, 747 (1999);
et al.Qing Zhong,
Complex and the DNA Damage Response
Association of BRCA1 with the hRad50-hMre11-p95
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Association of BRCA1 with the
and the DNA Damage Response
Qing Zhong, Chi-Fen Chen, Shang Li, Yumay Chen,
Chuan-Cheng Wang, Jun Xiao, Phang-Lang Chen,
Z. Dave Sharp, Wen-Hwa Lee*
BRCA1 encodes a tumor suppressor that is mutated in familial breast and
ovarian cancers. Here, it is shown that BRCA1 interacts in vitro and in vivo with
hRad50, which forms a complex with hMre11 and p95/nibrin. Upon irradiation,
BRCA1 was detected in discrete foci in the nucleus, which colocalize with
hRad50. Formation of irradiation-induced foci positive for BRCA1, hRad50,
hMre11, or p95 was dramatically reduced in HCC/1937 breast cancer cells
carrying a homozygous mutation in BRCA1 but was restored by transfection of
wild-type BRCA1. Ectopic expression of wild-type, but not mutated, BRCA1 in
these cells rendered them less sensitive to the DNA damage agent, methyl
methanesulfonate. These data suggest that BRCA1 is important for the cellular
responses to DNA damage that are mediated by the hRad50-hMre11-p95
BRCA1 is a tumor-suppressor gene linked to
familial breast and ovarian cancers (1). The
hallmarks of BRCA1 protein include an NH2-
terminal RING finger domain and BRCA1
COOH-terminal (BRCT) repeats that mediate
binding to CtIP (2). Several lines of evidence
have indicated that BRCA1 is involved in
DNA repair; BRCA1-deficient embryonic
stem cells are hypersensitive to ionizing ra-
diation and are defective in transcription-cou-
pled repair of oxidative DNA damage (3).
Upon DNA damage, BRCA1 becomes hyper-
phosphorylated and shows alterations in sub-
nuclear localization (4) and CtIP binding (2).
BRCA1 exon 11 deletion cells display a de-
fective G2/M checkpoint after ionizing radi-
ation and methyl methanesulfonate (MMS)
To determine potential binding partners
of BRCA1 that might elucidate its role in
DNA repair, we immunoprecipitated
methionine–labeled T24 human bladder
carcinoma cells with BRCA1 antibodies,
and three coprecipitated cellular proteins
(150, 95, and 84 kD) were revealed (6).
The largest (150-kD) protein was con-
firmed to be hRad50 by reprecipitation with
specific ?-hRad50 (7), co-migrating with
the immunoprecipitated and in vitro trans-
lated hRad50 (Fig. 1A). Following the cell
cycle, BRCA1 was coimmunoprecipitated
with hRad50, and this interaction appeared
to peak at 33 hours after release from den-
sity arrest (Fig. 1B). This corresponds to
late S and G2, a time when BRCA1 phos-
phorylation is maximal (6), suggesting that
BRCA1 may be involved in DNA recom-
bination during the normal cell cycle.
