Accelerat ing t he world's research.

DNA damage-induced G2–M

checkpoint activation by histone
H2AX and 53BP1
Julio C. Morales

Nature Cell Biology

Cite this paper Downloaded from Academia.edu 

Get the citation in MLA, APA, or Chicago styles

Related papers Download a PDF Pack of t he best relat ed papers 

Dist inct domains in Nbs1 regulat e irradiat ion-induced checkpoint s and apopt osis
Pet er McKinnon

Recognit ion, signaling, and repair of DNA double-st rand breaks produced by ionizing radiat ion in mam…
Larry T hompson

T he FHA and BRCT domains recognize ADP-ribosylat ion during DNA damage response
Chao-yie Yang
 brief communications

DNA damage-induced G2–M checkpoint

activation by histone H2AX and 53BP1
Oscar Fernandez-Capetillo*, Hua-Tang Chen*, Arkady Celeste*, Irene Ward†, Peter J. Romanienko‡,
Julio C. Morales§, Kazuhito Naka#, Zhenfang Xia§, R. Daniel Camerini-Otero‡, Noboru Motoyama#,
Phillip B. Carpenter§, William M. Bonner¶, Junjie Chen† and André Nussenzweig*||
*Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
†Division of Oncology Research, Mayo Clinic, 200 First Street, S. W., Rochester MN 55905, USA
‡Genetics and Biochemistry Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA
§Department of Biochemistry and Molecular Biology, Univeristy of Texas Health Sciences Center, Houston, TX 77030, USA
#Department of Geriatric Research, National Institute for Longevity Sciences (NILS), 36-3 Gengo, Morioka, Obu, Aichi 474-8522, Japan
¶Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
||e-mail: andre_nussenzweig@nih.gov

Published online: 25 November 2002, DOI: 10.108/ncb884

Activation of the ataxia telangiectasia mutated (ATM)
kinase triggers diverse cellular responses to ionizing radi-
ation (IR), including the initiation of cell cycle check-
points1. Histone H2AX, p53 binding-protein 1 (53BP1) and
Chk2 are targets of ATM-mediated phosphorylation2–5, but
little is known about their roles in signalling the presence
of DNA damage. Here, we show that mice lacking either
H2AX or 53BP1, but not Chk2, manifest a G2–M check-
point defect close to that observed in ATM−/− cells after
exposure to low, but not high, doses of IR. Moreover,
H2AX regulates the ability of 53BP1 to efficiently accu-
mulate into IR-induced foci. We propose that at threshold
levels of DNA damage, H2AX-mediated concentration of
53BP1 at double-strand breaks is essential for the ampli-
fication of signals that might otherwise be insufficient to
prevent entry of damaged cells into mitosis.
However, by counting the number of B lymphocytes in mitosis
under the microscope before and after irradiation, a milder G2
arrest in H2AX-deficient cells was apparent at the lowest dose
examined (0.75 Gy)13. To determine whether H2AX is involved in
the G2–M checkpoint, we exposed activated H2AX+/+, H2AX−/− and
ATM−/− splenic B cells to various doses of IR and measured the pro-
portion of mitotic cells by flow cytometry using anti-phospho-his-
tone H3 antibodies15. In the absence of IR, the frequency of mitot-
ic cells was comparable in H2AX+/+ and H2AX−/− B cells (Fig. 1a).
However, 1 h after exposure to 0.5 Gy, 60–80% fewer H2AX+/+ cells
entered mitosis, whereas almost no change was observed in the fre-
quency of H2AX−/− mitotic cells (Fig. 1a, b). H2AX−/− B cells had a
partially impaired G2–M checkpoint at doses up to 5 Gy (Fig. 1c),
beyond which a normal number of H2AX−/− cells arrested in G2. In
contrast, ATM−/− cells had a deficient G2–M checkpoint at both low
and high doses of IR16. The degree of the checkpoint defect seemed
to be less severe in H2AX−/− MEFs, compared with H2AX−/− B cells

