, mutations at none of the three phosphorylation sites (Ser-1387, Ser-1423, and Ser-1524) prevented YFP-BRCA1 from forming S-phase or DNA damage-induced nuclear foci (Fig. 3B). Taken together, these data suggest that ATM/ ATR-dependent phosphorylation of BRCA1 does not effect dynamic changes in BRCA1 subcellular localization in response to DNA damage.
We next sought to investigate whether BRCA1 phosphorylation is required for its adaptor function by comparing the effect of the BRCA1 phosphorylation mutant S1423A/S1524A to wild-type for its ability to affect p53 Ser-15 and Chk2 Thr-68 phosphorylation following UV-and IR-induced DNA damage. HCC1937 cells harbor a homozygous pathogenic mutation in the BRCA1 gene that renders the BRCA1 protein inactive (39). Therefore, we used HCC1937 cells to generate three stably transfected cell lines, which included vector, wild-type BRCA1, and BRCA1 S1423A/S1524A (20). Consistent with knocking out BRCA1 by siRNA (Fig. 2), p53 Ser-15 phosphorylation was reduced in the HCC1937 cell line stably transfected with vector only following UV-and IR-induced DNA damage (Fig. 3C). Importantly, this phosphorylation event was restored in the wild-type BRCA1-complemented HCC1937 cells. In contrast, HCC1937 cells stably expressing phosphorylation mutant BRCA1 S1423A/S1524A did not restore p53 Ser-15 phosphorylation (Fig. 3C). These findings suggest that phosphorylated BRCA1 is required to mediate efficient ATM/ATR-dependent phosphorylation of p53 Ser-15 .
siRNA-mediated Repression of BRCA1 Disrupts p21 Induction and the G 1 /S Checkpoint following IR-induced DNA Damage-Our finding that loss of BRCA1 expression disrupts p53 Ser-15 phosphorylation implicates BRCA1 in G 1 /S checkpoint control because DNA damage-induced phosphorylation of p53 Ser-15 is essential for its activity and contributes to a G 1 /S arrest (41,42). To establish a role for BRCA1 in the G 1 /S checkpoint, flow cytometric assays were performed by staining GFP and BRCA1 siRNA-transfected MCF-7 cells with BrdUrd in the absence and presence of the DNA-damaging agents IR (6 Gy) and UV radiation (50 J/m 2 ). In response to UV radiation, incorporation of BrdUrd was dramatically decreased in both GFP and BRCA1 siRNA-transfected cells compared with untreated cells (Fig. 4A), indicating that the depletion of BRCA1 expression does not affect UV-induced G 1 /S arrest. BrdUrd A, 293T cells were transfected with YFP-tagged BRCA1 wildtype (Wt) or the YFP-tagged BRCA1 phosphorylation mutants S1423A/ S1524A or S1387A/S1423A/S1524A. At 1 h post-irradiation with 10 Gy, cellular extracts were prepared and immunoblotted for BRCA1 levels with the anti-GFP antibody (which also detects the YFP tag) or for BRCA1 phosphorylation status using phosphospecific antibodies generated against residues Ser-1387, Ser-1423, and Ser-1524. B, COS-7 cells were transfected with YFP-BRCA1wt or YFP-BRCA1 S1387A/S1423AS/1524A . At 24 h post-transfection, cells were synchronized in mid S-phase by treating them with 2 g/ml aphidicolin for 16 h followed by a 4-h incubation at 37°C. The required cell samples were then irradiated (6 Gy) and incubated for 1 or 4 h before fixation for immunofluorescence microscopy to observe BRCA1 nuclear foci. Nuclei were visualized by staining the chromatin with Hoechst 33285 dye. Microscopy images illustrate that mutation of the BRCA1 phosphorylation sites do not impair the ability of BRCA1 to form S-phase and DNA damage-induced nuclear foci. C, cellular extracts were prepared from HCC1937 cells stably transfected with vector, wild-type BRCA1, or phospho-mutant BRCA1 S1423A/S1524A in the absence or presence of the indicated DNA-damaging agents. The phosphorylation status of p53 Ser-15 and Chk2 Thr-68 was assessed by immunoblotting at 1 h after DNA damage. Blots indicate that p53 Ser-15 phosphorylation is enhanced in cells stably expressing wildtype BRCA1 but not BRCA1 S1423A/S1524A following DNA damage. incorporation was also reduced by Ͼ50% in control cells (GFP siRNA) following exposure to IR compared with untreated cells, indicating an efficient arrest at the G 1 /S checkpoint. In contrast, BrdUrd incorporation was not significantly affected in BRCA1-depleted cells following exposure to IR