To delineate the specific binding sites of
hRad50 and BRCA1, we performed a gluta-
thione S-transferase (GST) pull-down assay
with in vitro translated hRad50 and various
bacterially expressed GST-BRCA1 fusion
proteins (Fig. 1, C through E). A fragment
containing amino acids from 341 to 748
(BRCA-Bgl in Fig. 1, C and D) was found to
bind to hRad50 specifically (Fig. 1E). A yeast
two-hybrid binding assay yielded similar re-
sults (Fig. 1F). The NH2-terminal half of
hRad50 was required for BRCA1 binding
Rad50, Mre11, and p95/nibrin form a
complex that functions in homologous re-
combination, nonhomologous end joining
(NHEJ), meiotic recombination, the DNA
damage response, and telomere mainte-
nance (8). Rad50 is a coiled-coiled struc-
tural maintenance of chromosomes–like
protein with adenosine 5?-triphosphate–de-
pendent DNA binding activity (9). Mre11
has been proposed to have both structural
(DNA end holding) and catalytic activities,
including DNA exo- and endonuclease ac-
tivities (10). Mutation of the NBS1 gene
encoding p95 is responsible for Nijmegen
Breakage Syndrome (NBS), a disease char-
acterized by an increased cancer incidence,
cell cycle checkpoint defects, and sensitiv-
ity to ionizing radiation (11, 12). A defi-
ciency of p95 in NBS cells abrogates the
formation of ionizing radiation–induced
hMre11-hRad50 foci (12). In normal hu-
man diploid fibroblasts, hMre11 localizes
to DNA breaks within 30 min of irradiation
(13). These observations have prompted
complex functions as a sensor of DNA
To examine BRCA1 and hRad50 interac-
tions upon DNA damage, we treated T24
cells with gamma irradiation or MMS and
?-BRCA1 6B4 or ?-hRad50 13B3 monoclo-
nal antibodies (mAb’s). As observed previ-
ously (2, 4), these treatments resulted in the
slower migration of BRCA1, consistent with
its phosphorylation (Fig. 2A). The treatments
did not appear to change the amount of the
BRCA1-hRad50 complex (Fig. 2A), suggest-
ing that it exists even in the presence of DNA
Considering that the level of their inter-
actions does not change after DNA damage,
as assessed by protein amounts in co-im-
munoprecipitates in Fig. 2A, relocalization
of the component proteins to sites of dam-
aged DNA may be a crucial aspect of
BRCA1 function during the repair process.
Both BRCA1 and hRad50 display discrete
nuclear foci after treatment of cells with
genotoxic agents (14, 15). The BRCA1 dot
pattern appears in untreated T24 cells.
Upon irradiation, the BRCA1 dots were
disrupted within 1 hour (4) then gradually
reassembled into bright foci. The BRCA1
irradiation-induced foci (IRIF) appear in 70
to 90% of nuclei at 6 to 8 hours after
irradiation and remain until 12 hours (16,
17). The hRad50 IRIF pattern is consistent
with the reported pattern, reaching its peak
at 6 to 8 hours and declining at 12 hours
after irradiation (15) (Table 1).
We tested whether radiation-induced
foci containing hRad50 colocalize with
those containing BRCA1. T24 cells irradi-
ated with 12-gray (Gy) gamma radiation
demonstrated the punctate pattern of immu-
nostaining for BRCA1 with mAb’s Ab-1
(4) or 17F8 (16, 18); this pattern overlaps
hRad50-containing foci identified with rab-
bit ?-hRad50 antiserum (15) (Fig. 2B).
Among cells displaying both hRad50 and
BRCA1 foci, ?90% showed substantial co-
localization. Irradiation-induced colocal-
ization of hRad51 and BRCA1 foci were
also observed (Fig. 2B), similar to the ob-
servation of the colocalization of these two
proteins upon hydroxyurea or ultraviolet
Cells appear to have one of two types of
BRCA1 foci: Most colocalize with hRad51,
and a portion of the cells colocalize with
hRad50. The percentage of BRCA1 foci-
containing cells associating with either
hRad50 or hRad51 foci varies after irradi-
ation (Table 1), and these two types of foci
appear to be mutually exclusive because
cells with both hRad50- and hRad51-asso-
Department of Molecular Medicine, Institute of Bio-
technology, University of Texas Health Science Cen-
ter at San Antonio, 15355 Lambda Drive, San Antonio,
TX 78245, USA.
*To whom correspondence should be addressed. E-
R E P O R T S
www.sciencemag.org SCIENCEVOL 28530 JULY 1999
on November 13, 2010
ciated foci have seldom been observed (15,
16). With the specific antibodies or antisera
(Fig. 3A), radiation-induced hMre11 and
p95 foci were also examined in T24 cells,
which display a pattern similar to that dis-
played by hRad50 foci (Fig. 3B) and colo-
calize with BRCA1 and hRad50 foci (16).
