Zhao, S. et al. Functional link between ataxia-telangiectasia and Nijmegen breakage syndrome gene products. Nature 405, 473-477

Department of Molecular Medicine/Institute of Biotechnology, The University of Texas Health Science Center at San Antonio, 78245-3207, USA.
Nature (Impact Factor: 41.46). 05/2000; 405(6785):473-477. DOI: 10.1038/35013083
Ataxia telangiectasia (A-T) and Nijmegen breakage syndrome (NBS) are recessive genetic disorders with susceptibility to cancer and similar cellular phenotypes. The protein product of the gene responsible for A-T, designated ATM, is a member of a family of kinases characterized by a carboxy-terminal phosphatidylinositol 3-kinase-like domain. The NBS1 protein is specifically mutated in patients with Nijmegen breakage syndrome and forms a complex with the DNA repair proteins Rad50 and Mre11. Here we show that phosphorylation of NBS1, induced by ionizing radiation, requires catalytically active ATM. Complexes containing ATM and NBS1 exist in vivo in both untreated cells and cells treated with ionizing radiation. We have identified two residues of NBS1, Ser 278 and Ser 343 that are phosphorylated in vitro by ATM and whose modification in vivo is essential for the cellular response to DNA damage. This response includes S-phase checkpoint activation, formation of the NBS1/Mre11/Rad50 nuclear foci and rescue of hypersensitivity to ionizing radiation. Together, these results demonstrate a biochemical link between cell-cycle checkpoints activated by DNA damage and DNA repair in two genetic diseases with overlapping phenotypes.


Available from: Jerry W Shay, Jun 24, 2014
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letters to nature
Functional link between
ataxia-telangiectasia and Nijmegen
breakage syndrome gene products
Song Zhao*
, Yi-Chinn Weng*
, Shyng-Shiou F. Yuan*
, Yi-Tzu Lin*
Hao-Chi Hsu*, Suh-Chin J. Lin*, Elvira Gerbino*, Mei-hua Song*,
gorzata Z. Zdzienicka§, Richard A. Gattik, Jerry W. Shay, Yael Ziv#,
Yosef Shiloh# & Eva Y.-H. P. Lee*
* Department of Molecular Medicine/Institute of Biotechnology, The University of
Texas Health Science Center at San Antonio, San Antonio, Texas 78245-3207,
§ MGC-Department of Radiation Genetics and Chemical Mutagenesis,
Leiden University, LUMC, Leiden, The Netherlands
k Department of Pathology, University of California Los Angeles, Los Angeles,
California 90095, USA
Department of Cell Biology, The University of Texas Southwestern Medical
Center, Dallas, Texas 75390-9039, USA
# Department of Human Genetics and Molecular Medicine, Sackler School of
Medicine, Tel Aviv University, Tel Aviv, Israel
These authors contributed equally to this work
Ataxia-telangiectasia (A-T) and Nijmegen breakage syndrome
(NBS) are recessive genetic disorders with susceptibility to
cancer and similar cellular phenotypes
. The protein product of
the gene responsible for A-T, designated ATM, is a member of a
family of kinases characterized by a carboxy-terminal phospha-
tidylinositol 3-kinase-like domain
. The NBS1 protein is speci-
®cally mutated in patients with Nijmegen breakage syndrome and
forms a complex with the DNA repair proteins Rad50 and
. Here we show that phosphorylation of NBS1, induced
by ionizing radiation, requires catalytically active ATM. Com-
plexes containing ATM and NBS1 exist in vivo in both untreated
cells and cells treated with ionizing radiation. We have identi®ed
two residues of NBS1, Ser 278 and Ser 343 that are phosphorylated
in vitro by ATM and whose modi®cation in vivo is essential for the
cellular response to DNA damage. This response includes S-phase
checkpoint activation, formation of the NBS1/Mre11/Rad50
nuclear foci and rescue of hypersensitivity to ionizing radiation.
Together, these results demonstrate a biochemical link between
cell-cycle checkpoints activated by DNA damage and DNA repair
in two genetic diseases with overlapping phenotypes.
The cellular response to DNA damage is complex and includes
cell-cycle checkpoint activation, DNA repair and changes in gene
. Cell lines representative of the inherited cancer-
prone human diseases ataxia-telangiectasia (A-T) and Nijmegen
breakage syndrome (NBS) are hypersensitive to ionizing radiation
and have defects in DNA-damage-activated cell-cycle checkpoints
Upon DNA damage, ATM phosphorylates p53 (refs 12, 13), and
Brca1 (ref. 14). ATM is required for phosphorylation of Chk2
and Rad51 (refs 16, 17) induced by ionizing radiation.