T
he ‘mean lethal’ dose of IR in humans is estimated to be 4 Gy,
which is defined as the dose that causes 50% mortality
(http://www.nrc.gov/). Among the cellular responses to IR, cell
cycle checkpoints function to protect cells from permanent damage
and transformation by delaying progression of the cell cycle until
the DNA is repaired. However, most of the current research proto-
cols used for the analysis of IR-induced cellular checkpoints are per-
formed at doses above 4 Gy. The large-scale DNA damage produced
by high-dose irradiation may be sufficient to activate redundant
checkpoint signalling cascades, and therefore may confound inter-
pretations concerning the role of a number of proteins in regulating
the response to DNA damage. For example, conflicting conclusions
have been drawn as to the nature of damage-induced cell cycle arrest
in cells from patients with Nijmegen breakage syndrome (NBS)6,7.
These data are not necessarily inconsistent, as several groups have
shown that NBS cells exhibit G1–S and G2–M checkpoint defects at
low, but not at high doses, of IR8–10. Similarly, Chk2−/− mouse embry-
onic fibroblasts (MEFs) were shown to have a dose-dependent G1–S
checkpoint defect11,12. Together, these results suggest that deficien-
cies in some ATM kinase substrates may result in intermediate
checkpoint defects.
Phosphorylation of histone H2AX (γ-H2AX) is among the ear-
liest ATM-dependent responses to DSBs2,3. Recently, it has been
demonstrated that loss of H2AX in mice results in genomic insta-
bility and defects in concentrating various DNA repair factors into
IR-induced nuclear foci13,14. No severe abnormalities in IR-induced
cell cycle checkpoints were observed in H2AX deficient cells13,14.
(Fig. 1c, d). However, an impaired IR-induced arrest in G2 was con-
sistently observed at the lowest IR doses in the absence of H2AX.
The tumour suppressor 53BP1 is a putative mammalian homo-
logue of scRad9, an important checkpoint regulator in yeast that
forms irradiation-induced foci which colocalize with γ-H2AX
foci4,5,17. To determine whether 53BP1 is involved in γ-H2AX for-
mation or the G2–M checkpoint, we analysed the response of
53BP1−/− cells (see Methods) to IR. In contrast to H2AX−/− cells,
which had impaired 53BP1 foci formation (see below)13, H2AX was
phosphorylated and γ-H2AX foci formed normally in 53BP1−/− B
cells (Fig. 2a). However, the G2–M checkpoint analysis showed that
similar to H2AX-deficient cells, 53BP1−/− cells were less responsive
to IR, exhibiting a fivefold greater threshold than normal before a
significant number of cells arrested in G2 (Fig. 2b). These results,
together with the finding that 53BP1−/− mice have an almost identi-
cal phenotype as H2AX−/− mice (including growth retardation, radi-
ation sensitivity and genomic instability (ref. 13; I.W. and J.C.,
unpublished observations), place 53BP1 downstream of H2AX in
the response to DNA damage.
ATM phosphorylates numerous targets that mediate cell cycle
checkpoints and DNA repair, including Chk2, Nbs1, 53BP1 and
H2AX3−5,18. In NBS cells, the G2–M checkpoint defect observed after
low-dose IR correlated with impaired ATM-dependent phosphoryla-
tion of Chk2 (ref. 9). Moreover, a recent study reported a modest
defect in the phosphorylation of Chk2 and Brca1 in human cells in
which 53BP1 expression was blocked with inhibitory RNA
(siRNA)19. To determine whether H2AX and 53BP1 affect the ability

NATURE CELL BIOLOGY VOL 4 DECEMBER 2002 www.nature.com/naturecellbiology 993

©2003 Nature Publishing Group

brief communications

a
Control IR (0.5 Gy)
104 104

103 2.3 103 0.5

b
102 102
H2AX+/+ 110

Percentage of P-H3 cells

101 101 100
90
80
100 100
100 101 102 103 104 100 101 102 103 104
70
60
104 104 50
40
103 1.9 103 1.8 30
20 H2AX–/–
H2AX–/– 10 H2AX+/+
102 102 0
0 20 40 60 80 100 120 140
101 101 Time (min)
P-H3