To explore the relation of BRCA1 to these
foci, we assayed, for IRIF (17), HCC1937
cells that express a COOH-terminally trun-
cated BRCA1 protein (19). BRCA1 foci were
diminished in these cells, and the nuclear
staining of BRCA1 was homogenous, albeit
much dimmer, in HCC1937 cells regardless
of treatment (Fig. 3B). Interestingly, hRad50,
hMre11, and p95 IRIF were dramatically re-
duced in HCC1937 cells. Most of the irradi-
ated cells displayed a diffuse nuclear pattern
of hRad50, hMre11, or p95 immunostaining
similar to that seen in untreated HCC1937
cells. In contrast, IRIF that were positive for
hRad51 antibodies were readily and efficient-
ly detected in both T24 and HCC1937 cells
HCC1937 also harbors many other genetic
changes (19). To determine whether the
BRCA1 deficiency was responsible for the
defect in IRIF formation, we transiently
transfected hemagglutinin (HA)–tagged wild-
type BRCA1 into HCC1937 cells and irradi-
ated cells 40 hours later. Of the transfected
cells, 18 to 28% reconstitute HA-BRCA1
foci, and among these cells, ?29 to 38% had
immunoreactive hRad50, hMre11, or p95
foci, colocalizing with BRCA1 foci (Fig.
3C). Cells mock transfected or transfected
with a vector showed no or very few foci
after radiation (Fig. 3C) (16). These results
indicate that BRCA1 is responsible for defec-
tive hRad50, hMre11, and p95 IRIF response
in HCC1937 cells.
hRad50-hMre11-p95 foci formation may be
due to mutated BRCA1 gene product in
HCC1937 cells, we examined the integrity of
the BRCA1-hRad50-hMre11-p95 complex in
HCC1937, and nuclear extracts prepared
from the cells that were untreated or treated
with MMS or gamma radiation (Fig. 3C)
were co-immunoprecipitated with ?-hRad50.
Both hMre11 and p95 were in the complex,
similar to T24 cells, but the truncated
BRCA1 (Fig. 3D), which is expressed at
detectable levels, was not. The disruption of
BRCA1-hRad50 interaction in HCC1937
cells may be due to conformation change,
lower expression, and possibly inefficient nu-
clear transportation (16).
To explore the biological consequence
of BRCA1 deficiency in HCC1937 cells,
we assayed cell survival after treatment
with MMS. Relative to T24 and another
breast cancer cell line, MCF7, both of
HCC1937 cells were hypersensitive to
MMS treatment (Fig. 4A). Transfection of
wild-type BRCA1, but not BRCA1 mutants
(Fig. 4B) with alterations at the NH2-ter-
minal RING finger domain (Cys613
Gly61) (20) or the COOH-terminal BRCT
domain (Ala17083 Glu1708) (21), substan-
tially increased the survival of MMS-treat-
ed HCC1937 cells (Fig. 4C). In contrast,
transfection with wild-type BRCA2 did not
affect cell survival under similar conditions
(Fig. 4C). The expression of these con-
structs was confirmed by protein immuno-
blot analysis with ?-BRCA1 COOH-termi-
nus antibody, C20 (Fig. 4D). These results
are consistent with a defective G2/M check-
point upon MMS treatment in BRCA1 exon
11–deficient cells (5), and they also sug-
gest that the BRCA1 RING finger domain
and the BRCT repeats may be critical for a
similar DNA damage response.
