ACCELERATED PUBLICATION
The Forkhead-associated Domain of NBS1 Is Essential for
Nuclear Foci Formation after Irradiation but Not Essential for
hRAD50·hMRE11·NBS1 Complex DNA Repair Activity*
Hiroshi
Tauchi
,
Junya
Kobayashi
,
Ken-ichi
Morishima
,
Shinya
Matsuura
,
Asako
Nakamura
,
Takahiro
Shiraishi
,
Emi
Ito
,
Debora
Masnada§,
Domenico
Delia§, and
Kenshi
Komatsu
¶
From the
Department of Radiation Biology,
Research Institute for Radiation Biology and Medicine, Hiroshima
University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8553, Japan and
§ Department of Experimental Oncology, Istituto Nazionale
Tumori, Via G. Venezian 1, 20133 Milano, Italy
Received for publication, August 25, 2000, and in revised form, October 17, 2000
 |
ABSTRACT |
NBS1 (p95), the protein responsible for
Nijmegen breakage syndrome, shows a
weak homology to the yeast Xrs2 protein at the N terminus region, known
as the forkhead-associated (FHA) domain and the BRCA1 C
terminus domain. The protein interacts with hMRE11 to form a complex
with a nuclease activity for initiation of both nonhomologous end
joining and homologous recombination. Here, we show in vivo
direct evidence that NBS1 recruits the hMRE11 nuclease complex into the
cell nucleus and leads to the formation of foci by utilizing different
functions from several domains. The amino acid sequence at 665-693 on
the C terminus of NBS1, where a novel identical sequence with yeast
Xrs2 protein was found, is essential for hMRE11 binding. The
hMRE11-binding region is necessary for both nuclear localization of the
complex and for cellular radiation resistance. On the other hand, the
FHA domain regulates nuclear foci formation of the multiprotein complex
in response to DNA damage but is not essential for nuclear
transportation of the complex and radiation resistance. Because the
FHA/BRCA1 C terminus domain is widely conserved in eukaryotic
nuclear proteins related to the cell cycle, gene regulation, and DNA
repair, the foci formation could be associated with many phenotypes of
Nijmegen breakage syndrome other than radiation sensitivity.
 |
INTRODUCTION |
NBS1 is a responsible gene for Nijmegen breakage syndrome
(NBS),1 a variant of
ataxia-telangiectasia. Disruption of NBS1 in NBS patients leads to
hypersensitivity to ionizing radiation, chromosomal instability, and a
predisposition to cancer (1-3). NBS1 (p95) protein shows a weak (29%)
homology to the yeast (Saccharomyces cerevisiae) Xrs2
protein only in the N terminus regions known as forkhead-associated
(FHA) domain and BRCA1 C terminus (BRCT) domain (1-3), which are
widely conserved in eukaryotic nuclear proteins related to the cell
cycle, gene regulation, or DNA repair (4, 5). The protein is known to
interact with hMRE11 to form a complex with a nuclease activity for
initiation of both nonhomologous end joining and homologous
recombination (6, 7). However, the function of most of the NBS1 protein
is still not understood, because about 70% of the NBS1 protein on the
C terminus end does not show any sequence homology to any known proteins including Xrs2 (1-3). Because the mutations found in NBS
patients all occur between codons 220 and 385 of the NBS1 gene (3) and
lead to proteins truncated downstream of the FHA/BRCT domain,
the C-terminal half of the protein must be associated with the crucial
phenotype of NBS, which may depend on nuclear localization of
hMRE11·hRAD50 (2). Recently, we reported that expression of the
full-length NBS1 protein complements multiple NBS phenotype
characteristics such as radiation sensitivity, the G2
checkpoint, and focus formation in nucleus after irradiation (8). These
findings enable us to analyze the functional domain of NBS1 using
deletion mutants of NBS1 transfected in NBS cells. By this approach, we
localized an essential domain at C terminus region of NBS1 for hMRE11
binding. Interestingly, we found that the FHA domain at N terminus of
NBS1 is not essential for restoration of radiation resistance of NBS
patient cells but is essential for nuclear foci formation after DNA
damage by radiation.