100 100
100 101 102 103 104 100 101 102 103 104

DNA content

c d
100 100
Percentage of P-H3 cells

Percentage of P-H3 cells

90 H2AX+/+ 90 H2AX+/+
80 H2AX–/– 80 H2AX–/–
70 ATM–/– 70
60 60
50 50
40 40
30 30
20 20
10 10
0 0
0 2 4 6 8 10 0 2 4 6 8 10
Dose (Gy) Dose (Gy)

Figure 1 H2AX is required for low-dose IR-induced G2 arrest. a, Flow cyto-
metric profiles of the cell cycle distribution of H2AX+/+ and H2AX−/− stimulated B
cells before and 1 h after treatment with 0.5 Gy IR. Propidium iodide staining for
DNA content (x axis) and for histone H3 phosphorylation (y axis) is used to distin-
guish mitotic cells from G2 cells. b, The percentage of mitotic cells positive for
phoshorylated histone H3 (P-H3) (irradiated/unirradiated) (as a function of time in
H2AX+/+ and H2AX−/− B cells after exposure to 0.5 Gy IR. c, The percentage of
mitotic cells before and after exposure to IR (0, 0.5, 1, 2.5, 5 and 10 Gy) was
analysed in H2AX+/+, H2AX−/− and ATM−/− stimulated B cells. d, The induction of the
G2–M checkpoint in H2AX+/+ and H2AX−/− MEFs was analysed 1 h after exposure to
different doses of IR, as described in c. Data are representative of at least three
independent experiments.

of ATM to be activated by DNA damage, we used western blotting scRad53 is essential for IR-induced arrest in Saccharomyces cerevisi-
to examine hyperphosphorylation of Chk2 in response to IR. After ae23. A recent study showed that decreased levels of 53BP1 in human
treating B cells with IR, we observed a dose-dependent decrease in cells results in an impaired G2 and intra-S phase checkpoint arrest
the mobility of Chk2 (Fig. 3a, b), which reflects its phosphorylation that correlates with a reduction in the Thr 68-phosphorylated form
state20. Comparison of the IR-induced mobility shift of Chk2 (or of Chk2 (Chk2-T68-P) and a failure of Chk2-T68-P to concentrate
Nbs1, data not shown)13 identified no large differences between in IR-induced foci19. To evaluate the role of Chk2 in the G2–M
H2AX+/+ versus H2AX−/− or 53BP1−/− versus 53BP1+/+ cells, even at checkpoint, we analysed the IR response of Chk2-deficient B cells
low IR doses. In marked contrast, no Chk2 or Nbs1 (data not and MEFs12 (Fig. 3d, e). In marked contrast to H2AX and 53BP1,
shown) migration delay was observed in ATM−/− B cells at doses up Chk2 seemed to be dispensable for a proper induction of the G2–M
to 5 Gy (Fig. 3a), demonstrating the ATM-dependence of these checkpoint at both low and high doses of radiation (Fig. 3d, e).
phosphorylation events at low doses of IR18. At 10 Gy, however, we To determine what aspects of 53BP1 function could be affected
did detect phosphorylation of Chk2 and H2AX in ATM−/− B cells by H2AX deficiency, we performed a detailed analysis of the
(Fig. 3a, c), consistent with previous studies suggesting the exis- responses of 53BP1 to IR in H2AX+/+ and H2AX−/− cells. As previ-
tence of redundant pathways for activation of these proteins at high ously reported for human cells4,5,17, 53BP1 formed nuclear foci very
doses20–22. However, ATM was required for phosphorylation of rapidly after IR in mouse cells, which colocalized with γ-H2AX foci
H2AX at low doses (Fig. 3c)3. Although we cannot rule out the pos- (Fig. 4a, b). 53BP1 foci were already detectable 5 min after irradia-
sibility that there are partial defects in phosphorylation of check- tion, but the average number of foci per cell increased markedly by
point kinase substrates in H2AX−/− and 53BP1−/− cells19 (see below), 1 h post-irradiation (Fig. 4c)17, which paralleled the onset of G2–M
our data indicate that a major impairment of ATM function is checkpoint activation (Fig. 1b). In contrast, 53BP1 foci formation
unlikely to be the primary cause of the G2–M checkpoint defect. was severely compromised in H2AX−/− cells (Fig. 4b)13. Although
scRad9-dependent phosphorylation of the Chk2 homologue H2AX did not affect the overall levels of 53BP1, phosphorylation of