In summary, our results suggest that
formation of the BRCA1-hRad50 complex
does not change in response to DNA dam-
age; rather, it is the nuclear partitioning of
the complex that changes. BRCA1 is
Fig. 1. BRCA1 inter-
acts with hRad50 in
vivo and in vitro. (A)
Lysates, labeled with
T24 cells were immuno-
precipitated with preim-
mune serum (lanes 1
and 4), ?-BRCA1 or
?-hRad50 (lanes 2 and
?-BRCA1 followed by
dissociation and re-
?-Rad50 (lane 3). In
hRad50 served as a
control (lane 6). Ar-
rows mark bands that
may contain p95 or
hMre11. (B) BRCA1 as-
sociates with hRad50 in
a cell cycle–dependent
ed T24 cells were re-
leased and collected
at the times indicated
(26) (U, unsynchro-
nized; G12, G1; G18,
G1/S; G24, S; G33, G2;
Nco, M; and G1, G0/
G1), and cell extracts
tated with ?-hRad50
and BRCA1 proteins
were detected by pro-
tein immunoblot anal-
cipitated BRCA1 peaks
in the late S and G2
phases. (C) Schematic
and (D) the expressed
and purified fusions
from Escherichia coli.
(E) In vitro translated
hRad50 and the bind-
ing results with the BRCA1-GST fusions indicated in (D). Only the BRCA-Bgl (amino acids 341
through 748) binds to hRad50 (lane 4). Lane 1 shows the total input of translated hRad50. (F)
BRCA1 interacts with hRad50 in a yeast two-hybrid assay. The indicated regions of BRCA1 were
fused to the DNA binding domain of GAL4 in pAS2-1. hRad50 was fused to the activation domain
of GAL4 in pGAD10. These plasmids were cotransformed from yeast strain Mav203 and (a) scored
for colony growth on Ura–/His–/Trp–/Leu–plates and (b) color assayed for ?-galactosidase activity.
BRCA-Bgl binds to hRad50. (G) NH2-terminus of hRad50 binds BRCA1 in a yeast two-hybrid assay.
NH2- or COOH-terminal fragments of hRad50 were fused to the transactivation domain of GAL4
in pGAD10, and these plasmids were cotransformed with the BRCA-Bgl fragment fused to the DNA
binding domain of GAL4 in pAS2-1 as in (F).
R E P O R T S
30 JULY 1999VOL 285SCIENCEwww.sciencemag.org
on November 13, 2010
present in both hRad50 and hRad51 foci
upon irradiation; however, cells containing
hRad50-hMre11-p95 foci have no detect-
able hRad51 foci, and vice versa (15, 16).
BRCA1 is crucial for hRad50-hMre11-p95
foci assembly but not for hRad51 foci in
HCC1937 cells. All these data suggest that
BRCA1 has distinct roles in each complex
in response to DNA damage.
The Rad50-Mre11-p95 complex partici-
pates in NHEJ or homologous recombina-
tion in DNA double-strand breaks. In ho-
mologous recombination, it is postulated
that the Rad50-Mre11-p95 complex is re-
sponsible for end processing, and Rad51 is
involved in strand exchange during a sub-
sequent phase. BRCA1 may facilitate the
coupling of these two steps. This hypothe-
sis is supported by evidence that BRCA2 is
associated with hRad51 (22) and that
BRCA1 interacts with BRCA2 (23). Also,
through interactions with the hRad50-
There is no evidence for a BRCA1-like
protein in the well-studied DNA repair
systems in yeast. It follows that BRCA1
may function as an accessory DNA repair
protein, perhaps in mammalian cells facil-
itating, coordinating, or sensing DNA dam-
Fig. 2. Co-immuno-
precipitation and co-
localization of BRCA1
were mock treated
(U) or treated with
0.05% MMS (M) for 1
hour or were exposed to 12-Gy gamma radiation (?), then har-
vested 1 hour later. The cell lysates were immunoprecipitated with
the indicated antibodies (IP, immunoprecipitate). BRCA1 and
hRad50 were detected by protein immunoblot analysis with 6B4 or
13B3, respectively. (B) Radiation-induced hRad51 foci [green (a and
e)] or hRad50 foci [green (i and m)] colocalize [merged images (c,
g, k, and o)] with BRCA1 foci [red (b, f, j, and n)]. T24 cells were
gamma irradiated with 12 Gy and stained at 8 hours after irradi-
ation with rabbit ?-hRad50 or ?-Rad51 antibodies, followed by
fluorescein isothiocyanate (FITC)–conjugated ?-rabbit antibody,
and ?-BRCA1 mAb Ab-1, followed by Texas Red–conjugated
?-mouse antibody (7, 17). Staining with 4?,6?-diamidino-2-phe-
nylindole (DAPI) was used to identify nuclei [blue (d, h, l, and p)].