 |
EXPERIMENTAL PROCEDURES |
Cloning of Chicken Nbs1 cDNA--
A partial fragment of the
chicken Nbs1 cDNA was obtained from chicken testis poly(A) RNA by
RT-PCR using Pyrobest DNA polymerase (TaKaRa) and
degenerate primer sets (5'-TACGTNGTNGGNMGNAARAA-3' and
5'-ATGARNGCRCADATNGTYTT-3'). A fragment representing the FHA/BRCT domains was cloned after two sets of PCR reactions, subcloned into a
pBluescript® II KS(
) vector (Stratagene), and sequenced. The
full-length cDNA was then cloned with the 3'- and 5'-rapid amplification of cDNA ends method.
Plasmid Construction--
Construction of the NBS1 expression
vector and its transfection into cells were performed as described
previously (8). Briefly, NBS1 cDNAs containing C-terminal deletions
were amplified by PCR using primers containing BamHI
sites and point mutations introducing in-frame termination codons (the
forward primer was 5'-AATATGGATCCTGGACCGATGTGGAAACTGCT-3', and reverse
primers were as follows: S744,
5'-ATAAGGGGATCCTCAAAAAAGATCATCAGCAAGAG-3'; S703, 5'-TAGATCGGATCCTCAAATGATGTGTGGAAGTTTTG-3'; S670,
5'-GCCAGGGATCCTTCAGGAAGTAGAGTTTTTAATCAC-3'; and S590,
5'-CCTTGTGGATCCTCAAACATTGACATCTTCCTC-3') and Pyrobest DNA
polymerase (TaKaRa). For construction of FHA or FHA/BRCT deletions, the
forward primer was substituted with FE3
(5'-TTAATGGGATCCACATGCAGAATGGCTTTTCCCG-3') or BRCT-d
(5'-TGCTGGATCCTTGTCATGGTATCAGTGAAA-3'), and a reverse primer for
full-length cDNA (8) was used in the PCR reaction. Amplified
cDNA was BamHI-digested and ligated into the
BamHI site of the pIRES-hyg vector
(CLONTECH). The entire cDNA insert was verified by sequencing.
For construction of yeast two-hybrid vectors, full-length NBS1 cDNA
or hMRE11 cDNA was ligated into pAS2-1
(CLONTECH) or into the GAL4-activating domain of
pACT2 (CLONTECH). Partial deletion mutant plasmids
were constructed by PCR using full-length constructs as a template,
Pyrobest DNA polymerase (TaKaRa), and oligonucleotides (22-24-mer)
designed to make an in-frame deletion. The PCR products were then
self-ligated, and the entire DNA sequence was verified.
Cell Culture and Transfection--
GM7166VA7 cells from an NBS
patient were used as an NBS cell line. HeLa cells were used as a
control cell line with normal radiation sensitivity. Cell cultures were
maintained in DMEM (Life Technologies, Inc.) supplemented with 10%
fetal bovine serum (HyClone). The vectors were transfected into
GM7166VA7 cells by electroporation using a GenePulser (Bio-Rad), and
stable transformants were selected by incubation in medium containing
200 µg/ml hygromycin B (Wako).
Cell Survival Assay--
Exponentially growing cells were
trypsinized, re-suspended in DMEM, and sealed in a glass
tube. The cells were then irradiated with 60Co
rays at a dose rate of 1.0 Gy/min. Immediately after irradiation, an
appropriate number of cells were plated in DMEM
supplemented with 10% fetal bovine serum and 10% fetal calf serum.
After 14 days of incubation, the cells were fixed with ethanol and
stained with a 4% Giemsa solution (Katayama Chemical). Surviving
fractions were calculated by comparing the number of colonies in the
experimental cells with the number of colonies formed in nonirradiated
control cells.
