Rapid Recruitment of BRCA1 to DNA Double-Strand Breaks Is ...

MOLECULAR AND CELLULAR BIOLOGY, Dec. 2008, p. 7380–7393 0270-7306/08/$08.00⫹0 doi:10.1128/MCB.01075-08 Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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Rapid Recruitment of BRCA1 to DNA Double-Strand Breaks Is Dependent on Its Association with Ku80䌤† Leizhen Wei,1 Li Lan,2 Zehui Hong,2 Akira Yasui,2 Chikashi Ishioka,1 and Natsuko Chiba1* Department of Clinical Oncology1 and Department of Molecular Genetics,2 Institute of Development, Aging and Cancer (IDAC), Tohoku University, 4-1 Seiryomachi Aoba-ku, Sendai 980-8575, Japan Received 9 July 2008/Returned for modification 29 August 2008/Accepted 7 October 2008

BRCA1 is the first susceptibility gene to be linked to breast and ovarian cancers. Although mounting evidence has indicated that BRCA1 participates in DNA double-strand break (DSB) repair pathways, its precise mechanism is still unclear. Here, we analyzed the in situ response of BRCA1 at DSBs produced by laser microirradiation. The amino (N)- and carboxyl (C)-terminal fragments of BRCA1 accumulated independently at DSBs with distinct kinetics. The N-terminal BRCA1 fragment accumulated immediately after laser irradiation at DSBs and dissociated rapidly. In contrast, the C-terminal fragment of BRCA1 accumulated more slowly at DSBs but remained at the sites. Interestingly, rapid accumulation of the BRCA1 N terminus, but not the C terminus, at DSBs depended on Ku80, which functions in the nonhomologous end-joining (NHEJ) pathway, independently of BARD1, which binds to the N terminus of BRCA1. Two small regions in the N terminus of BRCA1 independently accumulated at DSBs and interacted with Ku80. Missense mutations found within the N terminus of BRCA1 in cancers significantly changed the kinetics of its accumulation at DSBs. A P142H mutant failed to associate with Ku80 and restore resistance to irradiation in BRCA1-deficient cells. These might provide a molecular basis of the involvement of BRCA1 in the NHEJ pathway of the DSB repair process.

ious types of DNA damage result in hyperphosphorylation of BRCA1 and alterations in BRCA1 localization to nuclear foci (21, 41). Normally, BRCA1 redistributes to nuclear foci where multiple DNA repair factors accumulate. BRCA1 colocalizes with phosphorylated H2AX (␥H2AX), a protein that is rapidly phosphorylated at the sites of DSBs after DNA damage (39). Furthermore, BRCA1 colocalizes with Rad51, which mediates homologous recombination (HR), and/or with an MRN complex which is involved in both HR and nonhomologous end joining (NHEJ) (42, 57). These observations suggest that BRCA1 accumulates at sites of DNA damage, forms potentially distinct types of protein complexes, and functions in multiple aspects of DNA repair. DNA DSBs are produced directly by ionizing radiation and by some chemicals or indirectly by the blockage of replication forks. Repairing DSBs correctly is critical for maintaining genome integrity. NHEJ and HR are the major cellular mechanisms to repair DSBs. In the NHEJ pathway, the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and a Ku heterodimer of Ku80 and Ku70 are recruited to the sites of DNA DSBs, followed by subsequent recruitment of XRCC4 ligase IV. In contrast, Rad51 and replication protein A are the essential factors in the HR pathway. An MRN complex is involved at an early stage of both the HR and NHEJ pathways. Although BRCA1 is thought to be primarily involved in the HR pathway (46), it has been implicated in the NHEJ pathway as well (3, 5, 16, 55, 56). However, the precise mechanism by which BRCA1 accumulates at the sites of DSBs and what role BRCA1 plays in DSB repair pathways are not yet fully understood. We have recently established a laser light microirradiation system to create various types of DNA damage in living cells, including DSBs, single-strand breaks (SSBs), and base damage,

Germ line mutations in the breast cancer susceptibility gene BRCA1 predispose women to breast and ovarian cancers (26, 37). The human BRCA1 protein is composed of 1,863 amino acid (aa) residues and contains a RING domain in the N terminus. Two tandem BRCT domains, which are frequently found in DNA repair proteins and function as a binding module for phospho-serine peptides (34), are present in the C terminus. The N-terminal region of BRCA1 directly interacts with BARD1 (51), and the association with BARD1 enhances the ubiquitin polymerase activity of BRCA1 (2, 11, 33). BRCA1 is involved in many cellular processes, including DNA repair, transcription, cell cycle regulation, chromatin remodeling, and apoptosis. Several lines of evidences suggest that the involvement of BRCA1 in the DNA repair pathway is associated with the tumor suppressor activity of BRCA1. For example, mouse and human cells deficient in BRCA1 are more sensitive to DNA damage, including ionizing irradiation and drugs that produce double-strand breaks (DSBs) or interstrand cross-linking of DNA. Clinically observed missense mutations often result in a nonfunctional BRCA1 protein that has lost the ability to repair DNA damage (43). BRCA1 interacts with a number of DNA repair factors such as Rad51, the Mre11Rad50-Nbs1 (MRN) complex, BLM, and the DNA helicase BACH1 (also called BRIP1or FANCJ) (8, 42, 50, 57). BRCA1 localizes to nuclear foci during S phase of the cell cycle. Var* Corresponding author. Present address: Department of Molecular Immunology, Institute of Development, Aging and Cancer (IDAC), Tohoku University, 4-1 Seiryomachi Aoba-ku, Sendai 980-8575, Japan. Phone: 81-22-717-8478. Fax: 81-22-717-8482. E-mail: nchiba@idac .tohoku.ac.jp. † Supplemental material for this paper may be found at http://mcb .asm.org/. 䌤 Published ahead of print on 20 October 2008. 7380

