MOLECULAR AND CELLULAR BIOLOGY, Oct. 2004, p. 9207–9220
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 24, No. 20
Artemis Is a Phosphorylation Target of ATM and ATR and Is
Involved in the G2/M DNA Damage Checkpoint Response
Xiaoshan Zhang,1Janice Succi,1Zhaohui Feng,1† Sheela Prithivirajsingh,2
Michael D. Story,2and Randy J. Legerski1,3*
Department of Molecular Genetics,1Department of Experimental Radiation Oncology,2and Program in
Genes and Development,3The University of Texas M. D. Anderson Cancer Center, Houston, Texas
Received 16 March 2004/Returned for modification 13 April 2004/Accepted 20 July 2004
Mutations in Artemis in both humans and mice result in severe combined immunodeficiency due to a defect
in V(D)J recombination. In addition, Artemis mutants are radiosensitive and chromosomally unstable, which
has been attributed to a defect in nonhomologous end joining (NHEJ). We show here, however, that Artemis-
depleted cell extracts are not defective in NHEJ and that Artemis-deficient cells have normal repair kinetics
of double-strand breaks after exposure to ionizing radiation (IR). Artemis is shown, however, to interact with
known cell cycle checkpoint proteins and to be a phosphorylation target of the checkpoint kinase ATM or ATR
after exposure of cells to IR or UV irradiation, respectively. Consistent with these findings, our results also
show that Artemis is required for the maintenance of a normal DNA damage-induced G2/M cell cycle arrest.
Artemis does not appear, however, to act either upstream or downstream of checkpoint kinase Chk1 or Chk2.
These results define Artemis as having a checkpoint function and suggest that the radiosensitivity and
chromosomal instability of Artemis-deficient cells may be due to defects in cell cycle responses after DNA
Artemis is a member of the SNM1/PSO2 gene family, the
archetypical member of which was identified in budding yeast
(Saccharomyces cerevisiae) as a factor required for efficient
DNA interstrand cross-link repair (23, 53). Members of this
family, which in humans also include SNM1, SNM1B, ELAC2,
and CPSF73 (15, 27, 57), share a region of homology termed
the SNM1 domain, which contains a metallo-?-lactamase fold
and an appended ?-CASP (for metallo-?-lactamase-associated
CPSF Artemis SNM1/PSO2) domain that is a predicted nu-
cleic acid binding motif (7, 41). Outside the SNM1 domain, the
sequences of the yeast and human proteins are different. The
function of yeast Snm1 remains largely unresolved, although
several studies have indicated that it is involved in repairing
double-strand breaks (DSBs) resulting from processing of in-
terstrand cross-links (31, 35, 40). Artemis was originally iden-
tified molecularly as deficient in a human radiosensitive severe
combined immunodeficiency syndrome (RS-SCID) (41), which
is characterized by a defect in V(D)J recombination resulting
in premature arrest of both B- and T-cell maturation. In ad-
dition, patient cell lines exhibited greater sensitivity to ionizing
radiation (IR) than normal cells (9, 42, 44). RS-SCID resem-
bles murine SCID caused by defects in DNA-PK, a protein
complex involved in both V(D)J recombination and repair of
DSBs via the nonhomologous end-joining (NHEJ) pathway.
These findings have also been confirmed in a mouse model in
which the Artemis gene was disrupted by gene targeting (51).
Biochemical studies of Artemis have shown that it possesses a
5?33? exonucleolytic activity on single-stranded DNA, and
when complexed with DNA-PKcs, it acquires endonucleolytic
activity on 5? and 3? overhangs and the ability to open DNA
hairpins (34). This latter activity is consistent with the observed
defect in coding joint formation in Artemis-deficient cells. The
nuclease function of Artemis appears to reside in the con-
served SNM1 domain. In addition, it was shown that Artemis
is a substrate of the kinase activity of DNA-PKcs in vitro.
DNA-PKcs is a member of a family of large phosphatidylino-
sitol-3-OH kinase-like kinases (PIKKs) that includes the ataxia
telangiectasia mutated (ATM) and ataxia telangiectasia and
RAD3-related (ATR) gene products (reviewed in reference
16). These findings define a role for Artemis in V(D)J recom-
bination, and with the hypersensitivity of Artemis-deficient
cells to IR irradiation, also indicate a role for this gene in the
cellular response to DNA damage. It has been proposed, al-
though not formally demonstrated, that the radiosensitivity of
Artemis-deficient cells is due to a defect in NHEJ (34).