Western Blotting and Immunofluorescent Staining--
Whole-cell
extracts (from 2 × 106 cells) were prepared as
described (8), and 40 µg of total protein was applied to an 8% SDS
polyacrylamide gel. After electrophoresis, proteins were transferred to
a blotting membrane using an electroblot apparatus (ATTO), and
immunoblots were performed as described previously (8).
For immunofluorescent staining, cells grown on a glass slide were fixed
with cold methanol for 20 min, rinsed with cold acetone for several
seconds, and then air-dried. The slides were stained as described
previously (8). The primary antibodies used were as follows: anti-NBS1
(8), anti-hMRE11 (Novus Biologicals), and anti-hRAD50 (GeneTex).
Alexa-488-conjugated anti-rabbit IgG (Molecular Probes) was used for
visualization of NBS1 or hMRE11. Biotinylated anti-mouse IgG (Vector)
and Alexa-488-conjugated streptavidin (Molecular Probes) were used for
hRAD50. The 488-nm excited green fluorescence from the Alexa-488 dye
was visualized with a laser scanning microscope (Olympus).
Yeast Two-hybrid Analysis--
Full-length or mutated NBS1
cDNA was expressed as a fusion protein to a GAL4-DNA-binding domain
(BD) from pAS2-1 (CLONTECH) or to a
GAL4-activating domain (AD) from pACT2 (CLONTECH).
The full-length or mutated NBS1-BD (or -AD) plasmid was transfected into the yeast strain GC-1945 (CLONTECH), along
with a full-length hMRE11-AD (or -BD) plasmid. Interaction between the
expressed proteins was detected by growth on a synthetic dropout
(
Leu
Trp
His) plate and by
-galactosidase activity.
 |
RESULTS |
To locate the functionally important domain of the NBS1 gene
involved in the human NBS phenotype, we tried to determine which sequences were conserved in NBS1 in higher eukaryotes. Cloning and
sequencing of the chicken Nbs1 gene can be very useful for locating a
conserved domain, because a low homology of the NBS1 amino acid
sequence between the human sequence and the chicken sequence has been
suggested (2). The amino acid sequence of chicken Nbs1 shows apparent
homology with human and mouse NBS1 (62 and 63% identity, respectively)
at the N terminus 360 amino acids, which contains both the FHA/BRCT
domain and a phosphorylation site on a serine residue at 278 and 343 (Refs. 9 and 10 and data not shown). There is a region with low
homology from 360 to 630 (33% identity between chicken and human).
Another conserved sequence was observed at the region from 631 to 754 and the C terminus of the protein (Fig.
1). A novel identical sequence with yeast
Xrs2 protein was also found in this small region (Fig. 1). This is
consistent with the suggestion that the C-terminal half of NBS1 may
interact with hMRE11 (2). To confirm this, we generated various
C-terminal deletion mutants of NBS1 and tested their ability to bind to
hMRE11 using a yeast two-hybrid analysis. Significant interaction
between NBS1 and hMRE11 was detected only when codons 665-693 were
present in the construct even when N-terminal FHA/BRCT domains were
deleted (Fig. 2). This C-terminal region
contains a sequence highly conserved at codons 682-693 in the chicken, mouse, human NBS1, and yeast Xrs2 proteins, implying that the sequence
might be a critical region for hMRE11 binding.

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Fig. 1.
Comparison of amino acid sequence at the
C-terminal region in human, mouse, and chicken NBS1. Identical
amino acids among three organisms (white on black) and two
organisms (outlined) are indicated. A small identical
sequence with yeast (S. cerevisiae) Xrs2
protein is also shown at 678-694 of human NBS1.
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Fig. 2.
NBS1 constructs used in the present study and
their hMRE11 binding activity obtained from yeast two-hybrid
analysis. Left, a brief protein structure and designed
constructs of NBS1. FHA domain at 24-102, BRCT domain at 108-196, and
possible phosphorylation site at Ser278 or
Ser343 residues are indicated. Down arrows
represent the mutation position in NBS patients (1-3).