Nuclei containing both BRCA1 and hRad50 or hRad51 foci are
marked with arrows, whereas nuclei containing only BRCA1 foci are
marked with arrowheads. Panels (e through h) and (m through p)
are enlarged images from the boxed nuclei in panels (a through d)
and (i through l), respectively.
Fig. 3. BRCA1 is crucial for the formation of
hRad50-hMre11-p95 IRIF. (A) Antibodies spe-
cific for hRad50 [?-hRad50 mAb 13B3 (lane
1)], p95 [?-p95 polyclonal antiserum (lane 2)],
and hMre11 [?-hMre11 polyclonal antiserum
(lane 3) and ?-hMre11 mAb 12D7 (lane 4)] (7)
were tested by straight protein immunoblot
with T24 lysates. (B) hRad51, hRad50, hMre11,
p95, and BRCA1 subnuclear partitioning in
HCC1937 and T24 cells in response to gamma
irradiation. Cells were mock treated (columns
1, 3, 5, 7, and 9) or treated with 12-Gy gamma
rays (columns 2, 4, 6, 8, and 10) and stained
with indicated antibodies at 8 hours after
irradiation (17); rows 1 and 3 were stained
with FITC, and rows 2 and 4 were stained with
DAPI. HCC1937 cells contain hRad51 foci but
do not contain hRad50, hMre11, p95, and
BRCA1 foci. (C) Ectopic expression of BRCA1
restores formation of hRad50-hMre11-p95
IRIF in HCC1937 cells. Expression plasmid con-
(PcDNA3.1-HA) alone was transfected into
HCC1937 cells by lipofection (27). The cells
were treated with 12-Gy gamma rays and
stained with ?-hMre11 mAb, 12D7, and rabbit
?-HA (Y-11) (16, 17) as indicated. HA-BRCA1 foci colocalize with hMre11 foci in cells transfected with the HA-BRCA1 cDNA. (D) Formation
of hRad50-hMre11-p95 and BRCA1 complexes. Lysates from HCC1937 cells that were mock treated (lane 1), treated with 0.05% MMS for 1
hour (lane 2), or treated with 12-Gy gamma rays (lane 3) or lysates from untreated T24 cells (lane 4) were immunoprecipitated with ?-hRad50
mAb 13B3. The immunoprecipitates were analyzed by protein immunoblot probed with ?-BRCA1 mAb 6B4, ?-hRad50 mAb 13B3, ? -p95, and
? -hMre11, as indicated. BRCA1 is present in the hRad50-hMre11-p95 complex of T24 but not in HCC1937 cells. (E) Full-length or truncated
BRCA1 was detected by protein immunoblot with ?-BRCA1 mAb, 6B4, in lysates used in (D).
R E P O R T S
www.sciencemag.org SCIENCE VOL 28530 JULY 1999
on November 13, 2010
age. Efficient DNA double-strand break re-
pair is important because unrepaired le-
sions can lead to chromosome break, trans-
location, and other
instability seen in human cancer cells (24).
This notion is consistent with the dramati-
BRCA1-deficient cells (5). Further mecha-
nistic studies on BRCA1’s role in the DNA
damage response may lead to new thera-
peutic strategies for breast and ovarian can-
References and Notes
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5. X. L. Xu et al., Mol. Cell 3, 389 (1999).
6. Y. Chen et al., Science 270, 789 (1995); Y. Chen et al.,
Cancer Res. 56, 3168 (1996).