994 NATURE CELL BIOLOGY VOL 4 DECEMBER 2002 www.nature.com/naturecellbiology

©2003 Nature Publishing Group

 brief communications

Dose (Gy)
a
0 0.5 1 2.5 5 10
53BP1–/– γ-H2AX

b
Control IR

100

Percentage of p-H3 cells

90 53BP1+/+
53BP1+/+ 80 53BP1–/–
70
60
50
40
30
20
10
53BP1–/– 0
0 2 4 6 8 10

Dose (Gy)

γ-H2AX foci

Figure 2 53BP1 regulates the G2–M checkpoint. a, γ-H2AX formation was analysed by western blotting in 53BP1−/− stimulated B cells (top). The distribution of γ-H2AX
foci (green) was analysed in 53BP1+/+ and 53BP1−/− stimulated B cells before and after exposure to 1 Gy (bottom). DNA was counterstained with the TOPRO-3 dye (red).
Similar results were obtained after treatment with 3 Gy (data not shown). b, The percentage of mitotic cells positive for phoshorylated histone H3 (P-H3) before and after
exposure to IR was analysed in 53BP1+/+ and 53BP1−/− stimulated B cells, as described in Fig. 1. Data are representative of at least three independent experiments.
NIH3T3, No IR

NIH3T3, 6 Gy

Dose (Gy)
a b Dose (Gy)
0 0.5 1 2.5 5 10
0 0.25 0.5 1 2.5 5 10
Chk2PP
H2AX+/+ Chk2 Chk2PP
53BP1+/+
Chk2
Chk2PP
H2AX–/– Chk2
53BP1–/– Chk2PP
Chk2
Chk2PP
ATM–/– Chk2
d
Percentage of P-H3 cells

100
90 Chk2+/+
80 Chk2–/–
70
c Dose (Gy) 60
50
0 0.5 1 2.5 5 10 40
30
H2AX+/+ γ-H2AX 20
20
0
H2AX–/– 0 2 4 6 8 10
Dose (Gy)
e
ATM–/–
Percentage of P-H3 cells

γ-H2AX 100
90 Chk2+/+
80 Chk2–/–
70
60
50
40
30
20
20
0
0 2 4 6 8 10
Dose (Gy)

Figure 3 Chk2 phosphorylation in ATM−/−, H2AX−/− and 53BP1−/− cells and
G2–M checkpoint in Chk2−/−− cells. a, b, Lysates from wild-type, H2AX−/− and ATM−
/−
(a) or 53BP1+/+ and 53BP1−/− (b) stimulated B cells exposed to varying doses of IR
were used to analyse the phosphorylation status of Chk2 by western blotting. The IR-
induced migratory shift is shown for 3T3 cells. Chk2pp indicates the position of phos-
phorylated Chk2. c, γ-H2AX formation was analysed by western blotting in wild-type,
H2AX−/− and ATM−/− stimulated B cells exposed to several doses of IR, as described in
a. d, e, The induction of the G2–M checkpoint in Chk2+/+ and Chk2−/− stimulated B
cells (d) or MEFs (e) was analysed 1 h after exposure to different doses of radiation,
as described in Fig. 1. In both d and e, the percentage of mitotic cells are scored as
being positive for phosphorylated histone H3 (P-H3).