Right, the hMRE11 binding activity of various NBS1
constructs by means of yeast two-hybrid analysis. The columns
NBS1·MRE11 or MRE11·NBS1 represent that NBS1
fused to GAL4-DNA-BD (or NBS1 fused to GAL4-AD) was coexpressed with a
full-length hMRE11 fused to AD (or BD) in yeast strain GC-1945
(CLONTECH). -Galactosidase activities of each
transfectant are indicated by the following symbols: ++,
color change within 1 h; +, color change within 3 h; , no color change or weak color change over 12 h;
U, undetermined. Note that a weak color change for
S670-AD·hMRE11-BD (and for del 682-693) was detected after a 24-h
incubation. The black box represents the putative domain
that is essential for hMRE11 binding.
|
|
Because NBS1 is essential for the hMRE11·hRAD50·NBS1 complex to
express nuclease activity or ATP-dependent DNA unwinding
activity (11), the hMRE11-binding domain is probably necessary for the processing of damaged DNA. To assay the functions of the binding domain
in vivo, we subcloned mutant NBS1 constructs into expression vectors (shown in Fig. 2) and transfected them into the NBS cell line
GM7166VA7. All of the stable transformants expressed a significant amount of the mutant NBS1 protein (Fig.
3a). Although the expression of multiple smaller proteins were observed in the N-terminal-deleted mutants (Fig. 3a, FE3 and BRCT-d), the
expected mutant NBS1 proteins in the FE3 and BRCT-d clones were still
detected in their transformants (Fig. 3a). Because it is
known that NBS1 directly binds to hMRE11 and indirectly to hRAD50
through hMRE11 (2), we tested the ability of mutant NBS1 to form the
complex. Full-length and S703 mutant protein, containing C-terminal
conserved region, were able to form the triple-protein complex, because
they coimmunoprecipitated with hRAD50. On the other hand, S590 and
S670, in which the C-terminal conserves sequence was deleted, were not
able to form the complex (Fig. 3b). The result is consistent
with yeast two-hybrid experiment (Fig. 2), which demonstrated the
essential C-terminal domain for hMRE11 binding at 665-693. Although
the expected mutant protein from FE3 or BRCT-d was invisible in
NBS1-blot for hRAD50 precipitates (Fig. 3b), a very weak
signal was detected when the increased amount of the precipitate was
used for analysis (data not shown). Because the expected FE3 or BRCT-d
proteins were accompanied by the degraded small fragments (Fig.
3a), the N terminus mutant proteins could be unstable. The
absence of the N terminus region of the protein in the FE3 and BRCT-d
clones was confirmed by immunoblotting using antiserum that recognizes
the N-terminal end of NBS1 (data not shown). From these results, it
appears that the multiple proteins expressed in the FE3 and BRCT-d
mutants must contain functional hMRE11-binding domains at the
C-terminal NBS1 region.

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Fig. 3.
Expression of NBS1 protein in the
NBS1-transfected cell lines. a, immunoblot analysis of
NBS cells (GM7166VA7) and various NBS1 transfectants. b,
formation of the triple-protein complex in NBS cells or in
NBS1-transfected cells. Whole-cell extract was immunoprecipitated with
anti-hRAD50 antibody, and the precipitants were analyzed by
immunoblotting with anti-NBS1 (upper panel) or anti-hMRE11
antibody (lower panel). hMRE11 was always
coimmunoprecipitated with hRAD50, because they directly interact each
other. NBS1 was coimmunoprecipitated with hRAD50 when the mutant
protein contained an hMRE11-binding domain (Full,
S703, FE3, and BRCT-d).
|
|
Because restoration of radiation resistance was observed only when the
mutant proteins contained the hMRE11-binding domain at C terminus (Fig.
