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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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
25 MAY 2000
ATM phosphorylation of Nijmegen
breakage syndrome protein is
required in a DNA damage response
, 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
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,
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