NATURE CELL BIOLOGY VOL 4 DECEMBER 2002 www.nature.com/naturecellbiology 995

©2003 Nature Publishing Group

brief communications

a d H2AX+/+ H2AX–/–
0 1 3 0 1 3 (Gy)
53BP1S25P
53BP1

γ-H2AX 53BP1 Merge 0.7

Normalized 53BP1S25P
levels (arbitrary units)
0.6
0.5 H2AX–/–
b Control IR H2AX+/+
0.4
0.3
0.2
H2AX+/+ 0.1
0
0 1 3
Dose (Gy)

H2AX–/–
e Low number of DSBs

ATM
53BP1
H2AX γ-H2AX
c
γ-H2AX
45 Modified chromatin
Number of foci per cell

40
35
30
25
53BP1
20
15 Foci, signal amplification
10
5
0
0 5 10 60 16 hr
G2 Mitosis
Time (min)

Figure 4 H2AX regulates IR-induced 53BP1 foci formation. a, Colocalization
of γ-H2AX with 53BP1 foci. Wild-type MEFs were exposed to 1 Gy IR and then
incubated at 37 °C for 1 h before staining for γ-H2AX (red) and 53BP1 (green).
Images were merged to determine colocalization (yellow). b, Distribution of
53BP1 (green) was analysed in H2AX+/+ and H2AX−/− MEFs before and 1 h after
exposure to 1 Gy IR. Similar results were obtained after exposure to 3 Gy (data
not shown). DNA was counterstained with DAPI (blue). c, The number of foci per
cell (H2AX+/+ MEFs) averaged over 25 cells per data point. d, H2AX+/+ and H2AX−
/−
stimulated B cells were untreated or exposed to IR and nuclear extracts were
prepared 1 h after treatment. 200 µg of nuclear extracts were immunoprecipitat-
ed with a phospho-specific antibody (see Supplementary Information, Fig. S1) that
recognizes 53BP1 phosphorylated at Ser 25 (53BP1S25P)27,28. The immunoprecipi-
tates were analysed by Western blotting with 53BP1-specific antibodies. 15 µg of
these lysates were used for western blot analysis of 53BP1 levels. The ratio of
53BP1 phosphorylation on Ser 25 to the total amount of 53BP is plotted below
e, A model for the ATM/H2AX/53BP1-mediated regulation of the G2–M check-
point. In response to low-dose IR, activated ATM phosphorylates a wide range of
targets, including H2AX and 53BP1. H2AX is essential for the accumulation of
53BP1 and other factors into the chromatin microenvironment, within megabases
from the actual DSB. This localized concentration of signal transduction complex-
es would generate sufficient amplification of the initial signal to abort the entry of
G2 cells into mitosis.

53BP1 was reduced by 40% in H2AX−/− cells, relative to controls 1 h
after treatment with 3 Gy (Fig. 4d; also see Supplementary
Information, Fig. S1). Although it is unclear whether 53BP1 is
phosphorylated only when bound to damaged DNA, these results
suggest that H2AX may not be essential for the initial recruitment
of 53BP1 to sites of DSBs. Consistently, we have found that
although H2AX is a central regulator of IR-induced focus forma-
tion, the initial migration of 53BP1 (and other signalling proteins,
such as Nbs1) to DSBs is independent of H2AX (A. C., O. F-C. and
A.N., unpublished observations).
On the basis of these experiments, we conclude that abrogation
of the G2–M checkpoint in H2AX−/− cells in response to low-dose
IR is independent of Chk2, but is linked to the inefficient accumu-
lation 53BP1 at sites of damage. We propose the following model in
which ATM-mediated phosphorylation of histone H2AX results in
a large increase in the local concentration of 53BP1 in the chro-
matin region adjacent to the DSB (Fig. 4e). The accumulation of
53BP1 and other factors in close proximity to a DSB would func-
tion as a platform for interactions between multi-component sig-
nal transduction complexes, facilitating the activation of the check-
point. This model predicts that H2AX and 53BP1 deficiency should
result in impaired phosphorylation of downstream effectors.
Indeed, we have observed a partial defect in phosphorylation of
53BP1 in H2AX−/− cells and a recent study reported that 53BP1 reg-
ulates Brca1 phosphorylation19, which is essential for the G2–M
checkpoint15. H2AX-mediated concentration of signalling com-
plexes at DSBs would be critical under conditions in which there is
a low level of DNA damage. Above a threshold, there would be suf-
ficient activation of checkpoint effectors to propagate signalling
cascades along pathways independent of H2AX.