4), this suggests that the hMRE11-binding domain is essential for radiation resistance. This was supported by
finding that del 683-693 mutant lacking Xrs2-identical sequence could
not restore radiation resistance (data not shown). Interestingly, cells
transfected with mutant NBS1 lacking the FHA domain alone or lacking
both the FHA and BRCT domains (FE3 and BRCT-d) also became radiation
resistant to a degree similar to that seen in C-terminal end-deleted
mutant (S703 in Fig. 4). These results imply that the FHA/BRCT domain
is not essential for restoration of radiation resistance,
i.e. for DNA repair.

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Fig. 4.
Radiation sensitivity of parental NBS
(GM7166VA7)- and NBS1-transfected cells. Exponentially growing
cells were exposed to 4 Gy of rays, and survivals were determined
by colony formation. Each point represents mean ± S.D.
from at least five independent clones with duplicate experiments.
|
|
Subsequently, observations were made to see whether the various
truncated NBS1 proteins could restore the focus formation activity of
the hRAD50·hMRE11·NBS1 complex in NBS cells after irradiation,
because it has been reported that this triple-protein complex forms
foci at DNA double-strand breaks in the nucleus after exposure to
ionizing radiation (12). An absence of hMRE11 and hRAD50 in the nucleus
was observed in NBS cells (GM7166VA7), and foci did not form when cells
were irradiated with
rays (Fig. 5,
GM7166VA7; see Ref. 2). In contrast, nuclear localization of NBS1,
hMRE11, and hRAD50 was detected in NBS cells transfected with
full-length NBS1, and foci formation in the nucleus was clearly observed after irradiation (Fig. 5, +Full). A mutant of NBS1
containing the hMRE11-binding domain showed nuclear localization of
hMRE11 and hRAD50 (Fig. 5, +S703, +FE3, and
+BRCT-d), and the lack of the hMRE11-binding domain in the
S670, S590, or the del 682-693 clone resulted in the cytoplasmic
localization of the proteins. Because the cells in the absence of
triple complex in nuclei (S590, S670, and del 682-693) remained
radiation-sensitive (Fig. 4 and data not shown), these results suggest
that the nuclear localization of hMRE11 and hRAD50 is necessary to
restore the radiation resistance. Surprisingly, the FE3 and BRCT-d
cells could not form foci in response to DNA damage (Fig. 5, +IR
lanes, +FE3 and +BRCT-d) even though the
triple-protein complex was able to localize in the nucleus. To confirm
the inability of nuclear foci formation in FE3 mutant NBS1 protein, we
transfected this mutant cDNA into HeLa cells. Significant reduction
of foci formation after radiation was observed in FE3-expressed HeLa
cells, possibly by dominant negative effects (Fig.
6). This FE3-expressing HeLa cell clone showed no alteration of radiation sensitivity (data not shown), supporting the finding that FHA domain is not essential for restoration of radiation resistance.

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Fig. 5.
Localization and ionizing radiation-induced
foci formation of NBS1, hMRE11, and hRAD50 in NBS cells (GM7166VA7) or
in cells transfected with various mutants of NBS1.
Nonirradiated cells (IR ) or cells irradiated with
10 Gy of rays (IR+) were fixed at 3 h
post-treatment, and immunofluorescent staining with anti-NBS1,
anti-hMRE11, or anti-hRAD50 antibody was performed. For the del
682-693 clone, only hMRE11 localization in nonirradiated controls is
shown with 4,6-diamidino-2-phenylindole counter-staining for
identification of nucleus.
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Fig. 6.
Reduction of hRAD50·hMRE11·NBS1 focus
formation by expression of FHA-deleted NBS1. The FE3
construct was transfected in HeLa cells, and stable transfectants were
cloned. hMRE11 foci formation at 3 h after 10-Gy irradiation
was analyzed by immunofluorescent staining using anti-hMRE11 antiserum.