NBS1 is an integral component of the Mre11/Rad50/NBS1 nuclease
which is important in the repair of DNA double-strand
To examine whether a signalling cascade exists between ATM and
NBS1, we studied whether NBS1 was posttranslationally modi®ed
following treatment with ionizing radiation. The NBS1 monoclonal
antibody speci®cally recognized a protein with a relative molecular
mass of 95,000 (M
95K) in all human cell lines examined except
those established from NBS patients (data not shown). Although
the electrophoretic mobility of the 95K NBS1 protein was constant
throughout the cell cycle, treatment of cells with 10 Gy g-irradiation
resulted in a slower migrating form of NBS1 (Fig. 1a). The mobility
of the altered form of NBS1 immunoprecipitated from irradiated
cells reverted to that of NBS1 from undamaged cells upon incuba-
tion with phosphatase, indicating that NBS1 becomes phosphory-
lated in response to DNA damage by ionizing radiation (Fig. 1b,
compare lane 6 with lanes 2 and 5).
Cell lines lacking functional ATM protein were used to examine
whether ATM is required for phosphorylation of NBS1 after DNA
damage. The ionizing radiation- or bleomycin (0.1 U per ml)-
induced phosphorylation of NBS1 was not detected in these cells
Present address: Department of Obstetrics and Gynecology, Kaohsiung Medical University, No. 100,
Shih-Chuan 1st Road, Kaohsiung 80708, Taiwan.
Figure 1 ATM is required for DNA damage-induced phosphorylation of NBS1. a, Mobility
shift of NBS1 in response to DNA damage during the cell cycle. Synchronized T24 cells
were irradiated with 10 Gy g-irradiation. Lysates (50 mg protein) of untreated cells and
cells 1 h after ionizing radiation (IR) treatment were analysed by western blotting with anti-
NBS1 antibody. NBS1
, phosphorylated form of NBS1. Mre11 is included as a protein
loading control. b, Phosphorylation and mobility shift of NBS1. Lysates from untreated
(lanes 1±4) and IR-treated (lanes 5±8) human lymphoblast NAT10 cells were
immunoprecipitated with anti-NBS1 antibody. Immunoprecipitates were incubated with
phosphatase (PPase) in the absence or presence of phosphatase inhibitor. Lysates
from untreated (lane 1) and IR-treated cells (lane 8) were included as control.
c, Phosphorylation of NBS1 upon DNA damage and replication block in AT22IJE-T/pEBS7
(A-T) and AT22IJE-T/pEBS7-YZ5 (A-T cells complemented with ATM). d, Kinetics of IR-
induced modi®cation of NBS1. Cell lysates were prepared at the indicated time points after
IR treatment.
© 2000 Macmillan Magazines Ltd
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(Fig. 1c and data not shown). In contrast, the mobility shift induced
by treatment with either hydroxyurea (1 mM) or ultraviolet light
(10 J m
) was normal (Fig. 1c). Importantly, re-introduction of the
ATM gene into A-T cells
restored phosphorylation of NBS1
induced by ionizing radiation (Fig. 1c, bottom) showing that the
failure to phosphorylate NBS1 is directly related to a de®ciency of
the ATM protein. The absence of ATM does not abolish cellular
responses to DNA damage but it does result in a signi®cant delay in
these responses
. We therefore compared the kinetics of phosphor-
ylation of NBS1 induced by ionizing radiation in A-T cells, with that
in A-T cells that had also been complemented with the ATM
protein. Modi®cation of NBS1 was only detectable 6 h after ionizing
radiation in the absence of ATM (Fig. 1d). In contrast, the kinetics of
NBS1 phosphorylation in A-T cells complemented with ATM was
similar to that of T24 cells, with phosphorylation detectable within
30 min after ionizing radiation (Fig. 1d and data not shown). As the
ATM-related kinase, ATR, can phosphorylate p53 with delayed
kinetics in the absence of ATM
, we suggest that the delayed
phosphorylation of NBS1 in A-T cells may be attributable to ATR
or another related kinase.
The cellular amounts of the ATM and NBS1 proteins remained
unchanged following DNA damage (Fig. 2a, left panel). Although
ATM was co-immunoprecipitated by NBS1 antibody in extracts
from both treated and untreated cells, ionizing radiation increased
the amount of ATM in the NBS1-immunoprecipitates (Fig. 2a,
middle). Consistent with these results, NBS1 was co-immunopre-
cipitated by the ATM antibody from untreated cells and in increased
quantities from cells treated with ionizing radiation (Fig. 2a, right).