996 NATURE CELL BIOLOGY VOL 4 DECEMBER 2002 www.nature.com/naturecellbiology

©2003 Nature Publishing Group


Methods
Mice and cell lines
Generation of ATM−/−, H2AX−/− and Chk2−/− mice have been described elsewhere12,13,24. 53BP1−/− mice
were generated by gene targeting25. p53−/−H2AX−/− and Chk2−/− e13.5 MEFs were obtained by standard
procedures. Splenic B lymphocytes were isolated using CD19 microbeads (Miltenyi Biotec, Auburn,
CA) and stimulated for 48 h with lipopolysaccharide (LPS) and interleukin-4 (IL-4), as described13.
IR induced G2–M cell cycle checkpoint, immunofluorescence and western
blot analysis
For G2–M checkpoints, MEFs and activated B cells were mock-treated or irradiated, incubated at
37 °C for the indicated times, stained with antibodies recognizing phosphorylated histone H3 (1:50
dilution; Upstate Biotechnology, Waltham, MA) and the percentage of mitotic cells quantified by flow
cytometry, as previously described15. Primary antibodies used for immunofluorescence were rabbit anti
γ-H2AX (1:500), mouse anti-γ-H2AX (1: 500; Upstate Biotechnology) and rabbit anti-53BP1 (1:100)26.
All secondary antibodies conjugated with Alexa-568 or Alexa-488 (Molecular Probes, Eugene, OR)
were used at a dilution of 1:250. Immunofluorescence staining of B cells and MEFs performed as
described13. For western blotting, rabbit polyclonal Chk2 (1:5000, Upstate Biotechnology), Nbs1
(1:1000)13 and γ-H2AX (1:1000)13 were used. Anti-phospho-53BP1 antibodies (see Supplementary
Information, Fig. S1) were prepared by Bethyl Labs, Montgomery, TX. The peptide CLIIEDS*QPESQV,
where * represents phosphorylated Ser 25, was used to immunize rabbits. Antibodies that specifically
recognized the peptide in its phosphorylated form were separated from those that recognized other
epitopes in the peptide by affinity purification.
RECEIVED 14 OCTOBER 2002; REVISED 30 OCTOBER 2002; ACCEPTED 31 OCTOBER 2002;
PUBLISHED 25 NOVEMBER 2002.
1. Shiloh, Y. Biochem. Soc. Trans. 29, 661–666 (2001).
2. Rogakou, E. P., Pilch, D. R., Orr, A. H., Ivanova, V. S. & Bonner, W. M. J. Biol. Chem. 273,
5858–5868 (1998).
3. Burma, S., Chen, B. P., Murphy, M., Kurimasa, A. & Chen, D. J. J. Biol. Chem. 276, 42462–42467
(2001).
4. Anderson, L., Henderson, C. & Adachi, Y. Mol. Cell. Biol. 21, 1719–1729 (2001).
5. Rappold, I., Iwabuchi, K., Date, T. & Chen, J. J Cell Biol 153, 613–620 (2001).
6. Jongmans, W. et al. Mol. Cell. Biol. 17, 5016–5022 (1997).
7. Yamazaki, V., Wegner, R. D. & Kirchgessner, C. U. Cancer Res. 58, 2316–2322 (1998).
8. Girard, P. M., Riballo, E., Begg, A. C., Waugh, A. & Jeggo, P. A. Oncogene 21, 4191–4199 (2002).
NATURE CELL BIOLOGY VOL 4 DECEMBER 2002 www.nature.com/naturecellbiology
©2003 Nature Publishing Group
brief communications
9. Buscemi, G. et al. Mol. Cell. Biol. 21, 5214–5222 (2001).
10. Antoccia, A., Ricordy, R., Maraschio, P., Prudente, S. & Tanzarella, C. Int. J. Radiat. Biol 71, 41–49
(1997).