ATM and ATR are two central signaling kinases that medi-
ate pleiotropic response to DNA damage, including activation
of cell cycle checkpoints, DNA repair pathways, transcription,
and apoptosis (reviewed in references 1, 16, 56, and 63). Al-
though they have some functional redundancy, ATM and ATR
have specialized roles that appear to operate in parallel in the
response to DNA damage: ATM primarily responds to the
induction of DSBs, while ATR is activated by many forms of
DNA damage and replication inhibitors. Many downstream
phosphorylation targets of ATM and/or ATR have been iden-
tified, including p53, BRCA1, Nbs1, Smc1, FANCD2, Chk1,
Chk2, and Rad17. These proteins are mediators and transduc-
ers of the stress signal emanating from these two PIKKs. Some
of these substrates, as well as ATM and ATR, are found
together in a large multifactorial association of proteins re-
ferred to as the BRCA1-associated surveillance complex
* Corresponding author. Mailing address: Department of Molecular
Genetics, The University of Texas M. D. Anderson Cancer Center,
1515 Holcombe Blvd., Houston, TX 77030. Phone: (713) 792-8941.
Fax: (713) 794-4295. E-mail: email@example.com.
† Present address: Cancer Institute of New Jersey, University of
Medicine and Dentistry of New Jersey, New Brunswick, NJ 08903.
(BASC), which may function to recognize unusual or aberrant
DNA structures and to activate DNA repair and checkpoint
pathways (61). Thus, the BASC is thought to be a complex that
both senses and transduces the DNA damage signal.
In this report we demonstrate that Artemis-deficient cells
are not, in fact, significantly defective in NHEJ following ex-
posure to IR. Rather, Artemis is shown to interact with known
checkpoint proteins and to be phosphorylated by ATM and
ATR in vitro. In addition, Artemis is phosphorylated in vivo
after exposure of cells to genotoxic stress: this modification is
dependent upon both DNA-PK and ATM after IR and upon
ATR in response to UV radiation. Further, we show that
Artemis-deficient cells exposed to IR are defective in a G2/M
DNA damage checkpoint. These findings define a novel func-
tion for Artemis as a checkpoint protein and suggest a new
basis for the radiosensitivity and chromosomal instability of
MATERIALS AND METHODS
Antibodies. A fragment encoding amino acid residues 347 to 692 of Artemis
was fused to a hexahistidine tag by insertion into pET28 (Novagen). Purified
recombinant protein from Escherichia coli was used to raise antiserum in rabbits
using standard protocols. Antisera were affinity purified using antigen that had
been blotted and immobilized on nitrocellulose paper or by affinity chromatog-
Antibodies for Nbs1 (C-19), Rad17 (H-300), and cyclin B1 were purchased
from Santa Cruz Biotech. Antibodies for Mre11 (12 D7), ATM (2c-1), and
DNA-PKcs were from GeneTex. A monoclonal antibody for BRCA1 (Ab-1) was
from CalBiochem, and a polyclonal antibody for ATR (Ab-2) was from Onco-
gene. Polyclonal antibodies for phosphorylated Chk1 (phospho-Chk1) (Ser345)
and phospho-Chk2 (Thr68) and a monoclonal antibody for phospho-histone H3
(P-H3) (Ser10) were purchased from Cell Signaling. A monoclonal antibody for
?-H2AX (Ser139) was from Upstate. A monoclonal antibody for DNA-PKcs
(Ab-4) was from NeoMarkers. Monoclonal antibodies for ?-tubulin and Cdc2
were from Molecular Probes and Transduction Laboratories, respectively. Rad50
antibodies were provided by John Petrini.