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Figure 2 Interaction between ATM and NBS1, in vivo and in vitro phosphorylation of NBS1
by ATM. a, Interaction between ATM and NBS1. Left, direct immunoblotting using cell
lysates (100 mg) from mock-treated or 2 h after 10 Gy g-irradiated cells. T24, A-T
(GM09607) and NBS1-LBI cell lines were studied. Middle, co-immunoprecipitation of
ATM by anti-NBS1 antibody. Right, co-immunoprecipitation of NBS1 by anti-ATM
antibody. b, NBS1 structural domains and GST±NBS1 fusion proteins. Asterisk, serine
substituted with alanine. c, Expression of the recombinant ATM in human 293 cells 24 h
after transfection. Immunoblotting with anti-Flag antibody, M5 (Kodak). d, Autoradio-
grams showing ATM autophosphorylation (top) and phosphorylation of GST±NBS1 fusion
proteins by the ATM immunoprecipitated from either untreated or IR-treated cells
(bottom). Puri®ed fusion proteins were incubated with kinase buffer alone (lanes 1, 4, 7,
10, 13, 16 and 19) or kinase buffer and ATM. p53 is a known substrate of ATM.
e, Coomassie staining showing the amounts of substrates. f, Autoradiograms showing
Flag±ATM autophosphorylation (top) and phosphorylation of GST±NBS1 fusion proteins
by Flag-ATM and Flag-ATM
immunoprecipitates (bottom). g, Coomassie staining.
Arrowheads, GST fusion proteins; arrows, IgG.
wt wt wt wtwtS278A S343AS15A
wt wt wt wtwtS278A S343AS15A
1 2 3 4 5 6 7 8 9 101112 131415161718 192021
123456 78910 11121314 1516
123456 78910 11121314 1516
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Coomassie stain
Coomassie stain
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To determine whether ATM can phosphorylate NBS1 in vitro,
fragments of NBS1 expressed as glutathione S-transferase (GST)
fusion proteins in Escherichia coli were used as substrates in kinase
reactions (Fig. 2b) with either endogenous, recombinant Flag-
tagged wild-type ATM or a kinase-inactive form of ATM,
(Fig. 2c). Immunoprecipitated ATM was able to phos-
phorylate itself (Fig. 2d, top) and the GST±NBS1 fusion proteins
tested. All phosphorylation occurred within the amino-terminal
396 residues of NBS1 (Fig. 2d, bottom panel).
ATM phosphorylates Ser 15 of p53 (refs 8, 9). Consistent with this
result, GST±p53
, but not GST±p53
, was heavily phosphory-
lated by immunoprecipitated ATM in an in vitro kinase assay
(Fig. 2d, lanes 2 and 3). Examination of the amino-acid sequences
of NBS1 revealed several serine residues followed by glutamines (Q),
which are preferred sites for phosphorylation in p53 and c-abl by
. Alanine substitution of these potential phosphorylation
sites within the NBS1 protein identi®ed Ser 278 and Ser 343 as sites
phosphorylated by ATM in vitro (Fig. 2d, lanes 11, 12, 17 and 18).
The site(s) of ATM phosphorylation within amino-acid residues 1±
153 of NBS1 is not known but does not appear to be Ser 53 or Ser 58
in the FHA domain, as substitution of these residues with alanine
did not change the phosphorylation of GST±NBS1
(data not shown).
To provide further evidence that ATM phosphorylates Ser 278
and Ser 343 of NBS1, human 293 cells were transfected with either
Flag-tagged wild-type or mutant ATM complementary DNA, and
the recombinant proteins were immunoprecipitated with anti-Flag
antibody (Fig. 2c, f, top). As expected, wild-type ATM (Fig. 2f,
bottom, odd lanes) but not ATM
(Fig. 2f, bottom, even lanes)
in the anti-Flag immunoprecipitates could phosphorylate Ser 278
and Ser 343 of GST±NBS1 and Ser 15 of GST±p53.
To determine whether Ser 278 and Ser 343 of NBS1 are phos-
phorylated in vivo, NBS1-LBI, a T-antigen immortalized NBS cell
line established from a patient carrying the common founder
mutation 657del5 (ref. 23) in NBS1, was transfected with plasmids
encoding wild-type NBS1 (pCMV-NBS1
); NBS1
), NBS1
) or NBS1
). As expected, NBS1 protein was not
detectable by immunostaining of untransfected cells but was pre-
sent in the nuclei of cells transfected with either wild-type or mutant
NBS1 constructs (Fig. 3a). Expression of the 95K NBS1 protein was
con®rmed by immunoblotting of extracts from transfected cells
(Fig. 3b). About 20% of the cells were transfected as quanti®ed by
immunostaining (data not shown). The protein levels of wild-type
and NBS1
in the transfected cells are comparable (Fig. 3b, lanes
3±6), but NBS1
expression (Fig. 3b, lanes 9 and 10) was
consistently higher than that of the other constructs. Upon treat-
ment with ionizing radiation, the mobility of NBS1
, but not
, NBS1
or NBS1
, was altered (Fig. 3b,
compare lanes 5 and 6 with lanes 3 and 4 and 7±10). These data
indicate that Ser 278 and Ser 343 are phosphorylated in vivo.