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RAPID BRCA1 RECRUITMENT TO DNA DAMAGE DEPENDS ON Ku80

which has enabled us to detect BRCA1 accumulation at the site of DSBs (28, 29). In this study, we examined the real-time accumulation of endogenous BRCA1 and green fluorescent protein (GFP)-tagged BRCA1 at DSBs induced by laser irradiation. The behavior of various deletion and missense mutant forms of BRCA1 was also examined. The immediate recruitment of BRCA1 at DSBs after laser irradiation is mediated by an interaction of the N-terminal portion of BRCA1 with Ku80. MATERIALS AND METHODS Plasmid construction. Full-length pcDNA3-HA-BRCA1 and aa 1 to 302, aa 305 to 770, aa 775 to 1292, and aa 1527 to 1863 deletion mutant plasmids were constructed as described previously. Mutant plasmids with aa 305 to 770 and aa 775 to 1292 deleted contained the nuclear localization sequence (13). Full-length pEGFP-BRCA1 and ⌬1-302, ⌬305-770, ⌬775-1292, and ⌬1527-1863 mutant BRCA1-encoding plasmids were amplified by PCR from pcDNA3-HA-BRCA1 vectors with primers containing XhoI and NotI sites. PCR products were subcloned into the pEGFP-C1 vector (Clontech). The GFP-tagged aa 303 to 1526 fragment of BRCA1 was generated by digestion of the ⌬1-302 mutant with SacI and NotI; the resultant fragment was blunted and ligated. The GFP-tagged aa 1527 to 1863 fragment of BRCA1 was constructed by digestion of GFP-tagged full-length BRCA1 with XhoI and SacI; the resultant fragment was blunted and ligated. The aa 1 to 100, 1 to 200, 1 to 300, 101 to 200, 101 to 300, and 201 to 300 fragments of BRCA1 were amplified from pcDNA3-HA-BRCA1 with primers containing HindIII and EcoRI sites. The PCR products were cloned into pEGFP-C3 vectors (Clontech). BRCA1 missense mutations were generated by site-directed mutagenesis. For pCY4B-RAP80, the RAP80 fragment was amplified from MCF7 cell cDNA with primers that contained XhoI and NotI sites. PCR products were subcloned into the pCY4B-HA vector. The pCY4B vectors have been described previously (38). For p3XFLAG-BARD1, the BARD1 fragment was amplified from cDNA of HeLa cells as a template with primers that contained EcoRV and KpnI sites. The PCR product was then subcloned into p3XFLAG-CMV-14 (Sigma). For pEGFP-Ku80, the Ku80 fragment was amplified from HeLa cell cDNA with primers that contained XhoI and NotI sites. PCR products were subcloned into the pEGFP-C1 vector. To construct pCY4BFLAG-Ku80 and pCY4B-HA-Ku80, pEGFP-Ku80 was digested with XhoI and NotI. The purified Ku80 fragment was subcloned into the XhoI/NotI site of the pCY4B-FLAG or pCY4B-HA vector. To construct pCY4B-HA-BRCA1-1-304 and pCY4B-HA-BRCA1-1528-1863, aa 1 to 304 and 1528 to 1863 of BRCA1 were amplified from pcDNA3-HA-BRCA1 with primers containing XhoI and NotI sites. The PCR products were cloned into the pCY4B-HA vector. For p3XFLAG-BARD1, the BARD1 fragment was amplified from HeLa cell cDNA with primers that contained EcoRV and KpnI sites. The PCR product was then subcloned into the p3XFLAG-CMV-14 vector (Sigma). For pCMV-MycBARD1, the BARD1 fragment was amplified from p3XFLAG-BARD1 with primers that contained a 5⬘ SalI site and a 3⬘ XhoI site. The PCR product was subcloned into the pCMV-Myc vector (Clontech). pGEX-Ku80 was described previously (22). All constructs were verified by DNA sequencing. Cell lines and transfections. Saos-2 osteosarcoma cells, HEK 293T cells, AT1KY/T-n cells (ATM-deficient cell line), CHO9 cells (wild-type cells), XR-C1 cells (DNA-PKcs-deficient cell line), XR-1 cells (XRCC4-deficient cell line), XRV15B cells (Ku80-deficient cell line), XRV79B cells (parental cell line of XRV15B), H2AX-deficient mouse embryo fibroblasts (9), and HCC1937 breast cancer cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. For laser irradiation, cells were plated onto 35-mm glass bottom dishes (Matsunami Glass, Osaka, Japan) at 50% confluence 24 h before transfection (Fugene-6; Roche) and irradiated with laser light under a microscope 48 h after transfection. Microscopy and laser light irradiation. Fluorescent images were obtained and processed with an FV-500 confocal scanning laser microscopy system (Olympus). A 405-nm scan laser system (Olympus) for irradiation of cells in the epifluorescence path of the microscope system was used. One scan of the laser light at full power delivers approximately 1,600 nW. We scanned cells 500 times with the 405-nm laser at full power, which has been shown to induce primarily DSBs (28). The 405-nm laser light was focused through a 40⫻ objective lens. Cells were incubated with Opti-medium (Gibco) in glass bottom dishes and placed in a temperature-controlled (37°C) chamber. The fluorescence intensity at an irradiated site was measured with a laser power/energy monitor (Orion; Ophir Optronics, Israel). The mean intensity of each site was obtained after subtraction of the background intensity in the irradiated cell. Each experiment was done at least

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three times, and we observed 10 or more cells to obtain one result. Standard deviations were derived from at least three independent experiments. Chemicals. 5-Bromo-2⬘-deoxyuridine (BrdU; Roche) at a final concentration of 10 ␮M was added to the medium 8 h before laser irradiation. For photosensitization of cells, RO-19-8022 (Roche) at a final concentration of 250 nM was added to the medium and cells were incubated at 37°C for 5 min. For 1,5dihydroxyisoquinoline (DIQ) treatment, cells were incubated with DIQ (Sigma) in the medium at a final concentration of 500 ␮M for 1 h before laser irradiation. Immunocytochemistry. Saos-2 cells were laser light irradiated and stained with antibodies against human ␥H2AX and BRCA1. The cells were fixed with methanol-acetone (1:1) for 10 min at ⫺20°C at 2, 5, 8, 10, 60, 300, or 600 min after laser irradiation. Fixed cells were washed three times with phosphate-buffered saline (PBS) containing 0.05% Tween 20 (PBS-T). After blocking with PBS containing 1.5% skim milk at room temperature for 30 min, cells were incubated with anti-phosphorylated H2AX (␥H2AX) (1:200 dilution; Upstate Biotechnology) and anti-BRCA1 (1:1,000 dilution) antibodies diluted in PBS containing 1% bovine serum albumin at 37°C for 1 h. After washing with PBS-T, cells were incubated with Alexa Fluor 594-conjugated goat anti-rabbit immunoglobulin G (IgG) (Molecular Probes) and fluorescein isothiocyanate-conjugated goat antimouse IgG (Molecular Probes) at a dilution of 1:400 in PBS–1% bovine serum albumin at 37°C for 30 min. Cells were washed with PBS-T and then mounted in mounting buffer. Immunoprecipitation and Western analysis. Saos-2 and HEK 293T cells were transfected with expression vectors. At 2 days posttransfection, cell lysates were prepared in 1 ml of wash buffer (10 mM HEPES [pH 7.6], 250 mM NaCl, 0.1% Nonidet P-40, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride). For immunoprecipitation, 2 to 3 ␮l of anti-FLAG M2 (Sigma), anti-GFP (Covance), antihemagglutinin (anti-HA) (HA.11; Covance), anti-Myc (9E10; Covance), or anti-Ku80 (Santa Cruz) monoclonal antibodies and 20 ␮l of protein G-Sepharose beads (GE Healthcare Bio-Sciences) were added to each lysate. Mixtures were incubated at 4°C overnight with rotation, the supernatant was removed, and protein beads were washed three times with 0.4 ml of wash buffer. For Western blot analysis, samples were subjected to electrophoresis in 5% or 5 to 20% sodium dodecyl sulfate-polyacrylamide gels and immunoblotted with the polyclonal antibody specific to BRCA1 residues 397 to 1080s anti-BRCA1 antibodies H-100 (Santa Cruz) and C-20 (Santa Cruz), and anti-HA, anti-Ku80, anti-FLAG, anti-GFP, anti-BARD1 (H-300; Santa Cruz), anti-Myc, or antiactin (A2066; Sigma-Aldrich) antibodies. GST pull-down assay. Glutathione S-transferase (GST) recombinant protein for GST pull-down assay was expressed in BL21(DE3) bacterial cells. The bacterial cell pellets were lysed with sonication buffer (20 mM Tris-acetate [pH 7.9], 120 mM K-acetate, 10% glycerol, 1% Triton X-100, 1 mM EDTA, 1 mM dithiothreitol). The lysate was clarified by centrifugation. Three hundred microliters of the resultant supernatant was incubated with 20 ␮l of glutathioneSepharose beads (Amersham Biosciences) at 4°C. The beads were washed three times with 0.4 ml of sonication buffer. For binding experiments, glutathioneSepharose beads bound to GST fusion protein were incubated for 4 h at 4°C with lysate of HEK 293T cells. HEK 293T cells were transfected with HA-tagged wild-type or P142H mutant BRCA1 and treated with 10 Gy of ionizing radiation. Cell lysates were prepared immediately after irradiation. The beads were washed three times with 0.4 ml of wash buffer. For Western blot analysis, samples were subjected to electrophoresis in 5% sodium dodecyl sulfate-polyacrylamide gels and immunoblotted with anti-HA and anti-Ku80 antibodies. Colony formation assay. HCC1937 cells were transfected with HA-tagged wild-type or P142H mutant BRCA1. Forty-eight hours after transfection, cells were replated. Eight hours after replating, cells were exposed to irradiation as indicated and incubated for 10 days. Colonies were fixed and stained with 0.3% crystal violet and then counted. Survival (number of colonies) was expressed as a percentage of the nonirradiated colonies.