7. Polyclonal antibodies were obtained by using the
following bacterially expressed and purified GST
fusion proteins as antigens: GST-hRad50-15A5,
containing amino acids 211 through 575 of
hRad50; GST-MM, containing amino acids 82
through 582 of hMre11; GST-NBS1, containing
amino acids 12 through 754 of p95; GST-hRad51,
containing full-length hRad51; and GST alone (for
anti-GST mAb 8G11), to generate polyclonal or
monoclonal antibodies (25). BRCA1 mAb’s 6B4 and
17F8 were described in (18). BRCA1 mAb Ab-1 and
rabbit ?-hRad51 antibodies were from Oncogene
Research Product (Cambridge, MA). Affinity-puri-
fied rabbit ?-BRCA1 antibody (C-20) and ?-HA
antibody (Y-11) were from Santa Cruz Biotechnol-
ogy (Santa Cruz, CA).
8. J. E. Haber, Cell 95, 583 (1998).
9. W. E. Raymond and N. Kleckner, Nucleic Acids Res.
21, 3851 (1993).
10. T. T. Paull and M. Gellert, Mol. Cell 1, 969 (1998);
K. M. Trujillo, S. S. Yuan, E. Y. Lee, P. Sung, J. Biol.
Chem. 273, 21447 (1998); M. Furuse et al., EMBO J.
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11. Y. Shiloh, Annu. Rev. Genet. 31, 635 (1997); C. M.
Weemaes, D. F. Smeets, C. J. van der Burgt, Int. J.
Radiat. Biol. 66, S185 (1994); I. van der Burgt, K. H.
Chrzanowska, D. Smeets, C. Weemaes, J. Med.
Genet. 33, 153 (1996); R. Varon et al., Cell 93, 467
12. J. P. Carney et al., Cell 93, 477 (1998).
13. B. E. Nelms, R. S. Maser, J. F. MacKay, M. G. Lagally,
J. H. J. Petrini, Science 280, 590 (1998).
14. G. M. Dolganov et al., Mol. Cell. Biol. 16, 4832
15. R. S. Maser, K. J. Monsen, B. E. Nelms, J. H. J. Petrini,
ibid. 17, 6087 (1997).
16. Q. Zhong and W.-H. Lee, unpublished results.
17. The procedure for indirect immunofluorescence
staining was adopted and slightly modified from that
in (25). In a typical staining experiment, 500 to 1000
cells were counted. For the foci-containing cells, only
nuclei containing ?10 foci were counted as a foci-
containing cell. Each experiment was repeated at
least three times, and the results were reproducible
with the antibodies indicated here. The results did
not vary according to the conditions of fixation. The
foci formation studies were performed following dif-
ferent time courses after irradiation (at 1, 3, 6, 8, and
12 hours). Because all of the studied foci show rela-
tively high quality and quantity at 8 hours after
irradiation, the results at this time point are mostly
used as representatives here.
18. H. K. Chew, A. A. Farmer, W.-H. Lee, in Breast Cancer,
A. Bowcock, Ed. (Humana, Totowa, NJ, 1998), pp.
19. G. E. Tomlinson et al., Cancer Res. 58, 3237 (1998).
20. L. J. C. Wu et al., Nature Genet. 14, 430 (1996).
21. M. S. Chapman and I. M. Verma, Nature 382, 678
22. S. K. Sharan et al., ibid. 386, 804 (1997); P. L. Chen et
al., Proc. Natl Acad. Sci. U.S.A. 95, 5287 (1998).
23. J. Chen et al., Mol. Cell 2, 317 (1998).
24. C. Lengauer, K. W. Kinzler, B. Vogelstein, Nature 396,
643 (1998); H. Zhang, G. Tombline, B. Weber, Cell 92,
25. Q. Zhong et al., Cancer Res. 57, 4225 (1997).
26. Synchronization of T24 human bladder carcinoma
cells was performed by density arrest, then released
at time zero by replating at a density of 2 ? 106cells
per 10-cm plate. At various time points thereafter
(12 hours for G1,18 hours for G1/S, 24 hours for S, 33
hours for G2/M, and 1 hour for arrest in G0/G1), cells
were harvested. To obtain cells in M phase, we added
nocodazole (0.4 ?g/ml) to the culture medium for 10
hours before harvest.