11. Hirao, A. et al. Mol. Cell. Biol. 22, 6521–6532 (2002).
12. Takai, H. et al. EMBO J. 21, 5195–5205 (2002).
13. Celeste, A. et al. Science 296, 922–927 (2002).
14. Bassing, C. H. et al. Proc. Natl Acad. Sci. USA 99, 8173–8178 (2002).
15. Xu, B., Kim, S. & Kastan, M. B. Mol. Cell. Biol. 21, 3445–3450 (2001).
16. Zhou, B. B. et al. J. Biol. Chem. 275, 10342–10348 (2000).
17. Schultz, L. B., Chehab, N. H., Malikzay, A. & Halazonetis, T. D. J. Cell Biol. 151, 1381–1390 (2000).
18. Zhou, B. B. & Elledge, S. J. Nature 408, 433–439 (2000).
19. Wang, B., Matsuoka, S., Carpenter, P. B. & Elledge, S. J. Science Online (cited 3 October 2002)
C10.1126/science.1076182 (2002).
20. Matsuoka, S., Huang, M. & Elledge, S. J. Science 282, 1893–1897 (1998).
21. Cortez, D., Wang, Y., Qin, J. & Elledge, S. J. Science 286, 1162–1166 (1999).
22. Paull, T. T. et al. Curr. Biol. 10, 886–895 (2000).
23. Bartek, J., Falck, J. & Lukas, J. Nature Rev. Mol. Cell Biol. 2, 877–886 (2001).
24. Barlow, C. et al. Cell 86, 159–171 (1996).
25. Ward, I. et al. Mol. Cell. Biol. (submitted).
26. Ward, I. M., Wu, X. & Chen, J. J. Biol. Chem. 276, 47755–47758 (2001).
27. Iwabuchi, K. et al. J. Biol. Chem. 273, 26061–26068 (1998).
28. Canman, C. E. et al. Science 281, 1677–1679 (1998).
ACKNOWLEDGEMENTS
These studies were in part motivated by discussions with T. Halazonetis, who suggested examining the
effects of low-dose IR, and we thank T. Halazonetis for sharing unpublished results. We also thank M.
Lichten, J. Chung, A. Lee, S. Petersen and A. Singer for critical comments on the manuscript, and M.
Kruhlack for assistance with microscopy. P.B.C was supported by a grant from The Robert Welch
Foundation.
Correspondence and requests for material should be addressed to A.N.
Supplementary Information accompanies the paper on www.nature.com/naturecellbiology.
COMPETING FINANCIAL INTERESTS
The authors declare that they have no competing financial interests.
997
 supplementary infomation

Figure S1. Phosphorylation of 53BP1 serine residue (S25) in vitro and in

vivo in response to IR. a, ATM wild-type (WT) and kinase-defective (KD) phospho-
rylation of 1.0 µg purified GST fusion peptide (GFP) encompassing S25 (GFP-S25;
lanes 1, 2) or GFP with an S25A mutation (GFP-A25; lanes 3, 4). The sequence of
GFP-S25 is GSIIEDSQEAQRPHRD where the bold amino acids represent residues
derived from the pGEX-4T vector and therefore do not naturally occur in 53BP1.
ATM assays were performed as described28. b, In vivo specificity of anti-pS25 anti-
body and DNA-damage inducible phosphorylation of 53BP1 at S25 in human 293T
cells. 293T cells were transfected with a vector expressing either wild-type 53BP1
(pCMH6K53BP1)27 or a S25A mutant . After 48 hours the samples were either left
untreated or treated with 10 Gy IR and processed for immunoprecipitations with
either anti-phospho S25 (top) or anti-HA (bottom) followed by Western blotting with
anti-HA antibody (Covance).

©2003 Nature Publishing Group