At least 150 hMRE11 foci-positive cells (>7 foci) were randomly
observed under a fluorescent microscope, and the numbers of hMRE11 foci
per cell were calculated. The values are expressed as mean ± S.D.
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 |
DISCUSSION |
NBS1 is reported to be essential for nuclear localization of
hRAD50·hMRE11 complex (2) and to enhance the nuclease activity of the
complex (7, 11). These observations suggest the function of NBS1 as a
key regulator of both localization and activity of the triple-protein
complex. We identified the essential region at codons 665-693 of NBS1
for hMRE11 binding. The present results also showed that the FHA/BRCT
domain of NBS1 is essential for nuclear foci formation after DNA damage
but not for cellular survival after irradiation. This is confirmed by
evidence that foci formation was repressed in FE3-transfected HeLa
cells. Zhao et al. (10) reported that the alteration
of the phosphorylation site at both Ser273 and
Ser343 residues markedly reduced the foci formation and
radiation resistance, suggesting phosphorylation of NBS1 is necessary
for both foci formation and DNA repair after irradiation. It is
consistent with our result that the expression of a mutant NBS1 protein
lacking both phosphorylation site and FHA/BRCT domain in GM7166VA cells was unable to complement not only nuclear foci formation of the complex
but also radiation resistance of the cells (data not shown). Therefore,
we conclude that FHA/BRCT domain, possibly sole FHA domain, is
essential for the nuclear foci formation of the triple-protein complex
together with the presence of both hMRE11-binding domain and the serine
residues for phosphorylation.
A number of DNA repair-related proteins are known to form nuclear foci
in response to DNA damage, such as RAD51, BRCA1, and BLM, as
well as the hRAD50·hMRE11·NBS1 complex (1, 8, 12, 13), and these
might be affected or regulated by phosphorylation signals. In view of
this, the failure of the triple-protein complex in the FE3 and BRCT-d
clones to form nuclear foci supports the putative functions of the FHA
and BRCT domains, namely the FHA domain motif is for protein-protein
interactions that recognize the phosphorylation state of the target
protein (14), and the BRCT domain might provide a DNA-binding domain
for repair-related proteins (15). The results shown here indicate that
FHA/BRCT domain is essential for nuclear foci formation activity
following DNA damage, even though they are not directly related to the
DNA repair ability itself. This finding is consistent with the fact that most of the DNA double-strand breaks are rejoined within the first
hour after irradiation (16), but foci formation persists even 5-8 h
after irradiation (8, 12). Taken together, it is suggested that the
nuclear foci formation is not a strict hallmark of DNA repair. Because
the FHA/BRCT domain is conserved in eukaryotic NBS1 homologue, they
might be involved in other crucial phenotypes of NBS, such as in
insuring the fidelity of the rejoined DNA.
 |
ACKNOWLEDGEMENT |
We are grateful to Dr. L. N. Kapp at
University of California, San Francisco, for editing the manuscript and
Dr. S. Takeda and Dr. M. Takata at Kyoto University and
Dr. N. Tsuyama at Radiation Effects Research Foundation, Hiroshima,
Japan for helpful comments. We also thank Taeko Jo, Miki Ueda,
Aoi Kodama, and Aya Okamoto for laboratory assistance.
 |
FOOTNOTES |
*
This work was supported in part by the Ministry of
Education, Science, Sports and Culture of Japan.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF230342.
¶
To whom correspondence should be addressed. Tel.:
81-82-257-5809; Fax: 81-82-256-7101; E-mail:
komatsu@hiroshima-u.ac.jp.
Published, JBC Papers in Press, November 2, 2000, DOI 10.1074/jbc.C000578200
 |
ABBREVIATIONS |
The abbreviations used are:
NBS, Nijmegen
breakage syndrome;
FHA, forkhead-associated;
BRCT, BRCA1 C terminus;
PCR, polymerase chain reaction;
DMEM, Dulbecco's modified Eagle's
medium;
Gy, gray;
BD, binding domain;
AD, activating domain;
del, deletion.
 |
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