RESULTS BRCA1 and ␥H2AX accumulate at laser-microirradiated sites with different kinetics. We recently reported that microirradiation of living cells by scanning 500 times with a 405-nm laser light through a confocal microscope can generate DSBs and that GFP-tagged BRCA1 visibly accumulates at laserirradiated sites (28). The present study first examined whether endogenous BRCA1 accumulates at the irradiated sites simi-

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FIG. 1. Accumulation of endogenous ␥H2AX and BRCA1 at laser-microirradiated sites. (A) Immunochemical detection of ␥H2AX and BRCA1 in Saos-2 cells after 500 scans of 405-nm laser irradiation. Cells were fixed at the indicated time points after laser irradiation and stained with antibodies against ␥H2AX and BRCA1. (B) Comparison of the fluorescence intensities of ␥H2AX and BRCA1 at DSB sites. Standard deviations were derived from at least three independent experiments.

larly to GFP-BRCA1. The localization of ␥H2AX was also examined. Human Saos-2 cells were fixed and processed for double immunofluorescence at 2, 5, 8, 10, 60, 300, or 600 min after DSBs were introduced into the nuclei of the cells by laser microirradiation (Fig. 1A). ␥H2AX was detected as bright fluorescence along the line of irradiation as early as 2 min after irradiation and reached its maximum level at 10 min. The accumulation of ␥H2AX persisted for up to 60 min but had decreased by 300 min and was not observed at 600 min. In contrast, the accumulation of BRCA1 along the line of irradiation was rather gradual and was not observed at 2 min. The fluorescent lines of BRCA1 were detected at 5 min and reached maximum intensity at 60 min. In striking contrast to the accumulation of ␥H2AX, the accumulation of BRCA1 persisted for at least 600 min. The time course of accumulation of fluorescence intensity for each protein is shown in Fig. 1B. These quantitative analyses confirmed the qualitative description of the kinetics of accumulation of the proteins along the line of irradiation. Thus, both the accumulation and clearance of BRCA1 were slower than those of ␥H2AX, suggesting that

distinct mechanisms mediate the mobilization of these two proteins. We next examined the real-time localization of GFP-tagged BRCA1 in living cells after laser irradiation. GFP-BRCA1 was transfected into Saos-2 cells, and the cells were then laser irradiated. As shown in Fig. 2A (control), GFP-BRCA1 clearly accumulated at the irradiated sites. The mean intensity of GFP-BRCA1 at the accumulation site was quantified, and the kinetics after irradiation are shown in Fig. 2B (control). Accumulation of BRCA1 at the irradiated sites was gradual for 10 min. Furthermore, we analyzed the accumulation kinetics of GFP-tagged BRCA1 over a longer time course in Saos-2 cells (see Fig. S1A in the supplemental material). The fluorescence intensity of GFP-BRCA1 at the irradiated sites reached a maximum at 60 min and then decreased but persisted for 600 min, similarly to endogenous BRCA1. The results indicate that the accumulation kinetics of overexpressed GFP-BRCA1 mimics that of endogenous BRCA1. BRCA1 accumulation at the laser-irradiated site is a response to DNA DSBs. Laser irradiation with a very low dose of UVA radiation might damage DNA through the production of

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FIG. 2. GFP-tagged BRCA1 accumulates at DSB sites. (A) Accumulation of GFP-tagged full-length BRCA1 in Saos-2 cells in the presence or absence of BrdU. Arrows indicate sites of irradiation. (B and C) Kinetics of GFP-tagged full-length BRCA1 accumulation after laser irradiation in Saos-2 cells incubated in the presence or absence of BrdU (B) or RO-19-8022 (C).

reactive oxygen species, which cause DSBs, SSBs, and oxidative base damage in DNA. We previously demonstrated by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay that our laser light microirradiation system induced DNA strand breaks in cells (23). Here, we examined whether the BRCA1 accumulation observed under our experimental conditions targets DSBs. BrdU-incorporated DNA is sensitive to laser irradiation and prone to DSBs (14, 31, 53). Cells were preincubated in the presence of BrdU and then scanned 500 times with a 405-nm laser light. The BrdU treatment significantly increased the mean fluorescence intensity of GFP-BRCA1 at the accumulation sites, compared to the non-BrdU treatment (BrdU in Fig. 2A and B). A photosensitizer, RO-19-8022, exacerbates laserinduced oxidative base damage. Treatment of cells with RO19-8022 significantly enhanced the fluorescent intensity of GFP-tagged OGG1, which is a DNA glycosylase for repair of oxidative base damage, at laser-irradiated sites (see Fig. S1B in the supplemental material). On the other hand, treatment of cells with this agent did not cause any significant change in BRCA1 accumulation (Fig. 2C). Poly (ADP-ribose) polymerase1 (PARP1) and PARP2 are known to play key roles in SSB repair. Similar patterns of BRCA1 accumulation were observed regardless of the presence or absence of DIQ, which inhibits PARPs and their SSB repair activity, suggesting that the accumulation is not involved in PARP-dependent repair pathways (see Fig. S1C in the supplemental material). Taken together, the above findings suggest that the accumulation of

GFP-tagged BRCA1 at laser-irradiated sites observed here reflects a response to DNA DSBs. The number of X-ray-induced DSBs in human cells can be estimated by counting the nuclear foci detected by ␥H2AX staining (40). We compared the number of foci induced by X-ray exposure with the number of foci induced by laser irradiation in our system and deduced that the 500-scan irradiation used throughout our experiments generates approximately 100 DSBs per cell (data not shown). BRCA1 localizes at DSBs via its N- and C-terminal regions. We observed a real-time accumulation of GFP-BRCA1 at laser-induced DSBs. We next identified the regions in BRCA1 that mediate this accumulation. Several GFP-tagged deletion mutant forms of BRCA1 were constructed (Fig. 3A). Fulllength BRCA1 and the ⌬305-770, ⌬775-1292, ⌬1-302, and ⌬1527-1863 deletion mutant forms were transfected into Saos-2 cells. Protein expression was confirmed by Western blot assays with antibodies against BRCA1 (Fig. 3B). Figure 3C shows the GFP fluorescence of living cells before and after the introduction of DSBs. Because the RING domain is located in aa 1 to 302 and the BRCT domains are located in aa 1527 to 1863, we hypothesized that deletion of either of these two regions would suppress BRCA1 accumulation at laser-irradiated sites. However, each of the four deletion mutant BRCA1 proteins accumulated at DSBs. Therefore, the aa 303 to 1526 BRCA1 fragment was constructed, which contained only the central region of BRCA1 and lacked both the N- and Cterminal regions. This BRCA1 fragment did not accumulate at

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FIG. 3. Accumulation of GFP-tagged deletion mutant forms of BRCA1 at DSBs. (A) Diagram of GFP-tagged full-length and deletion mutant forms of BRCA1 and their accumulation at DSBs showing the locations of the GFP tag. (B) Expression of GFP-tagged full-length and deletion mutant forms of BRCA1 in HEK 293T cells detected by Western blot assay. Lanes 2 to 7, GFP-tagged full-length and mutant forms of BRCA1.