27. Constructs based on plasmid pcDNA3.1 (Invitrogen,
San Diego, CA) were used for lipofectin-mediated
transfection of HCC1937 cells with BRCA1 cDNA.
Cells were harvested after 48 hours for immunopre-
cipitation and protein immunoblot analysis. Parallel
cultures were treated with 0.1% MMS for 50 min, and
surviving cells were counted after 8 days. The exper-
iments were repeated at least three times, and the
results were consistent.
28. We thank J. Petrini for rabbit antisera to hRad50, P.
Garza and D. Jones for antibody production, and
N. Ting and T. Boyer for critical reading. Supported
by grants from NIH (CA 58183 and CA 30195), the
McDermott endowment (W.H.L.), and the Susan
G. Komen Foundation for Breast Cancer Research
30 March 1999; accepted 25 June 1999
Table 1. Focus formation and colocalization of BRCA1, hRad51, and hRad50 after gamma irradiation. A
cell nucleus displaying ?10 foci was counted as a foci-containing cell. At least 500 cells, irradiated by
12-Gy gamma rays, were analyzed for each experiment, and results were summarized from three
Foci contained in cells (%)
BRCA1 (total)hRad50 BRCA1 and hRad50*hRad51 BRCA1 and hRad51†
37 ? 8
13 ? 6
43 ? 7
81 ? 13
79 ? 11
1 ? 1
2 ? 2
17 ? 5
30 ? 7
13 ? 6
0 ? 0
1 ? 1
15 ? 3
28 ? 6
11 ? 5
7 ? 2
10 ? 2
20 ? 6
53 ? 6
74 ? 4
3 ? 2
7 ? 3
17 ? 5
49 ? 4
64 ? 612
*Among cell nuclei containing both BRCA1 and hRad50 foci, 85 to 95% of cells show ?50% of colocalization.
cell nuclei containing both BRCA1 and hRad51 foci, 75 to 83% of cells show ?50% of colocalization.
Fig. 4. Ectopic expression of BRCA1 in
HCC1937 confers resistance to MMS. (A) Hy-
persensitivity of HCC1937 to MMS. T24,
MCF7, and HCC1937 cells were treated with a
dose of MMS (indicated by the x axis) for 50 min, and the number of surviving cells was
counted by trypan blue exclusion assay with hematocytometry 48 hours after treatment.
These experiments were repeated three times. Error bars indicate SD. (B) Schematic diagrams
of the BRCA1 cDNA used to rescue resistance of HCC1937 to MMS. These cells express a
COOH-terminally truncated BRCA1 lacking a portion of the BRCT domain, as indicated. Two of
the constructs contain familial missense mutations (asterisks) [Cys613 Gly61(C61G) and
Ala17083 Glu1708(A1708E)] in the RING and BRCT domains, respectively. (C) Graphic
summary of cell survival in response to 0.1% MMS treatment. Parallel cultures of transfected,
empty vector, or untransfected cells (none) were treated with 0.1% MMS for 50 min (27). The
surviving cells were counted and plotted (y axis). Only transfection of cells with wild-type
BRCA1 restored resistance to MMS. Error bars indicate SD. (D) Expression of exogenous BRCA1
in transfected cells. Lysates from parallel cultures of BRCA1-transfected cells after 48 hours
were prepared and immunoprecipitated by rabbit ?-BRCA1 antibody, C20, which recognizes
full-length but not COOH-terminally truncated BRCA1, and detected by ?-BRCA1 mAb 6B4.
The expected 220-kD BRCA1 full-length or mutant proteins are indicated.
R E P O R T S
30 JULY 1999VOL 285 SCIENCEwww.sciencemag.org
on November 13, 2010