Next we examined the effect of NBS1 phosphorylation on cell-
cycle checkpoints activated by DNA damage. Both A-T and NBS
cells display radioresistant DNA synthesis (RDS)
. RDS is likely to
be comparable to the DNA damage- but not replication block-
induced S-phase checkpoint. These two distinct checkpoint path-
ways have been found in various eukaryotes including ®ssion and
budding yeast, and mammals
. Plasmids encoding either wild-
type or mutant NBS1 were co-transfected with an enhanced green
¯uorescent protein (EGFP) expression plasmid into the NBS1-LBI
cells. 25-bromodeoxyuridine (BrdU) was added to cell cultures
immediately after mock or ionizing radiation treatment. The ratio
of BrdU-positive/EGFP-positive cells to EGFP-positive cells was
determined in untreated and treated cells to assess the effect of
phosphorylation of NBS1 on RDS. In comparison with wild-
type NBS1, NBS1 mutants with one serine residue replaced with
alanine were signi®cantly less effective at inhibiting RDS, and an
NBS1 mutant with the double alanine substitution was completely
ineffective (Fig. 4a).
We examined the effect of NBS1 phosphorylation on cellular
sensitivity to ionizing radiation by counting viable cells 72 h after
5Gy g-irradiation. Transfection of NBS1-LBI cells with pCMV±
resulted in a signi®cant reduction of cellular sensitivity to
ionizing radiation compared with the control vector (Fig. 4b). The
reduction in cellular sensitivity was abolished by mutation of both
the Ser 278 and Ser 343 phosphorylation sites. Interestingly, the
sensitivity of the pCMB-NBS1
and -NBS1
mutants to
ionizing radiation was intermediate, indicating that both phosphor-
ylation events contribute to cell sensitivity. Although stable protein
complexes of Mre11/Rad50/NBS1 exist in untreated or ionizing
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Mock wt S278A S343A S278A/S343A
10 11 12
S343A wt S278A
Figure 3 Expression of the full-length wild-type and NBS1 mutant proteins lacking
phosphorylation site(s) in NBS1-LBI cells. a, Immunostaining of NBS1 proteins in mock or
NBS1 expression-plasmid-transfected NBS1-LBI cells. Cells were immunostained with
anti-NBS1 monoclonal antibody, MHN1, 36 h after transfection. b, Immunoblotting
analysis of NBS1 protein in transfected cells. Cells were transfected with expression
plasmids (10 mg DNA per 2 3 10
cells). Cell treatment is as described in Fig. 2a and cell
lysate (100 mg protein) was analysed. Cell lysates from T24 cells were included as
control. Longer exposure is shown to illustrate recombinant NBS1 expression in the
transfected NBS1-LBI cells. Shorter exposures are shown for NBS1 and Mre11 in T24
cells, and Mre11 in NBS1-LBI cells.
© 2000 Macmillan Magazines Ltd
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radiation-treated cells
, it has been reported that Mre11, Rad50,
NBS1 and Brca1 all co-localize within nuclear foci induced only
after treatment with ionizing radiation
. Because NBS1 is required
for the formation of foci at DNA damage sites following ionizing
, we examined the role of NBS1 phosphorylation in foci
formation. Consistent with previous studies
, 26.6% of NBS1-LBI
cells transfected with pCMV±NBS1
were foci-positive (Fig. 4c). In
contrast, ionizing radiation did not induce foci formation in cells
transfected with pCMV±NBS1
(6.0% foci-positive cells
after ionizing radiation, 5.4% foci-positive cells in untreated cells).
Similar to the result of the ionizing radiation sensitivity assays,
inactivation of either serine phosphorylation site partially restored
the ability of NBS1 to form foci in response to ionizing radiation.