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the irradiated sites. When cells into which the aa 303 to 1526 fragment had been introduced were laser irradiated, fixed, and immunostained, ␥H2AX was clearly detected at the irradiated sites (Fig. 3D). These data suggest that either the N- or the C-terminal region alone is sufficient for BRCA1 accumulation at DSBs. Subsequently, aa 1 to 304 and aa 1528 to 1863 fragments were constructed (Fig. 3A and B) and each fragment was shown to accumulate at laser-induced linear DSBs (Fig. 3C). Thus, each of the N- and C-terminal BRCA1 fragments possessed independent potential to accumulate at laser-irradiated sites. In line with this notion, we confirmed the interaction of each fragment with BARD1 and RAP80, respectively (see Fig. S2 in the supplemental material). The mean intensity of GFP-BRCA1 at the accumulation site was quantified, and the kinetics of accumulation of full-length BRCA1 and the aa 1 to 304, aa 303 to 1526, and aa 1528 to 1863 fragments were examined for 600 s after irradiation (Fig. 3E). Interestingly, the N-terminal (aa 1 to 304) fragment rapidly and maximally accumulated at the DSBs within 20 s, although endogenous BRCA1 accumulated at irradiated sites 5 min after irradiation (Fig. 1). We confirmed that this very fast accumulation of the N terminus indeed targeted DSBs by pretreatment of cells with BrdU as in Fig. 2B (see Fig. S3 in the supplemental material). On the other hand, the C-terminal (aa 1528 to 1863) fragment slowly and gradually accumulated, reaching a plateau at 360 s (Fig. 3E). Thus, the observed differential kinetics suggest that the N- and C-terminal regions of BRCA1 may accumulate at DSBs through different mechanisms. The full-length BRCA1 protein that accumulated appeared to consist of two populations. One was rapidly recruited within 20 s, whereas the other accumulated gradually. Although rapid accumulation of BRCA1 may be contributed by a partial population of whole BRCA1 molecules, its accumulation was as fast as that of the N terminus. Thus, the kinetics of full-length BRCA1 likely reflects a combination of rapid accumulation via its N terminus and gradual accumulation via its C terminus. Presumably, endogenous BRCA1 was not detected by immunostaining, since it was under the detection limit within 5 min after irradiation. Next, we compared the clearance kinetics of the N- and C-terminal fragments of BRCA1 from DSBs (Fig. 3F and quantification in panel G). The accumulation of the N-terminal fragment along the line of irradiation was detected 10 min after laser irradiation but was almost completely lost by 2 h after irradiation. In contrast, the fluorescence of the C-terminal fragment at DSBs was present at 2 h after irradiation. These results indicate that the C-terminal region is unique in

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its ability to remain at the DSBs and that BRCA1 displays domain-specific kinetics in terms of its initial accumulation and subsequent retention at the damaged sites. Accumulation of the N-terminal region of BRCA1 at DSBs requires the presence of Ku80. Next, we examined whether BRCA1 accumulation at DSBs is dependent on specific DNA repair factors, with factor-deficient cells. BRCA1 is phosphorylated by ataxia-telangiectasia mutated (ATM) kinase after ionizing radiation (12, 15, 18). GFP-tagged BRCA1 N- and C-terminal fragments were transfected into human ATM-deficient AT1KY/T-n cells. The N- and C-terminal fragments accumulated at laser-induced DSBs in these cells (Fig. 4A). Thus, phosphorylation of BRCA1 by ATM kinase is not required for BRCA1 accumulation at irradiated sites. In H2AX⫺/⫺ mouse embryo fibroblast cells, both N- and C-terminal fragments of BRCA1 accumulated at DSBs. Thus, accumulation of BRCA1 is not likely to be dependent on H2AX, even though ␥H2AX was detected at the irradiated sites before BRCA1 (Fig. 1). Similarly, in CHO-derived CHO9, XR-C1 (DNA-PKcs⫺/⫺), and XR-1 (XRCC4⫺/⫺) cells, the N- and C-terminal fragments of BRCA1 accumulated at laser-irradiated sites (Fig. 4A). However, in XRV15B (Ku80⫺/⫺) cells, the N-terminal fragment of BRCA1 failed to accumulate at DSB sites, whereas the C-terminal fragment did accumulate. In the XRV79B cell line, which is Ku80 proficient and is the parental cell line of XRV15B, the N terminus of BRCA1 accumulated at DSBs (see Fig. S4 in the supplemental material). Therefore, accumulation of the N-terminal fragment, but not the C-terminal fragment, of BRCA1 at DSBs is Ku80 dependent. Next, we examined the localization of Ku80 itself in Saos-2 cells. As shown in Fig. 4B, GFP-Ku80 accumulated at laserinduced DSBs. To confirm that the accumulation of the N terminus of BRCA1 at DSBs is dependent on the presence of the Ku80 protein, we examined whether the GFP-tagged N terminus of BRCA1 accumulates at laser-irradiated sites in XRV15B cells reconstituted with HA-tagged Ku80. XRV15B cells were transfected with vectors to express the GFP-tagged N terminus of BRCA1 and HA-tagged Ku80 or the control vector. Ten minutes after laser irradiation, cells were fixed and stained with anti-HA. As shown in Fig. 4C, the N terminus of BRCA1 accumulated at laser-irradiated sites in cells that express the Ku80 protein but not in cells transfected with the control vector. Taken together, the results show that the N terminus of BRCA1 accumulates at DSBs dependent on the presence of the Ku80 protein. The N-terminal region of BRCA1 is associated with Ku80. BRCA1 functions in the NHEJ pathway, as well as the HR

Lanes 1 to 7 were probed with a polyclonal antibody specific for BRCA1 residues 397 to 1080. Lanes 8 and 9 were probed with BRCA1-H100, an antibody specific for the N terminus of BRCA1. Lanes 10 and 11 were probed with BRCA1-C20, an antibody specific for the C terminus of BRCA1. (C) Accumulation of GFP-tagged full-length and mutant forms of BRCA1 in Saos-2 cells. Arrows indicate sites of irradiation. (D) Immunostaining with ␥H2AX of Saos-2 cells expressing the GFP-tagged aa 303 to 1526 fragment of BRCA1 after laser irradiation. Saos-2 cells were transfected with GFP-tagged BRCA1 aa 303-1526. Two days after transfection, cells were laser irradiated and then fixed. Cells were immunostained with anti-␥H2AX antibody. (E) Kinetics of accumulation of full-length BRCA1 and the aa 1 to 304, aa 303 to 1526, and aa 1527 to 1863 fragments after laser irradiation in Saos-2 cells. Standard deviations were derived from at least three independent experiments. (F) Accumulation of the N terminus and the C terminus of BRCA1 at DSBs 10 min or 2 h after laser irradiation of Saos-2 cells. Arrows indicate sites of irradiation. (G) Mean fluorescence intensity of the GFP-tagged N terminus or C terminus of BRCA1 at laser-irradiated sites 10 min and 2 h after irradiation. Standard deviations were derived from at least three independent experiments.

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FIG. 4. Accumulation of BRCA1 at DSBs in cell lines defective for DNA repair proteins. (A) Accumulation of the GFP-tagged N terminus or C terminus of BRCA1 at DSBs in cell lines deficient in ATM, H2AX, Ku80, DNA-PKcs, or XRCC4 and in CHO9 cells. Arrows indicate sites of irradiation. (B) Accumulation of GFP-tagged Ku80 at DSBs in Saos-2 cells. Arrows indicate sites of irradiation. (C) Accumulation of the GFP-tagged N terminus of BRCA1 and HA-tagged Ku80 at DSBs in Ku80-deficient XRV15B cells reconstituted with HA-tagged Ku80. XRV15B cells were transfected with the GFP-tagged N terminus of BRCA1 and the vector to express HA-tagged Ku80 or the control vector. Ten minutes after laser irradiation, cells were fixed and stained with anti-HA antibody. Arrows indicate sites of irradiation. (D) Endogenous BRCA1 is associated with endogenous Ku80. HEK 293T cells were treated with 10 Gy of ionizing radiation. Lysates were immunoprecipitated (IP) with a control irrelevant IgG (lane 1) or anti-Ku80 (lane 2) antibody, and Western blots (WB) were probed with anti-BRCA1 or anti-Ku80 antibody. Input samples were analyzed as indicated. (E) BRCA1 with the N terminus deleted does not associate with Ku80. HEK 293T cells were transfected with plasmids for the expression of HA-tagged full-length BRCA1 (lanes 2, 4, and 7), HA-tagged ⌬1-302 mutant BRCA1 (lane 5), HA-tagged ⌬1527-1863 mutant BRCA1 (lane 6), and FLAG-tagged Ku80 (lanes 3 to 7) and treated with 10 Gy of ionizing radiation (IR) (lanes 1 to 6). Lysates were immunoprecipitated with anti-FLAG antibody (middle panel), and immunoblots were probed with anti-HA antibody. Input samples were analyzed in the top and bottom panels. (F) BRCA1 associates with Ku80 via its N terminus. HEK 293T cells were transfected with plasmids to express HA-tagged aa 1 to 304 of mutant BRCA1 (lane 1), HA-tagged aa 1528 to 1863 of mutant BRCA1 (lane 2), and FLAG-tagged Ku80 (lanes 1 and 2) and treated with 10 Gy of ionizing radiation. Lysates were immunoprecipitated with anti-FLAG antibody, and immunoblots were probed with anti-HA antibody. Input samples were analyzed as indicated.

pathway, and yet interaction of BRCA1 with proteins involved in NHEJ has not been reported. To examine directly whether BRCA1 interacts with Ku80, we performed coimmunoprecipitation assays. HEK 293T cells were treated with 10 Gy of ionizing radiation, and 2 h after irradiation, cell lysates were immunoprecipitated with control IgG or anti-Ku80 antibody.

