MINIREVIEW
Dual role of Nbs1 in the ataxia telangiectasia mutated-
dependent DNA damage response
Joo-Hyeon Lee and Dae-Sik Lim
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Guseong-D, Yuseong-G, Daejeon, Korea
Eukaryotic cells have evolved a signaling pathway that
is activated by DNA damage. The primary function of
this pathway is to sense DNA strand breaks and then
to amplify the initial signal and convey it to down-
stream effectors that regulate cell cycle checkpoints
and DNA repair [1]. Activation of the DNA damage
signaling pathway by DNA double-strand breaks thus
leads either to arrest of cell cycle progression and
repair of the DNA breaks or, if the damage is too
extensive, to death of the cell by apoptosis, thus ensur-
ing the maintenance of genomic stability. Dysfunction
of this pathway has potentially severe consequences,
such as the development of cancer or other conditions
related to genomic instability [2].
Among many proteins that participate in the DNA
damage signaling pathway, ataxia telangiectasia
mutated (ATM) plays a central role. This serine–thre-
onine kinase is rapidly activated in response to DNA
strand breakage and phosphorylates many targets
important in DNA repair or cell cycle checkpoint acti-
vation [3,4]. The ATM gene was found to be mutated
in individuals with ataxia telangiectasia (AT), a rare
autosomal-recessive disorder with pleiotropic clinical
phenotypes, including progressive neuronal degener-
ation, oculocutaneous telangiectasia, immune dysfunc-
tion, cancer predisposition and premature aging. Cells

Abbreviations
AT, ataxia telangiectasia; ATLD, AT-like disorder; ATM, ataxia telangiectasia mutated; BRCT, Brca1 COOH-terminus; Chk2, checkpoint kinase
2; FHA, forkhead associated; IR, ionizing radiation; MRN, Mre11–Rad50–Nbs1; NBS, Nijmegen breakage syndrome; RDS, radioresistant DNA
synthesis.
1630 FEBS Journal 273 (2006) 1630–1636 ª 2006 The Authors Journal compilation ª 2006 FEBS
cell cycle, radiation hypersensitivity and an increased
frequency of chromosome breakage.
A complex of Mre11, Rad50 and Nbs1 proteins (the
so-called MRN complex) is another key player in the
DNA damage signaling pathway [5]. The MRN com-
plex is the primary constituent of nuclear foci that
form rapidly after exposure of cells to ionizing radi-
ation (IR) and which represent sites of ongoing sensing
or repair of DNA double-strand breaks. Hypomorphic
mutations of the Nbs1 gene in humans give rise to
Nijmegen breakage syndrome (NBS), which is charac-
terized by microcephaly, immunodeficiency, chromoso-
mal instability, predisposition to cancer and cells that
show hypersensitivity to IR and abnormal S-phase
checkpoint control [6,7]. Germline hypomorphic muta-
tions of the Mre11 gene also result in an AT-like dis-
order (ATLD) [8]. The phenotypic similarities among
AT, NBS and ATLD indicate that the MRN complex
functions in the ATM-dependent signaling pathway
activated by DNA damage [9]. In this review, we will
discuss recent advances in our understanding of Nbs1
function in the ATM-dependent DNA damage signa-
ling pathway.
Functional domains of Nbs1 relevant to
the DNA damage signaling pathway

the BRCT domain was essential for radiation resist-
ance [13]. These discrepancies are probably caused by
differences in the doses of radiation, in Nbs1 muta-
tions, or in Nbs1 expression levels among the studies.
The FHA and BRCT domains participate in the
interaction of Nbs1 with the phosphorylated histone,
c-H2AX, which occurs near sites of DNA strand
breakage [14]. Mouse cells that lack c-H2AX do not
form Nbs1 foci after exposure to IR, suggesting that
the direct interaction of Nbs1 with c-H2AX is required
for foci formation by the MRN complex [15]. Cell
cycle checkpoint control appears largely intact in the
c-H2AX-deficient cells, however, suggesting that foci
formation is not directly related to checkpoint func-
tion. The MRN-interacting protein, MDC1, was
recently shown to contribute to the formation of foci
containing Nbs1, 53BP1 and Brca1, on the basis of the
observation that down-regulation of MDC1 prevented
the formation of such foci in response to IR [15–18].
Whether or not the FHA or BRCT domains of Nbs1
directly interacts with MDC1 remains unclear.
Although the functional relevance of the FHA and
BRCT domains of Nbs1 appears to differ among stud-
ies, we can conclude that both domains are required
for recruitment of the MRN complex to DNA lesions
(possibly through interaction with c-H2AX or MDC1)
and consequent foci formation, as well as for cell sur-
vival after exposure to IR.
Two serine residues at positions 278 and 343 of
human Nbs1 are phosphorylated by ATM on exposure