Our results indicate that phosphorylation of Ser 278 and Ser 343 in
NBS1 contributes to both cellular resistance to ionizing radiation
and formation of foci that contain NBS1 in response to ionizing
An enhanced interaction between ATM and NBS1 upon DNA
damage was seen using different antibodies in the co-immunopre-
cipitation experiments. However, we did not detect increased co-
fractionation of ATM and NBS1 upon DNA damage using gel-
®ltration chromatography (data not shown). It is unclear whether
this is due to the transient nature of the enhanced interaction or
whether the increase in complexed ATM and NBS1 is the result of
conformational changes induced in the protein complex upon DNA
damage. We have mapped two residues of NBS1, Ser 278 and
Ser 343, which are phosphorylated by ATM. Inactivation of these
phosphorylation sites abolishes NBS1 function in DNA damage-
activated cell-cycle checkpoints and DNA repair. Phosphorylation
of NBS1 by ATM may be required for signal transduction to
downstream effectors of DNA damage-induced S-checkpoint acti-
vation, such as Chk1 and Chk2 (refs 8±11). We speculate that
phosphorylation of NBS1 is also critical for the assembly of
repair proteins at the sites of DNA damage. This is supported by
the observation that inactivation of the Ser 278 and Ser 343 phos-
phorylation sites in NBS1 abolished the formation of Mre11/Rad50/
NBS1 foci upon treatment with ionizing radiation. The functional
interaction between ATM and NBS1 provides a molecular explana-
tion for similar defects in cell-cycle checkpoints and DNA repair
exhibited by NBS and A-T cells. Moreover, these studies provide
new insights into the complex relationship between DNA damage-
activated cell-cycle checkpoints and DNA repair mechanisms that
are involved in the cellular response to DNA double-strand
Immunoblotting and immunoprecipitation
Cells were lysed in EBC buffer supplemented with protease inhibitors (10 mgml
aprotinin, 50 mgml
leupeptin, 1 mM phenylmethylsulphonyl ¯uoride, 100 mM NaF and
). We determined protein concentration by Bradford assay (Biorad). Cell
lysates containing 20±100 mg protein were mixed with SDS sample buffer before
separation by SDS±7.5% polyacrylamide gel. Proteins were transferred to Immobilon P
(Millipore) or nitrocellulose membrane (Schleicher and Schuell). Anti-NBS1 monoclonal
antibodies were generated from mice immunized with GST±NBS1
. Anti-Mre11
antibodies were generated from mice immunized with puri®ed full-length Mre11
expressed in Sf9 insect cells. For co-immunoprecipitation, we incubated protein lysate (2±
3 mg) with primary antibodies overnight at 4 8C as described
except that we used
secondary antibodies cross-linked to magnetic beads (Dynal).
Phosphatase treatment
Cell lysates containing 1 mg of protein from untreated and ionizing radiation-treated
human lymphoblast NAT10 cells were incubated with anti-NBS1 antibody. Immuno-
precipitates were washed with EBC buffer and were resuspended in l phosphatase buffer
(New England Biolab) with or without 10 mM phosphatase inhibitor, b-sodium glycerol
Kinase assay
ATM was immunoprecipitated from non-irradiated or irradiated HeLa cells and used in
kinase assays as described
. GST fusion proteins (2±10 mg) were added to the ATM±
protein G sepharose beads and the kinase reaction was carried out in 20 ml reaction volume
containing kinase buffer and 10 mCi of g
P-ATP. Recombinant ATM was immunopre-
cipitated from transfected 293 cells 24 h after transfection using anti-Flag M2 antibody
Plasmid construction
Full-length NBS1 cDNA and the cDNA fragment encoding amino acids 343±754 were
generated by polymerase chain reaction with reverse transcription (RT-PCR) using RNA
isolated from human Raji cells. The full-length NBS1 cDNA was cloned into a pCMV
vector. Site-speci®c mutagenesis was performed using a QuickChange site-directed
mutagenesis kit (Stratagene) and was con®rmed by DNA sequencing. To construct the
pCMV±Flag±ATM expression vector, a 9.2-kilobase (kb) SalXhoI fragment containing
full-length ATM cDNA tagged with a Flag epitope and a His
sequence was excised from
pFB-YZ5 (ref. 19) and subcloned into a XhoI-linearized pCMV vector. Site-speci®c
mutagenesis was performed as described above using a 1.6-kb fragment of the ATM open
reading frame as the template. GST±NBS1 and GST±P53 fusion proteins were generated
by PCR using full-length cDNAs as templates. The PCR product was cloned into pGEX-
4T-3 vector (Pharmacia).
Cell culture and transfection
Primary NBS ®broblasts from Coriell Institute were immortalized with telomerase. The
NBS ®broblast cell line NBS1-LBI has been described
. Other cell lines were from the
American Type Culture Collection. Cells were cultured in Dulbecco's Modi®ed Eagle
medium supplemented with 10% fetal bovine serum (Gibco). Human bladder carcinoma
T24 cells were synchronized by density arrest. Cells at various cell cycle stages were
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VOL 405
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Cellular sensitivity to IR
26.6±3.6 15.5±1.3 16.3±0.8 6.0±0.3% foci
Figure 4 Radioresistant DNA synthesis (RDS) and sensitivity to ionizing radiation by
expression of wild-type and NBS1 mutants lacking serine phosphorylation site(s). a, RDS.