As shown in Fig. 4D, endogenous BRCA1 coimmunoprecipitated with endogenous Ku80. Next, HEK 293T cells were cotransfected with HA-tagged wild-type BRCA1 and FLAGtagged Ku80. After exposure to ionizing radiation, cell extracts were prepared and immunoprecipitated with an anti-FLAG antibody and the precipitates were probed with anti-HA anti-

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FIG. 5. Accumulation of GFP-tagged aa 1 to 100, 1 to 200, 1 to 300, 101 to 200, 101 to 304, and 201 to 304 fragments of BRCA1 at DSBs. (A) Diagram of GFP-tagged BRCA1 fragments and results of accumulation at laser-irradiated sites. (B) Accumulation of GFP-tagged BRCA1 fragments at DSBs in Saos-2 cells. Arrows indicate sites of irradiation. (C) Kinetics of GFP-tagged BRCA1 fragment accumulation after laser irradiation. Standard deviations were derived from at least three independent experiments. (D) GFP-tagged BRCA1 aa 1 to 100 and 101 to 200 fragments associate with Ku80. HEK 293T cells were transfected with vectors for the expression of GFP-tagged BRCA1 aa 1 to 100 (lanes 1 and 4), GFP-tagged BRCA1 aa 101 to 200 (lanes 2 and 5), or GFP-tagged BRCA1 aa 201 to 304 (lanes 3 and 6) and FLAG-tagged Ku80 (lanes 4 to 6) and treated with 10 Gy of ionizing radiation (IR). Lysates were immunoprecipitated (IP) with anti-FLAG antibody (middle and bottom panels), and Western blots (WB) were probed as indicated. Input samples were analyzed in the top panels. (E) Accumulation of GFP-tagged BRCA1 aa 1 to 100 and 101 to 200 fragments at DSBs in parental cells and Ku80⫺/⫺ cells. Arrows indicate sites of irradiation.

body. A substantial amount of BRCA1 coprecipitated with Ku80 from the irradiated cells, whereas only a small amount of BRCA1 coprecipitated with Ku80 from cells that were not irradiated (Fig. 4E, compare lanes 4 and 7), suggesting that the BRCA1 association with Ku80 is significantly enhanced by irradiation. To identify the region of BRCA1 responsible for the Ku80 interaction, N- and C-terminal deletion mutant constructs were transfected into HEK 293T cells. The C-terminal deletion mutant (Fig. 4E, lane 6), but not the N-terminal deletion mutant (Fig. 4E, lane 5), coprecipitated with Ku80. When the N- and C-terminal fragments alone were transfected into HEK 293T cells, the N-terminal fragment of BRCA1 associated with Ku80 whereas the C-terminal fragment did not (Fig. 4F). These results indicate that the N-terminal region of BRCA1 is responsible for the interaction with Ku80. To examine whether BRCA1 directly binds with the Ku80 protein, we purified the GST-tagged Ku80 protein and the His-tagged N-terminal part of the BRCA1 protein and analyzed the direct

interaction of these proteins. However, in our experiment, we could not detect direct binding of the N-terminal region of BRCA1 with the Ku80 protein (data not shown). The aa 1 to 100 and aa 101 to 200 regions of BRCA1 accumulate at DSBs and interact with Ku80 independently. We next investigated which motifs in the N terminus of BRCA1 mediate Ku80-dependent accumulation at DSBs. To examine the role of the RING domain, which is located at aa 24 to 64, we constructed several GFP-tagged fragments of BRCA1 including aa 1 to 100, 1 to 200, 1 to 304, 101 to 200, 101 to 304, and 201 to 304 (Fig. 5A). As shown in Fig. 5B, the aa 1 to 100, 1 to 200, and 1 to 304 fragments, each of which contains the RING domain, accumulated at laser-induced DSBs. However, the aa 101 to 200 and 101 to 304 fragments, which lack the RING domain, also accumulated at DSBs. The aa 201 to 304 fragment was the only one that did not accumulate at DSBs. These results indicate that, in addition to the aa 1 to 100 region, where the RING domain is located, the aa 101 to 200

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region alone can also accumulate at DSBs. The accumulation kinetics were quantified for each of these N-terminal fragments of BRCA1 (Fig. 5C). Interestingly, all of the fragments, with the exception of the aa 201 to 304 fragment, showed similar kinetics, with a rapid (within 20 s) accumulation at DSBs after laser irradiation. These data suggest that the aa 1 to 100 and 100 to 200 regions of BRCA1 use similar mechanisms to accumulate at DSBs. As the N-terminal aa 1 to 302 region of BRCA1 interacted with Ku80 and accumulated at DSBs in a Ku80-dependent manner, we tried to further identify the specific region of the fragment responsible for the interaction with Ku80. HEK 293T cells were cotransfected with the GFP-tagged aa 1 to 100, 101 to 200, or 201 to 304 fragment of BRCA1 together with FLAGKu80 and irradiated. Cell extracts were prepared, and antiFLAG immunoprecipitates were probed with anti-GFP (Fig. 5D). Interestingly, the aa 1 to 100 and aa 101 to 200 fragments, but not the aa 201 to 304 fragment, coimmunoprecipitated with Ku80. Localization of the GFP-tagged aa 1 to 100 and aa 101 to 200 fragments in Ku80-deficient cells was also examined. As shown in Fig. 5E, neither the aa 1 to 100 nor the aa 101 to 200 fragment accumulated at DSBs in Ku80-deficient XRV15B cells, but they did accumulate at DSBs in Ku80-proficient XRV79B cells. These data indicate that the aa 1 to 100 and aa 101 to 200 regions alone are each able to interact with Ku80 and accumulate at irradiated sites in a Ku80-dependent manner. Missense mutations in aa 101 to 200 change the accumulation kinetics of BRCA1 at DSBs and abolished the association with Ku80. Although the aa 101 to 200 fragment of BRCA1 alone accumulated at laser-irradiated sites, there do not appear to be any known domains or motifs in this region responsible for its accumulation. Thus, we examined whether missense mutations would affect the accumulation of BRCA1 at DSBs. Four missense mutations, Y105C, P142H, E143K, and Y179C, were selected on the basis of their relatively frequent occurrence in the Breast Cancer Information Core database (www.nhgri.nih.gov). However, the pathological significance of these mutations has not been verified. GFP-BRCA1 constructs (aa 101 to 200), each harboring one of the missense mutations, were generated. Each of the four mutants accumulated at the irradiated sites, but the fluorescence intensity, especially that of P142H, was markedly diminished relative to that of the wild-type BRCA1 fragments (Fig. 6A). All mutant constructs were expressed at levels similar to the wild-type BRCA1 fragments, as confirmed by Western blot analysis (see Fig. S5A in the supplemental material). Next, we analyzed the accumulation kinetics of GFP-tagged full-length BRCA1 containing these missense mutations. As shown in Fig. 6B, the four mutants of BRCA1 accumulated at the irradiated sites, but the accumulation kinetics of these mutants were distinct from those of wild-type BRCA1. These mutants did not show the rapid accumulation shown by wildtype BRCA1. The patterns of accumulation of these mutants were similar to that of the C terminus of BRCA1 (Fig. 3E). Especially the P142H mutant, which showed the most significant reduction in accumulation as analyzed with the aa 101 to 200 fragment, completely lost rapid accumulation and accumulated slowly at laser-irradiated sites. These results also suggest