compared with untreated cells (Fig. 4A), implicating BRCA1 in the IR-induced G 1 /S checkpoint. As a result, unlike control cells, which arrested at the G 1 /S checkpoint, BRCA1-deficient cells accumulated at the next DNA damage checkpoint, G 2 /M, in the 16-h period after exposure to IR (Fig. 4B). Similar results were obtained in the U2OS cell line (data not shown). Because we observed differences in the ability of BRCA1-depleted cells to arrest at the G 1 /S checkpoint following exposure to IR and UV radiation, we examined the levels of the CDK inhibitor p21 in GFP and BRCA1 siRNA-transfected cells in response to DNA damage (IR and UV) because it lies downstream and is induced by p53 following its activation. It is interesting to note that in the absence of DNA damage the level of p21 expressed was markedly reduced in BRCA1-depleted cells compared with control cells (Fig. 4C, GFP siRNA). This result is consistent with previous findings implicating BRCA1 in the induction of p21 (43,44). As expected, p21 levels increased in control cells (GFP siRNA-transfected) 4 h following exposure to IR (Fig. 4C). In contrast, p21 was not induced in BRCA1-depleted cells following IR (Fig. 4C), which is consistent with these cells having a defective G 1 /S checkpoint. In response to UV radiation, however, neither GFP nor BRCA1 siRNA-transfected cells induced p21, and instead a significant reduction in p21 levels was evident (Fig. 4C), despite their ability to arrest at the G 1 /S checkpoint. This result is consistent with previously published data demonstrating that cells exposed to UV radiation and not IR are able to arrest at the G 1 /S checkpoint via a p53-and p21-independent pathway, which is activated by ATR (31). Collectively, these findings support a role for BRCA1 in the IRbut not UV-induced G 1 /S checkpoint. DISCUSSION In the present study, we have demonstrated that the BRCA1-BARD1 tumor suppressor complex acts as an important mediator in the DNA damage response. We show that the BRCA1-BARD1 complex is not required for the activation of ATM kinase function. In addition, BRCA1 is dispensable for the phosphorylation of H2AX, which is rapidly targeted following DNA double strand breaks and replication stress by ATM (32) and ATR (33), respectively. These findings indicate that the BRCA1-BARD1 complex acts downstream in the DNA damage response. A recent study has reported that a subset of ATM and ATR substrates requires BRCA1 for phosphorylation (45). The graph demonstrates that GFP siRNA-transfected cells effectively arrest in G 1 after IR or UV radiation; however, BRCA1-depleted cells cannot arrest in G 1 following exposure to IR. B, fluorescence-activated cell sorter profiles of propidium iodide-stained cells of a representative experiment, described in A, are shown and indicate that BRCA1-depleted cells accumulate in G 2 /M following exposure to IR but not UV light, compared with control cells, which arrest in G 1 . C, MCF-7 cells were transfected with siRNAs targeting GFP and BRCA1. At 72 h post-transfection, cells were treated with the indicated DNA-damaging agents then incubated for 4 h before cellular extracts were prepared and immunoblotted for p21 levels using anti-p21 antibodies. Cellular extracts were also immunoblotted for ␥-tubulin, which served as a loading control.

FIG. 5.
A model illustrating the role of BRCA1-BARD1 dimers in the G 1 /S checkpoint. Stimulation of ATM activity by autophosphorylation following IR-induced DNA damage results in phosphorylation of BRCA1 (cycle 1), which is bound to BARD1. This phosphorylation event allows BRCA1-BARD1 complexes to act as an adaptor for p53, enabling it to be targeted for phosphorylation by ATM at Ser-15 (cycle 2). Consequently, the transcriptional activity of p53 is enhanced to induce p21, causing a G 1 /S arrest. In contrast, BRCA1-BARD1 complexes are not required for a UV-induced G 1 /S arrest. Instead this pathway involves activation of ATR, which consequently phosphorylates the kinases Chk1 and Chk2, resulting in their activation. The Cdc25A phosphatase is then targeted by these kinases for phosphorylation and is subsequently degraded, preventing it from dephosphorylating/activating CDK2, which causes a G 1 /S arrest.