induced foci formation and radiation resistance. Phosphorylation
of Ser278 and Ser343 by ataxia telangiectasia mutated (ATM) is
essential for activation of the S-phase checkpoint. The Mre11-bind-
ing domain is responsible for binding to Mre11 during formation of
the Mre11–Rad50–Nbs1 (MRN) complex. The ATM-binding domain
at the COOH-terminus binds to ATM and mediates recruitment of
ATM to IR-induced foci.
J H. Lee and D S. Lim Role of Nbs1 in ATM-dependent DNA damage signaling
FEBS Journal 273 (2006) 1630–1636 ª 2006 The Authors Journal compilation ª 2006 FEBS 1631
[19–21]. This finding might explain, in part, the failure
of cells from patients with AT or NBS to arrest DNA
synthesis in response to IR (radioresistant DNA syn-
thesis, RDS). However, it remains controversial
whether Nbs1 phosphorylation is also required for
radiation resistance or IR-induced formation of MRN
foci [19–21].
The Mre11-binding domain of human Nbs1 has
been localized to amino acids 682–693 in the COOH-
terminal region of the protein. Deletion of this region
of Nbs1 results in a cellular phenotype virtually identi-
cal to that of NBS, including defective formation of
MRN foci, radiation hypersensitivity and the impair-
ment of checkpoint control [10,22]. These observations
suggest that the association of Nbs1 with Mre11–
Rad50 is essential for its role in the DNA damage
response. In addition, the extreme COOH-terminal
region (amino acids 734–754) of Nbs1 mediates the
interaction of Nbs1 with ATM and the recruitment of
ATM to sites of DNA damage, thereby promoting
ATM-dependent signaling [23]. Domains similar to

Nbs1 is essential for S-phase checkpoint control. The
observation that the RDS phenotype of NBS cells is
less pronounced than that of AT cells suggested that
the S-phase checkpoint might also be regulated in an
Nbs1-independent manner [27]. Indeed, the kinase,
checkpoint kinase 2 (Chk2) was shown to be a target
of ATM in S-phase checkpoint control, indicating that
ATM regulates two parallel pathways to achieve such
control. However, phosphorylation of Chk2 by ATM
also requires Nbs1 in cells subjected to low-dose irradi-
ation (1–2 Gy); it does not require Nbs1 in those
exposed to high-dose radiation (> 4 Gy) [10,28].
Together, these various observations suggest that sign-
aling by ATM and Nbs1 may differentially influence
SMC1 or Chk2 in S-phase checkpoint control, depend-
ing on the extent of DNA damage.
The contribution of Nbs1 to the G1 and G2 ⁄ M
checkpoints remains controversial. NBS cells have
been found to be defective in the induction of p53 and
in G1 checkpoint control in some studies, but not in
others [19,29–32]. A partial defect in G1 checkpoint
control, and in the induction of p53 and p21, was
apparent in NBS cells exposed to low-dose radiation,
but not in those subjected to high-dose irradiation. In
addition, the activation of Chk2 in the G2 ⁄ M check-
point was found to be impaired in NBS cells after low-
dose irradiation [28], but G2 ⁄ M checkpoint control in
NBS cells was found to be normal in other studies
[10,28,33]. Similar discrepancies have arisen in studies
of mice with mutations in the Nbs1 gene [9]. Most