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that the N-terminal fragment of BRCA1 is responsible for the rapid accumulation of BRCA1. Furthermore, we tested whether the P142H mutant is associated with the Ku80 protein. HEK 293T cells were cotransfected with the GFP-tagged aa 101 to 200 fragment of wildtype or P142⌯ mutant BRCA1 together with FLAG-Ku80. After exposure to ionizing irradiation, cell lysates were immediately prepared and anti-FLAG immunoprecipitates were probed with anti-GFP and anti-FLAG antibodies. As shown in Fig. 6C, the aa 101 to 200 fragment of P142⌯ mutant BRCA1 was not associated with the Ku80 protein. Next, we examined the association of HA-tagged full-length P142H mutant BRCA1 with the FLAG-Ku80 protein. The association of the P142⌯ mutant protein with Ku80 significantly decreased compared to that of wild-type BRCA1. We observed the weak association of the full-length P142H mutant with Ku80. This is consistent with the finding that the aa 1 to 100 fragment of BRCA1 was also associated with Ku80 (Fig. 5D). When cell lysates were prepared 30 min and 2 h after irradiation, we could not detect a clear difference in association between the wild-type and mutant proteins (data not shown). Next, we examined whether this mutation of BRCA1 affects the interaction with the Ku80 protein by GST pull-down assay. GST or a GST-Ku80 fusion protein was incubated with lysates of HEK 293T cells, which were transfected with HA-tagged full-length wild-type or P142H mutant BRCA1 and treated with ionizing radiation. As shown in Fig. 6E, the GST-Ku80 fusion protein specifically bound wild-type but not P142H mutant BRCA1. HCC1937 is a BRCA1-deficient breast cancer cell line that is sensitive to DNA damage. Exogenous expression of BRCA1 restores resistance to DNA damage in these cells (30, 43, 57). We compared P142H mutant BRCA1 with wild-type BRCA1 for the ability to confer resistance to ionizing irradiation. HCC1937 cells were transfected with HA-tagged wild-type or P142H mutant BRCA1, and the expression of HA-BRCA1 was confirmed by Western blot assay (see Fig. S5B in the supplemental material). Cells were irradiated with various doses of irradiation, and their ability to form colonies was assessed (Fig. 6F). Under conditions of wild-type BRCA1 expression, HCC1937 cells were more resistant to irradiation. In contrast, expression of P142H mutant BRCA1 did not appear to affect sensitivity to irradiation. These results suggest that BRCA1Ku80 association is involved in the repair following ionizing irradiation and mediates the resistance to DNA damage induced by irradiation. Therefore, the aa 101 to 200 fragment indeed is a functional protein interaction domain and especially the P142H mutation of BRCA1 is likely to be a pathological mutation. The accumulation kinetics of the N-terminal region of BRCA1 at DSBs differs from that of BARD1. Since the accumulation of the aa 1 to 304 fragment of BRCA1 at DSBs required Ku80 and GFP-Ku80 accumulated at laser-induced DSBs (Fig. 4 and 5), we analyzed the accumulation kinetics of Ku80. Ku80 was rapidly recruited to DSBs after irradiation, with kinetics similar to those of the aa 1 to 304 fragment of BRCA1 (Fig. 7A). BARD1 colocalizes with BRCA1 at irradiation-induced nuclear foci (1, 21, 41) and also accumulates at UV laser-induced DSBs (19). Thus, we examined the localization of BARD1 in

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FIG. 6. Accumulation at DSBs and association with Ku80 of missense mutant forms of BRCA1. (A) Comparison of the accumulation kinetics of GFP-tagged BRCA1 fragments containing missense mutations in Saos-2 cells. Standard deviations were derived from at least three independent experiments. (B) Comparison of the accumulation kinetics of GFP-tagged full-length BRCA1 containing missense mutations in Saos-2 cells. Standard deviations were derived from at least three independent experiments. (C) Association of the aa 101 to 200 BRCA1 fragment containing a P142H mutation with Ku80. HEK 293T cells were transfected with plasmids for the expression of GFP-tagged wild-type (WT) BRCA1 aa 101 to 200 (lanes 1 and 2) or GFP-tagged P142⌯ mutant BRCA1 aa 101 to 200 (lane 3) and FLAG-tagged Ku80 (lanes 1 to 3) and treated with 10 Gy of ionizing radiation (IR) (lanes 1 to 3). Lysates were immunoprecipitated (IP) with anti-FLAG antibody, and Western blots (WB) were probed with anti-GFP and anti-FLAG antibodies. Input samples were analyzed as indicated. (D) Association of full-length BRCA1 containing a P142H mutation with Ku80. Full-length P142H mutant BRCA1 does not associate with Ku80. HEK 293T cells were transfected with vectors for the expression of HA-tagged wild-type BRCA1 (lanes 1 and 2) or HA-tagged P142⌯ mutant BRCA1 (lane 3) and FLAG-tagged Ku80 (lanes 1 to 3) and treated with 10 Gy of ionizing radiation (lanes 1 to 3). Lysates were immunoprecipitated with anti-FLAG antibody, and immunoblots were probed with anti-HA and anti-FLAG antibodies. Input samples were analyzed as indicated. (E) Association of full-length BRCA1 containing a P142H mutation with GST-tagged Ku80. The P142H mutant protein does not associate with the GST-tagged Ku80 protein. Glutathione-Sepharose beads bound to GST or GST-Ku80 protein were incubated with lysates of HEK 293T cells that were transfected with HA-tagged wild-type or P142H mutant BRCA1 and irradiated. The bound proteins were analyzed by immunoblotting with anti-HA and anti-Ku80 antibodies. (F) Colony formation assay of HCC1937 cells expressing HA-tagged wild-type and P142H mutant BRCA1. HCC1937 cells were transfected with vectors for the control or HA-tagged wild-type or P142H mutant BRCA1. Cells were replated 48 h after transfection, and then 8 h later, cells were exposed to X-ray irradiation as indicated. Ten days after irradiation, colonies were counted. Error bars represent standard error of triplicate measurement.

our experimental system. As shown in Fig. 7B, GFP-BARD1 accumulated at laser-irradiated sites in Saos-2 cells. The accumulation kinetics of BARD1 was, however, quite distinct from that of the aa 1 to 304 fragment of BRCA1 (Fig. 7A). The fluorescence intensity of the aa 1 to 304 BRCA1 fragment reached a maximum level within 40 s, whereas the fluorescence intensity of BARD1 was only one-third of its maximum level

after 2 min. The distinct kinetics of BRCA1 and BARD1 in the early phase of accumulation were also confirmed by comparing full-length BRCA1 and BARD1 (Fig. 7C). These results suggest that the rapid accumulation of BRCA1 is independent of BARD1. BRCA1-Ku80 and BRCA1-BARD1 interactions are independent of each other. In the N terminus of BRCA1, the aa 1 to

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FIG. 7. Accumulation of BARD1 at DSBs. (A) Comparison of the accumulation kinetics of GFP-tagged Ku80, BARD1, and the N terminus of BRCA1 (aa 1 to 304) after laser irradiation at DSBs. Standard deviations were derived from at least three independent experiments. (B) Accumulation of GFP-tagged BARD1 and at DSBs in Saos-2 cells. Arrows indicate sites of irradiation. (C) Comparison of the accumulation kinetics of GFP-tagged BARD1 and full-length BRCA1 after laser irradiation at DSBs. Standard deviations were derived from at least three independent experiments.