These substrates include Chk2, CtIP, Nbs1, p53, and c-jun. The findings published by Foray et al. (45) are based on the use of the BRCA1 mutant cell line HCC1937 stably transfected with either vector or wild-type BRCA1. HCC1937 is a tumor cell line that contains mutations in multiple genes other than BRCA1, for example, p53 and PTEN (39). Our experiments, however, using a distinct siRNA methodology to deplete BRCA1 function in cells, are not consistent with the previous study. By using this system, we have demonstrated that BRCA1 is required for p53 Ser-15 phosphorylation following UV-and IR-induced DNA damage. In addition our study provides evidence for the first time that ATM/ATR-mediated phosphorylation of BRCA1 at Ser-1423 or Ser-1524 is required for its adaptor function for p53. However, Chk1, Chk2, and c-jun phosphorylation occurs in the absence of BRCA1. Consistent with our findings, earlier studies have reported that the Chk1 and Chk2 mobility shift, which is indicative of phosphorylation, is not affected in the HCC1937 cell line (46,47). Heterodimerization of BRCA1 and BARD1 protects both proteins from degradation (26,27), thus explaining why Ͼ75% of the cellular pool of BRCA1 is complexed with BARD1 in vivo (22). Therefore, it is not surprising that BARD1 depletion by siRNA also reduced p53 Ser-15 phosphorylation and, like BRCA1 siRNA, had no effect on Chk2 and c-jun phosphorylation. These findings suggest that BRCA1-BARD1 complexes act as an adaptor for p53, enabling it to be targeted for ATM/ATR-directed phosphorylation following IR/ UV-induced DNA damage. An analogous model consistent with these data has been demonstrated for the budding yeast proteins Rad9 and Rad53 (17,48). In this case, Rad9 acts as an adaptor for Rad53, enabling it to be phosphorylated by Mec1. However, for Rad9 to recruit Rad53, it must first be phosphorylated by Mec1.
p53 is the main effector target of the G 1 /S checkpoint. It is rapidly turned over in normal cells, and thus for cells to arrest at the G 1 /S border, p53 needs to be stabilized and activated in response to DNA damage. Phosphorylation of p53 at Ser-15 is mediated by ATM rapidly after exposure to IR (49 -51) and by ATR rapidly following UV exposure (29). This phosphorylation event is essential for enhancing p53 activity as a transcription factor to induce the CDK2-cyclin E inhibitor, p21, which causes cell cycle arrest (41,42). Consistent with these findings, the depletion of BRCA1 not only disrupted p53 Ser-15 phosphorylation but also compromised p21 induction and G 1 /S checkpoint arrest following IR (Fig. 4). In contrast, in response to UV radiation, BRCA1-depleted cells were able to arrest at the G 1 /S checkpoint, despite a reduction in p53 Ser-15 phosphorylation and p21 levels (Fig. 4). UV radiation induces cytotoxic DNA lesions such as cyclobutane-pyrimidine dimers and 6 -4 photoproducts, whereas IR induces DNA double strand breaks. Therefore, the differences we observed suggest that different types of damage activate different signaling pathways to effect cell cycle progression and further suggest that cells can utilize a pathway independent of p53 and p21 to induce a G 1 /S arrest in response to UV radiation. This is consistent with a recent report demonstrating that the UV-induced G 1 /S checkpoint is not compromised in p21 null cells, whereas the IR-induced G 1 /S checkpoint is defective (52). Bendjennat et al. (52) also report that unlike IR, p21 is degraded following low dose exposure to UV radiation. In addition, some recent investigations (4,53) have elucidated that UV-induced G 1 /S arrest in human cells results from ATR activation, which mediates the phosphorylation and consequent activation of the Chk1 and Chk2 kinases. The phosphatase Cdc25A is a downstream target of these kinases and when phosphorylated is targeted for degradation, thus preventing it from dephosphorylating/activating CDK2 and thus resulting in a G 1 /S arrest (31). This supports our findings that BRCA1 is not necessary for Chk1/Chk2 phosphorylation, because BRCA1 depletion does not compromise the UV-induced degradation of Cdc25A and subsequent G 1 /S arrest.
Like BRCA1-BARD1 dimers, other proteins involved in the DNA damage response have also been shown to play an adaptor role, i.e. they present substrates to ATM/ATR for phosphorylation in response to DNA damage. Nbs1, a member of the MRN complex, has been shown to be required for the phosphorylation of ATM downstream targets (54 -56). Two other proteins, namely MDC1 and 53BP1, have also been implicated as adaptor proteins for ATM-dependent phosphorylation events (34,(57)(58)(59)(60)(61)(62). Like BRCA1, Nbs1, MDC1, and 53BP1 contain at least one BRCT domain and are phosphorylated by ATM in response to DNA damage. In addition, all of these proteins are recruited to nuclear foci following DNA damage, suggesting that these foci are concentrated pools of adaptor proteins and ATM/ATR substrates to allow fast and efficient transfer of DNA damage signals to mediate checkpoint control. In conclusion, we provide evidence that the BRCA1-BARD1 tumor suppressor complex acts as an adaptor to mediate G 1 /S checkpoint control following exposure of cells to IR but is dispensable for G 1 /S arrest in response to UV radiation (Fig. 5). Collectively, these findings highlight the importance of the BRCA1-BARD1 complex in the DNA damage response to maintain genomic stability.