The ratio of BrdU- and EGFP-double positive cells to EGFP-positive cells is determined in
untreated and IR-treated cells, respectively. Bar represents the ratio of the IR-treated cells
divided by that in the untreated cells multiplied by 100 (.200 ¯uorescent cells counted
per experiment). The mean and s.d. of three experiments is shown. b, Cellular sensitivity
to IR. Cellullar sensitivity to g-irradiation (5 Gy) was calculated by dividing the number of
cells recovered from mock treated cells by the number of cells recovered from the IR-
treated cells 72 h after treatment. The mock treated cells have a cellular sensitivity of 1
(.1,000 transfected cells counted per experiment). The mean and s.d. of three
experiments is shown. c, Formation of IR-induced NBS1 foci in the transfected cells.
Three hours after IR (15 Gy), cells were stained with anti-NBS1 monoclonal antibody,
MHN1, followed by rhodamine-conjugated anti-mouse antibody. Staining with 49,69-
diamidino-2-phenylindole (DAPI) identi®es nuclei (blue). Bottom, ´ 500 original
magni®cation; top two panels, ,´ 31. Percentages of transfected cells with foci are
shown. Similar results were obtained with anti-Mre11 antibody.
© 2000 Macmillan Magazines Ltd
Page 4
collected as described
. Cells were irradiated using a
Cs g-irradiator at 2.55 Gy per min
(Shepherd). Ultraviolet irradiation was performed using UV Stratalinker 2400 (Strata-
gene). Hydroxyurea (Sigma) was used at 1 mM. Cell lysates were prepared 1 h after
treatment with ionizing radiation or ultraviolet unless otherwise speci®ed and after 24 h of
hydroxyurea treatment. Transfection was performed using Lipofectamine (Gibco)
according to the manufacturer's instructions.
Radioresistant DNA synthesis
NBS1 expression plasmids were co-transfected with pEGFP-N1 (Clontech) in a ratio of
10:1 into the NBS1-LBI cells. Cells were treated with 15 Gy g-irradiation and immediately
incubated in medium containing BrdU for 2 h. We detected BrdU incorporation into DNA
using a monoclonal antibody speci®c for BrdU (Becton-Dickinson) following the
immunostaining procedures described below.
Sensitivity to ionizing radiation
Plasmids containing NBS1 cDNA were co-transfected with pEGFP-N1 at a ratio of 10:1
into the NBS1-LBI cells. Cells were treated with 5 Gy g-irradiation 36 h after transfection.
The viable GFP positive cells within 2.9 cm
were counted at 72 h after ionizing radiation.
Immunostaining was carried out as described
. Rhodamine-conjugated second antibody
was obtained from Jackson Research Laboratories. Slides were mounted in Lipshaw
mountant (Immunon) and staining was analysed with a Zeiss AXIOPHOT ¯uorescence
microscope. Images were processed using the software program Adobe Photoshop 5.0.
Received 22 November 1999; accepted 27 March 2000.
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response. Science 285, 747±750 (1999).
27. Nelms, B. E., Maser, R. S., MacKay, J. F., Lagally, M. G. & Petrini, J. H. In situ visualization of DNA
double-strand break repair in human ®broblasts. Science 280, 590±592 (1998).
28. Ziv, Y., Banin, S., Lim, D.-S., Kastan, M. B. & Shiloh, Y. in Expression and Assay of Recombinant ATM
Kinase (ed. Keyse, S.) (Humana, New Jersey, 1999).
29. Chen, P. L., Scully, P., Shew, J. Y., Wang, J. Y. & Lee, W. H. Phosphorylation of the retinoblastoma gene
product is modulated during the cell cycle and cellular differentiation. Cell 58, 1193±1198 (1989).
We thank W.-H. Lee, A. Tomkinson and members of their laboratories for discussion;
A. Tomkinson, T. Boyer and P. Sung for critical reading; S.-Y. Lee for assistance on the
manuscript; M. Chen for site-speci®c mutagenesis of ATM; Q. Du for the initial studies of
ATM kinase; L. Zheng for technical advice; and S. Deb for the full-length human p53
cDNA. E.L. is supported by grants from NIH NINDS, Texas Advanced Research/Advanced
Technology Program and NCI P01. S.Z. is supported by a DOD training grant.
Correspondence and requests for materials should be addressed to E.L.