Regulation of ATM targets, such as Chk2 and SMC1,
has consistently been found to be largely dependent on
the MRN complex, even though Nbs1 is not abso-
lutely required for Chk2 or SMC1 phosphorylation
by ATM in cells exposed to high doses of IR
[10,11,25,28,39]. Together, the available data suggest
that Nbs1 functions as a downstream target of ATM,
as well as a modulator of ATM activity, facilitating
ATM activation and ATM-dependent phosphorylation
of many downstream substrates in the ATM-depend-
ent DNA damage signaling pathway [10,11,39].
Given that ATM is a central player in the cellular
response to DNA strand breakage, it is important to
understand the mechanisms both by which it is activa-
ted and by which it signals to downstream effectors in
cells with DNA strand breaks. To date, more studies
have focused on the identification of downstream tar-
gets of ATM [3] than on the molecular mechanism of
ATM activation. Important insight into the mechanism
of ATM activation has been provided by a recent
study [40] showing that ATM exists as a catalytically
inactive dimer or higher-order multimer in the absence
of DNA damage. In response to DNA damage, how-
ever, ATM undergoes rapid autophosphorylation on
Ser1981, resulting in dissociation of the inactive
homodimers or multimers to yield active monomers.
This autophosphorylation of ATM is triggered in cells
within minutes after low-dose irradiation, or even in
the presence of two exogenous DNA strand breaks per
cell [40]. In addition to DNA damage, chromatin

DNA strand breaks [41]. Under these conditions, an
MRN complex containing an Nbs1 protein in which
Ser343 is replaced with alanine failed to stimulate
ATM activity, suggesting that both the presence of
Nbs1 and its phosphorylation by ATM are required
for stimulation of ATM activity by the MRN complex.
In contrast to the lack of a requirement of DNA
strand breaks for ATM activation in this latter study,
highly purified inactive ATM dimers or multimers were
found, in the second study, to be activated by the
MRN complex only in the presence of DNA strand
ends, resulting in the phosphorylation of downstream
targets [42]. Furthermore, both Nbs1 and the unwind-
ing of DNA ends by Mre11–Rad50 were found to be
sufficient for stimulating ATM activity in vitro. The
presence of both the MRN complex and DNA strand
breaks thus appeared to result in the efficient convers-
ion of inactive ATM dimers or multimers to active
monomers. Consistent with this result, the extreme
COOH-terminal region of Nbs1 is responsible for
association with ATM and the recruitment of ATM to
sites of DNA strand breakage [23].
Surprisingly, mutation of the autophosphorylation
site of ATM (Ser1981 to alanine) affected neither the
dimer-to-monomer transition of ATM nor the stimula-
tion of its kinase activity induced by the MRN com-
plex in the presence of DNA strand breaks in vitro
[42], suggesting that autophosphorylation of ATM on
Ser1981 is not required for ATM activation induced
by the MRN complex and DNA breaks. This conclu-

erally accepted that Nbs1 plays a dual role both as a
downstream target and an upstream regulator of ATM
(Fig. 2). The role of Nbs1, as an upstream regulator of
ATM, appears both to depend on the dose of radi-
ation to which cells are exposed as well as to be differ-
entially affected by Nbs1 gene mutations. Cells from
NBS or ATLD patients, with hypomorphic mutations
in the corresponding genes, still manifest partial ATM
activity as a result of the expression of truncated Nbs1
or Mre11, respectively; such cells thus exhibit only par-
tial checkpoint defects after exposure to low doses of
radiation. In cells subjected to low-dose irradiation,
Nbs1 is required for both activation of ATM and its
recruitment to sites of DNA damage. In contrast,
Nbs1 is no longer necessary for ATM activation and
subsequent checkpoint control (with the exception of
the intra S-phase checkpoint) in cells exposed to high
doses of radiation. High doses of IR may generate
more DNA strand breaks and abnormal chromatin
structures that exceed a threshold for the activation of
ATM in the absence of Nbs1.
Acknowledgements
D S.L. was supported by the National Research
Laboratory Program and the 21st Century Frontier
Functional Human Genome Project of Korea.
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