109 region is responsible for binding to BARD1 (36). Whether the aa 101 to 200 fragment of BRCA1 contributes to the BARD1 interaction is unknown. The GFP-tagged aa 1 to 100, 1 to 200, and 101 to 200 fragments of BRCA1 were cotransfected with FLAG-BARD1 into HEK 293T cells. Cell lysates were immunoprecipitated with anti-GFP antibody, and the precipitates were probed with anti-BARD1 antibody. As shown in Fig. 8A, regardless of cell irradiation, BARD1 did not coimmunoprecipitate with the aa 101 to 200 fragment (lanes 4 and 7) whereas BARD1 did coprecipitate with the aa 1 to 100 and 1 to 200 fragments (positive controls; lanes 2, 3, 5, and 6). These results suggest that the rapid accumulation of BRCA1 at DSBs is not dependent on the BARD1 interaction. HA-tagged wild-type or C61G mutant BRCA1, in which binding to BARD1 is abolished, was cotransfected with FLAGKu80 into HEK 293T cells. Two hours after irradiation, cell extracts were prepared and the immunoprecipitates obtained with anti-FLAG antibody were probed with anti-HA antibody. As shown in Fig. 8B, not only wild-type BRCA1 but also C61G mutant BRCA1 coprecipitated with Ku80 (lanes 3 and 4). These results indicate that the BRCA1-Ku80 interaction is independent of the BRCA1-BARD1 interaction. Based on the above observations, we hypothesized that a putative BRCA1-Ku80 complex would be distinct from a BRCA1-BARD1 complex. To examine this possibility, we overexpressed HA-BRCA1, Myc-BARD1, and FLAG-Ku80 in HEK 293T cells, irradiated the cells, and examined the molecular interactions by coimmunoprecipitation. As shown in Fig. 8C, when the extract was immunoprecipitated with anti-HA antibody, both BARD1 and Ku80 coprecipitated with BRCA1 (lanes 1 and 2). When anti-FLAG antibody was used, BRCA1, but not BARD1, coprecipitated with Ku80 (lanes 3 and 4). Immunoprecipitation with anti-Myc antibody revealed that

BRCA1, but not Ku80, coprecipitated with BARD1 (lanes 5 and 6). Expression of HA-BRCA1 was lower in the absence of Myc-BARD1. This result is consistent with the previous report that BRCA1 and BARD1 stabilize each other (20, 52). Finally, the BRCA1-BARD1 and presumptive BRCA1Ku80 interactions were examined for endogenously expressed proteins. HEK 293T cells were treated with ionizing radiation, and lysates were immunoprecipitated with control IgG (lanes 2, 4, and 6), anti-BRCA1 (lane 3), anti-BARD1 (lane 5), or anti-Ku80 (lane 7, Fig. 8D) antibody. BRCA1 was detected in the anti-Ku80 and anti-BARD1 precipitates. On the other hand, the BRCA1/Ku80 precipitates did not contain BARD1 and the BRCA1/BARD1 precipitates did not contain Ku80. Thus, the BRCA1/BARD1-containing complex is distinct from the BRCA1/Ku80-containing complex. Taken together, these data suggest that Ku80, and not BARD1, is involved in the rapid accumulation of the N-terminal region of BRCA1 at laser-induced DSBs.

DISCUSSION In this study, we demonstrated that both the N- and Cterminal regions of BRCA1 are recruited at DSBs induced by laser irradiation. The N terminus of BRCA1 accumulated at laser-irradiated sites immediately and transiently after laser irradiation, whereas the C-terminal region accumulated slowly and remained associated with the irradiated sites for longer times (Fig. 3E, G, and H). We propose that the N-terminal region may function to guide BRCA1 to DSBs in the early phase of the repair process and that the C-terminal region may be responsible for its accumulation in the later phase of repair and for its sustained retention.

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FIG. 8. BRCA1-Ku80 and BRCA1-BARD1 interactions. (A) GFP-tagged BRCA1 aa 1 to 100 and 1 to 200 fragments associate with BARD1. HEK 293T cells were transfected with plasmids for the expression of GFP-tagged BRCA1 aa 1 to 100 (lanes 2 and 6), GFP-tagged BRCA1 aa 1 to 200 (lanes 3 and 6), or GFP-tagged BRCA1 aa 101 to 200 (lanes 4 and 7) and FLAG-tagged BARD1 (lanes 1 to 7). Lysates were immunoprecipitated (IP) with anti-GFP antibody, and immunoblots were probed as indicated. Input samples were analyzed by probing Western blots (WB) with anti-BARD1 antibody. Arrowheads indicate GFP and GFP-tagged BRCA1 fragments. IR, ionizing radiation. (B) HA-tagged missense mutant BRCA1 associates with Ku80. HEK 293T cells were transfected with vectors for the expression of full-length HA-tagged wild-type BRCA1 (lanes 1 and 3), full-length HA-tagged C61G mutant BRCA1 (lanes 2 and 4), and FLAG-tagged Ku80 (lanes 3 and 4) and treated with 10 Gy of ionizing radiation. Lysates were immunoprecipitated with anti-FLAG antibody (middle and bottom panels), and immunoblots were probed as indicated. Input samples were analyzed in the top panels. (C) The BRCA1-BARD1 and BRCA1-Ku80 interactions are independent. HEK 293T cells were transfected with vectors for the expression of HA-tagged BRCA1 (lanes 2 to 6), FLAG-tagged Ku80 (lanes 1, 2, and 4 to 6), and Myc-BARD1 (lanes 1 to 4 and 6) and treated with 10 Gy of ionizing radiation. Lysates were immunoprecipitated with the indicated antibodies, and immunoblots were probed as indicated. Input samples were analyzed as indicated. (D) Endogenous BRCA1-BARD1 and BRCA1-Ku80 interactions. HEK 293T cells were treated with 10 Gy of ionizing radiation. Lysates were immunoprecipitated with control irrelevant IgG (lanes 2, 4, and 6), anti-BRCA1 (lane 3), anti-BARD1 (lane 5), or anti-Ku80 (lane 7) antibody. The input sample was analyzed in lane 1. Immunoblots were probed with anti-BRCA1, anti-BARD1, or anti-Ku80 antibody.

Au et al. have reported that the N and C termini of BRCA1 cooperate in the localization of BRCA1 at irradiation-induced nuclear foci and that the N terminus of BRCA1 itself did not form nuclear foci (1). Greenberg et al. suggested that the accumulation of BRCA1 at laser-irradiated sites and its localization at nuclear foci after ionizing irradiation reflect different phenomena (19). The accumulation of BRCA1 at laser-irradiated sites was observed within several minutes after laser irradiation, demonstrating the acute response to laser-induced DSBs. In contrast, the localization of BRCA1 at the nuclear foci was found hours after exposing cells to ionizing irradiation. We found that the N terminus of BRCA1 could accumulate at laser-induced DSBs. This is probably because the N terminus of BRCA1 remains at DSBs only transiently and its fluorescence is not detected any longer when nuclear foci are formed. Furthermore, we also observed BRCA1 accumulation at laser-irradiated sites, even in H2AX⫺/⫺ cells, consistent with the previous report that BRCA1 accumulates at laser-induced DSBs in H2AX⫺/⫺ cells (9). In contrast, BRCA1 does not localize at nuclear foci after irradiation in the same H2AX⫺/⫺ cells (4, 10). Interestingly, in H2AX⫺/⫺ cells, 53BP1 localized

at irradiation-induced nuclear foci and laser-irradiated sites in the early phase but not in the late phase of repair (53BP1 accumulated in both the early and late repair phases in H2AX⫹/⫹ cells) (9). It might be that the accumulation of BRCA1 at DSBs is also transient in the absence of H2AX. H2AX could be involved in the retention of BRCA1, analogous to H2AX-mediated 53BP1 retention in the late phase. The N terminus of BRCA1 was associated with Ku80 and rapidly accumulated at DSBs in a Ku80-dependent manner (Fig. 4). We have shown for the first time that BRCA1 is associated with NHEJ factor. We could not detect direct interaction of BRCA1 with Ku80 protein in our experiment in vitro, suggesting that the direct binding of BRCA1 with Ku80 requires some protein modification or the presence of some other factors. Similar to the N terminus of BRCA1, Ku80 accumulated at DSBs rapidly after laser irradiation (Fig. 4B and 7B), consistent with previous reports (27, 35, 47). The accumulation of BRCA1 did not depend on the presence of other NHEJ factors, DNA-PKcs and XRCC4, which function in the downstream part of the NHEJ pathway. Interestingly, DNA-PKcs also accumulates at laser-irradiated DSBs in a Ku80-dependent manner (47). Kim et al. have reported that