letters to nature
VOL 405
25 MAY 2000
| 477
ATM phosphorylation of Nijmegen
breakage syndrome protein is
required in a DNA damage response
Xiaohua Wu*
, Velvizhi Ranganathan
§, David S. Weisman
Walter F. Heine
§, David N. Ciccone
§, Ted B. O'Neill
Kindra E. Crick*
, Kerry A. Pierce, William S. Lane, Gary Rathbun
David M. Livingston*
& David T. Weaver
* Dana Farber Cancer Institute, 44 Binney Street, Boston, Massachusetts 02115,
Center for Blood Research, 200 Longwood Avenue, Boston, Massachusetts 02115,
Departments of Genetics and Medicine, § Department of Microbiology and
Molecular Genetics and k Department of Pediatrics, Harvard Medical School,
Boston, Massachusetts 02115, USA
Harvard Microchemistry Facility, Harvard University, Cambridge,
Massachusetts 02138, USA
Nijmegen breakage syndrome (NBS) is characterized by extreme
radiation sensitivity, chromosomal instability and cancer
. The
phenotypes are similar to those of ataxia telangiectasia mutated
(ATM) disease, where there is a de®ciency in a protein kinase that
is activated by DNA damage, indicating that the Nbs and Atm
proteins may participate in common pathways. Here we report
that Nbs is speci®cally phosphorylated in response to g-radiation,
ultraviolet light and exposure to hydroxyurea. Phosphorylation of
Nbs mediated by g-radiation, but not that induced by hydroxy-
urea or ultraviolet light, was markedly reduced in ATM cells. In
vivo, Nbs was phosphorylated on many serine residues, of which
S343, S397 and S615 were phosphorylated by Atm in vitro. At least
two of these sites were underphosphorylated in ATM cells.
Inactivation of these serines by mutation partially abrogated
Atm-dependent phosphorylation. Reconstituting NBS cells with
a mutant form of Nbs that cannot be phosphorylated at selected,
ATM-dependent serine residues led to a speci®c reduction in
clonogenic survival after g-radiation. Thus, phosphorylation of
Nbs by Atm is critical for certain responses of human cells to DNA
The Atm protein kinase is activated in response to genotoxic
stresses. Its substrates include many checkpoint-determining and
regulatory proteins, for example, p53, Chk2 and BRCA1 (refs 2±7),
indicating its central position in DNA-damage-activated signalling
pathways. We investigated whether Nbs acts in the same pathway(s)
as Atm and, if so, whether it operates downstream of the latter.
Human cells from many tissues, but not NBS cells, synthesize intact
Nbs (Fig. 1a). Following g-radiation, slower SDS±polyacrylamide
gel electrophoresis (SDS±PAGE) mobility was observed for Nbs in
several tumour-derived and primary ®broblast cell lines (Fig. 1b,
© 2000 Macmillan Magazines Ltd
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    • "Interruption of the S343 phosphorylation site has also been shown to compromise survival and radiosensitivity after DNA damage but not affect the stability of the MRN complex or capacity to form foci [112,113]. On the other hand Zhao et al. [110] found that phosphorylation at S278 and S343 was essential for the cellular response to DNA damage including S phase checkpoint activation, formation of nuclear foci and rescue of hypersensitivity to ionising radiation. In contrast to that report a later study employing expression of NBN, containing mutations in the ATM-targeted phosphorylation sites (S278,S343), did not resolve S phase checkpoint control but did resolve the ability of radiation to activate Chk2, induce nuclear foci formation and normalize radiosensitivity in NBS cells [114]. "
    [Show abstract] [Hide abstract] ABSTRACT: The recognition, signalling and repair of DNA double strand breaks (DSB) involves the participation of a multitude of proteins and post-translational events that ensure maintenance of genome integrity. Amongst the proteins involved are several which when mutated give rise to genetic disorders characterised by chromosomal abnormalities, cancer predisposition, neurodegeneration and other pathologies. ATM (mutated in ataxia-telangiectasia (A-T) and members of the Mre11/Rad50/Nbs1 (MRN complex) play key roles in this process. The MRN complex rapidly recognises and locates to DNA DSB where it acts to recruit and assist in ATM activation. ATM, in the company of several other DNA damage response proteins, in turn phosphorylates all three members of the MRN complex to initiate downstream signalling. While ATM has hundreds of substrates, members of the MRN complex play a pivotal role in mediating the downstream signalling events that give rise to cell cycle control, DNA repair and ultimately cell survival or apoptosis. Here we focus on the interplay between ATM and the MRN complex in initiating signaling of breaks and more specifically on the adaptor role of the MRN complex in mediating ATM signalling to downstream substrates to control different cellular processes.
    Preview · Article · Oct 2015
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    • "The patient was re-evaluated and a rare mutation in the NBN gene was found, resulting in the final diagnosis of NBS [76]. The molecular pathways of these two diseases clearly overlap [135,137]. "
    [Show abstract] [Hide abstract] ABSTRACT: Nijmegen breakage syndrome (NBS) is a rare autosomal recessive syndrome of chromosomal instability mainly characterized by microcephaly at birth, combined immunodeficiency and predisposition to malignancies. Due to a founder mutation in the underlying NBN gene (c.657_661del5) the disease is encountered most frequently among Slavic populations. The principal clinical manifestations of the syndrome are: microcephaly, present at birth and progressive with age, dysmorphic facial features, mild growth retardation, mild-to-moderate intellectual disability, and, in females, hypergonadotropic hypogonadism. Combined cellular and humoral immunodeficiency with recurrent sinopulmonary infections, a strong predisposition to develop malignancies (predominantly of lymphoid origin) and radiosensitivity are other integral manifestations of the syndrome. The NBN gene codes for nibrin which, as part of a DNA repair complex, plays a critical nuclear role wherever double-stranded DNA ends occur, either physiologically or as a result of mutagenic exposure. Laboratory findings include: (1) spontaneous chromosomal breakage in peripheral T lymphocytes with rearrangements preferentially involving chromosomes 7 and 14, (2) sensitivity to ionizing radiation or radiomimetics as demonstrated in vitro by cytogenetic methods or by colony survival assay, (3) radioresistant DNA synthesis, (4) biallelic hypomorphic mutations in the NBN gene, and (5) absence of full-length nibrin protein. Microcephaly and immunodeficiency are common to DNA ligase IV deficiency (LIG4 syndrome) and severe combined immunodeficiency with microcephaly, growth retardation, and sensitivity to ionizing radiation due to NHEJ1 deficiency (NHEJ1 syndrome). In fact, NBS was most commonly confused with Fanconi anaemia and LIG4 syndrome. Genetic counselling should inform parents of an affected child of the 25% risk for further children to be affected. Prenatal molecular genetic diagnosis is possible if disease-causing mutations in both alleles of the NBN gene are known. No specific therapy is available for NBS, however, hematopoietic stem cell transplantation may be one option for some patients. Prognosis is generally poor due to the extremely high rate of malignancies. Zespół Nijmegen (Nijmegen breakage syndrome; NBS) jest rzadkim schorzeniem z wrodzoną niestabilnością chromosomową dziedziczącym się w sposób autosomalny recesywny, charakteryzującym się przede wszystkim wrodzonym małogłowiem, złożonymi niedoborami odporności i predyspozycją do rozwoju nowotworów. Choroba występuje najczęściej w populacjach słowiańskich, w których uwarunkowana jest mutacją założycielską w genie NBN (c.657_661del5). Do najważniejszych objawów zespołu zalicza się: małogłowie obecne od urodzenia i postępujące z wiekiem, charakterystyczne cechy dysmorfii twarzy, opóźnienie wzrastania, niepełnosprawność intelektualną w stopniu lekkim do umiarkowanego oraz hipogonadyzm hipogonadotropowy u dziewcząt. Na obraz choroby składają się także: niedobór odporności komórkowej i humoralnej, który jest przyczyną nawracających infekcji, znaczna predyspozycja do rozwoju nowotworów złośliwych (zwłaszcza układu chłonnego), a także zwiększona wrażliwość na promieniowanie jonizujące. Wyniki badań laboratoryjnych wykazują: (1) spontaniczną łamliwość chromosomów w limfocytach T krwi obwodowej, z preferencją do rearanżacji chromosomów 7 i 14, (2) nadwrażliwość na promieniowanie jonizujące lub radiomimetyki, co można wykazać metodami in vitro, (3) radiooporność syntezy DNA, (4) hipomorficzne mutacje na obu allelach genu NBN, oraz (5) brak w komórkach pełnej cząsteczki białka, nibryny. Małogłowie i niedobór odporności występują także w zespole niedoboru ligazy IV (LIG4) oraz w zespole niedoboru NHEJ1. Rodzice powinni otrzymać poradę genetyczną ze względu na wysokie ryzyko (25%) powtórzenia się choroby u kolejnego potomstwa. Możliwe jest zaproponowanie molekularnej diagnostyki prenatalnej jeżeli znane są obie mutacje będące przyczyną choroby. Nie ma możliwości zaproponowania specyficznej terapii, ale przeszczep szpiku może być alternatywą dla niektórych pacjentów. Generalnie prognoza nie jest pomyślna z uwagi na wysokie ryzyko rozwoju nowotworu.
    Full-text · Article · Feb 2012 · Orphanet Journal of Rare Diseases
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    • "Several investigations suggest that the Mre11-Rad50-Nbs1 (MRN) complex is involved in ATM activation and recruitment to the sites of DSBs (Uziel et al, 2003; Cerosaletti & Concannon, 2004), because attenuated activation and no recruitment of ATM to DSBs upon damage were found in Mre11-and Nbs1-deficient cell lines. Earlier studies have shown that MRN lies downstream of the ATM mediated DNA damage signalling pathway because ATM can phosphorylate the components of the MRN complex in response to IR (Lim & Ki, 2000; Wu & Ranganathan, 2000; Zhao & Weng, 2000). However, further analyses demonstrate that the MRN complex is more like an upper actor of ATM pathway (Uziel et al, 2003;Difilippantonio et al, 2005; Carson et al, 2003). "
    Full-text · Chapter · Aug 2011
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