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Ku70 and DNA-PKcs are only transiently localized to laserirradiated sites (25). In our analysis, the N terminus of BRCA1 also accumulated only transiently (Fig. 3G and H), indicating that BRCA1 interacts with NHEJ factors in the early phase of the repair process and dissociates from the site of DSBs together with NHEJ factors. Breast cancer risk has been correlated with single-nucleotide polymorphisms in NHEJ factor genes (17). Furthermore, the BRCA1 genotype significantly affects the degree of this risk, if the NHEJ factors are a high-risk genotype (5). There have been conflicting reports on the role of BRCA1 in NHEJ, including promotion, suppression, or no effect. The existence of two subpathways has been recently proposed for NHEJ, namely, errorfree and error-prone NHEJ (6, 54). Moreover, recent reports have indicated that BRCA1 functions to promote error-free NHEJ or to inhibit error-prone NHEJ (49, 58). Therefore, our results demonstrate the molecular basis of the involvement of BRCA1 in the NHEJ pathway. It is possible that the above facilitation and/or suppression of NHEJ might be mediated by the Ku80-dependent accumulation of BRCA1 at DSBs. We found that both the aa 1 to 100 and aa 101 to 200 fragments accumulated at DSBs. Interestingly, these two Nterminal fragments of BRCA1 exhibited similar kinetics of accumulation. This result suggests that the two fragments may use similar mechanisms to localize to DSBs. Indeed, we demonstrated that each of these fragments interacts with Ku80 and that the accumulation of each fragment at DSBs is Ku80 dependent. This mechanism is analogous to the independent interactions of estrogen receptor ␣ with the aa 1 to 100 and aa 100 to 200 fragments of BRCA1 (32). There may be novel sequences or motifs in the aa 100 to 200 region of BRCA1 that are responsible for these protein-protein interactions. The Y105C, P142H, E143K, and Y179C missense mutations of the aa 101 to 200 fragment of BRCA1 significantly reduced BRCA1 accumulation at DSBs. These mutations in full-length BRCA1 reduced or diminished its rapid accumulation at laserirradiated sites. Furthermore, we showed that one of the missense mutations, P142H, abolished the association with the Ku80 protein and failed to restore resistance to ionizing irradiation in BRCA1-deficient cells. The pathological significance of mutations found in the BRCA1 N terminus has been evaluated only around the RING domain by examining BARD1 association and ubiquitination activity. In our analysis, the Y105C, P142H, E143K, and Y179C mutant proteins were able to interact with BARD1 and exhibited ubiquitination activity (unpublished data). Therefore, we showed for the first time that these missense mutations in aa 101 to 200 could be pathological mutations. These data suggest that rapid accumulation of BRCA1 at DSBs via its N terminus is important for its tumor suppressor activity. BARD1 accumulated at the irradiated sites, but with kinetics distinct from that of BRCA1. BRCA1 rapidly accumulated at the irradiated sites within 20 s, whereas BARD1 accumulated gradually (Fig. 7). C61G mutant BRCA1, which is unable to interact with BARD1, interacts with Ku80. This suggests that the association of the N terminus of BRCA1 with Ku80 is independent of BARD1. Furthermore, the BRCA1/BARD1containing complex was distinct from the BRCA1/Ku80-containing complex. Since BARD1 accumulated slowly at DSBs (Fig. 7B and C), the BRCA1/BARD1 complex may be re-

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cruited to DSBs through interactions between the C terminus of BRCA1 and unidentified factors. According to recent reports, the RAP80-Abraxas/CCDC98 complex interacts with the BRCT domains of BRCA1 and is required to recruit BRCA1 to DNA damage sites (24, 45, 48). BARD1 is also reported to be associated with the BRCT domains, as well as the RING domain of BRCA1 (44). Therefore, it might be either the RAP80-Abraxas/CCDC98 complex or BARD1 that contributes to the gradual accumulation of BRCA1 via its C terminus. In any case, a BRCA1/Ku80-containing complex may accumulate rapidly at DSBs via the N terminus of BRCA1, function in the early phase of the repair process, and then dissociate rapidly, whereas a BRCA1/BARD1-containing complex may function later in the repair process. Bekker-Jensen et al. reported that BRCA1 accumulates in two distinct nuclear compartments, the DSB-flanking chromatin marked by H2AX and single-stranded DNA microcompartments (7). Downregulation of the checkpoint mediator Mdc1/ NFBD1 dissociates BRCA1 from the DSB-flanking chromatin but not from single-stranded DNA microcompartments. This observation suggests that BRCA1 may form different types of protein complexes and that each complex may localize to distinct structures near sites of DNA damage. Our results indicate that BRCA1 forms distinct protein complexes by using different intramolecular regions. Taken together, BRCA1 may contribute to multiple protein complexes whose constitution is spatiotemporally and dynamically regulated and which function in various phases of DNA DSB repair processes. ACKNOWLEDGMENTS This study was supported by grants-in-aid from the Ministry of Education, Science, Sports and Culture of Japan (N.C. and C.I.), the Gonryo Medical Foundation (N.C. and C.I.), the HIROMI Medical Research Foundation (N.C.), and a Genome Network project grant from the Ministry of Education, Science, Sports and Culture of Japan (A.Y.). REFERENCES 1. Au, W. W., and B. R. Henderson. 2005. The BRCA1 RING and BRCT domains cooperate in targeting BRCA1 to ionizing radiation-induced nuclear foci. J. Biol. Chem. 280:6993–7001. 2. Baer, R., and T. Ludwig. 2002. The BRCA1/BARD1 heterodimer, a tumor suppressor complex with ubiquitin E3 ligase activity. Curr. Opin. Genet. Dev. 12:86–91. 3. Baldeyron, C., E. Jacquemin, J. Smith, C. Jacquemont, I. De Oliveira, S. Gad, J. Feunteun, D. Stoppa-Lyonnet, and D. Papadopoulo. 2002. A single mutated BRCA1 allele leads to impaired fidelity of double strand break end-joining. Oncogene 21:1401–1410. 4. Bassing, C. H., K. F. Chua, J. Sekiguchi, H. Suh, S. R. Whitlow, J. C. Fleming, B. C. Monroe, D. N. Ciccone, C. Yan, K. Vlasakova, D. M. Livingston, D. O. Ferguson, R. Scully, and F. W. Alt. 2002. Increased ionizing radiation sensitivity and genomic instability in the absence of histone H2AX. Proc. Natl. Acad. Sci. USA 99:8173–8178. 5. Bau, D. T., Y. P. Fu, S. T. Chen, T. C. Cheng, J. C. Yu, P. E. Wu, and C. Y. Shen. 2004. Breast cancer risk and the DNA double-strand break end-joining capacity of nonhomologous end-joining genes are affected by BRCA1. Cancer Res. 64:5013–5019. 6. Bau, D. T., Y. C. Mau, and C. Y. Shen. 2006. The role of BRCA1 in non-homologous end-joining. Cancer Lett. 240:1–8. 7. Bekker-Jensen, S., C. Lukas, R. Kitagawa, F. Melander, M. B. Kastan, J. Bartek, and J. Lukas. 2006. Spatial organization of the mammalian genome surveillance machinery in response to DNA strand breaks. J. Cell Biol. 173:195–206. 8. Cantor, S. B., D. W. Bell, S. Ganesan, E. M. Kass, R. Drapkin, S. Grossman, D. C. Wahrer, D. C. Sgroi, W. S. Lane, D. A. Haber, and D. M. Livingston. 2001. BACH1, a novel helicase-like protein, interacts directly with BRCA1 and contributes to its DNA repair function. Cell 105:149–160. 9. Celeste, A., O. Fernandez-Capetillo, M. J. Kruhlak, D. R. Pilch, D. W. Staudt, A. Lee, R. F. Bonner, W. M. Bonner, and A. Nussenzweig. 2003. Histone H2AX phosphorylation is dispensable for the initial recognition of DNA breaks. Nat. Cell Biol. 5:675–679.

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