Immunoprecipitation and dephosphorylation assays. For coimmunoprecipi-
tation experiments, cells were grown in 100-mm-diameter plates, washed with
cold phosphate-buffered saline (PBS) twice, and lysed by adding 400 ?l of EBC
buffer (50 mM Tris-HCl [pH 8.0], 120 mM NaCl, 0.5% Nonidet P-40 [NP-40], 1
mM phenylmethylsulfonyl fluoride [PMSF]) on ice for 20 min. In some cases,
cells were transfected with a construct (pDEST27-Artemis) expressing a gluta-
thione S-transferase (GST)–Artemis fusion protein and incubated for 2 days
prior to extract preparation. The lysates were spun for 15 min at 8,200 ? g, and
the supernatants were mixed with 2 volumes of Net-N buffer (20 mM Tris [pH
8.0], 100 mM NaCl, 1 mM EDTA, 0.5% NP-40) plus the indicated antibodies and
incubated at 4°C for 60 min. For some experiments, extracts were pretreated with
DNase I (0.5 mg/ml) for 10 min at room temperature. Fifteen microliters of
protein A–Sepharose CL-4B beads (Amersham Pharmacia Biotech) equilibrated
with Net-N buffer was then added, and the mixture was incubated for 60 min. The
beads were washed five times with Net-N buffer, and bound proteins were eluted
in sodium dodecyl sulfate (SDS) sample buffer and separated by SDS-polyacryl-
amide gel electrophoresis for immunoblotting. For some experiments, HeLa
whole-cell extracts prepared by the method of Manley et al. (36) were used.
For dephosphorylation experiments, Artemis protein was immunoprecipitated
from cell lysates as described above and washed with 1? alkaline phosphatase
buffer (provided by the manufacturer). Protein A-Sepharose beads with bound
protein were incubated with 20 U of alkaline phosphatase (Boehringer Mann-
heim) at 30°C for 30 min, with or without 10 mM Na3VO4, and washed twice with
Net-N buffer. The proteins were eluted in SDS sample buffer and separated by
SDS-polyacrylamide gel electrophoresis for immunoblotting.
In vitro kinase assay. Kinase assays were performed essentially as described
previously (8). The substrate for these experiments was recombinant Artemis
prepared as described above for antibody preparation. This truncated fragment
contains all the Arteims SQ motifs but is lacking the two TQ motifs in the amino
terminus of the protein. The concentration of recombinant Artemis was 0.9 ?M.
Cell-free nonhomologous end-joining assay. Immunodepletion of Artemis was
achieved by mixing 15 ?l of equilibrated protein A-Sepharose beads and 20 ?l of
Artemis antiserum with 400 ?l of Net-N buffer and then incubating the mixture
at 4°C for 1 h. After the beads were washed three times with Net-N buffer, they
were mixed with 100 ?g of HeLa whole-cell extract prepared as described
previously (5) and incubated at 4°C for 2 h. The beads were spun down, and the
supernatant was removed for immunoblot analysis and the end-joining assay.
The end-joining assays were performed as previously described (5).
In vivo assay for DSB rejoining after IR. DNA DSB rejoining, as measured by
residual lesions remaining after IR, was determined by pulsed-field gel electro-
phoresis as previously described (11, 29).
Preparation of ATR-deficient cells. To prepare ATR?/?cells, ATRflox/?
HCT116 cells in which one allele of ATR is disrupted and the other contains loxP
sites flanking exon 2 (12) were seeded at a density of 8 ? 105per 60-mm-
diameter plate and infected with an adenovirus encoding Cre recombinase at a
multiplicity of infection of 150. After 8 h of incubation, fresh medium was added,
and the plates were incubated for 48 h. Cells were then exposed to IR or UV, and
the lysates were prepared as described above for immunoblotting.
Inhibition of expression by siRNA. The sequence of the coding strand of the
ATM small interfering RNA (siRNA) was GCACCAGUCCAGUAUUGGC.
The sequence of the Artemis-1 and Artemis-2 siRNAs were CUGAAGAGAG
CUAGAACAG and UUAGGAGUCCAGGUUCAUG, respectively, and the
sequence of the DNA-PKcs siRNA was UGGGCCAGAAGAUCGCACC. The
53BP1 siRNA was previously described (59).
Cell cycle analysis and phospho-histone H3 staining. Cells were exposed to IR
or UV or left untreated, incubated for various times (indicated in the figures),
harvested by trypsinization, and fixed with 70% ethanol. Cells were then perme-
abilized with 0.15% Triton 100 in PBS containing 4% bovine serum albumin for
30 min and then incubated with phospho-histone H3 antibody for 60 min. Cells
were washed with PBS and incubated with fluorescein isothiocyanate (FITC)-
conjugated goat anti-mouse immunoglobulin G (IgG) for 20 min. After the cells
were washed with PBS, they were stained with 50 ?g of propidium iodide per ml,
treated with 10 ?g of DNase-free RNase per ml, and analyzed by flow cytometry.
Artemis is not essential for NHEJ. The findings that human
cells defective for Artemis are deficient in V(D)J recombina-
tion and hypersensitive to IR have led to the conclusion that,
as in mice with SCID, these cells have a defect in NHEJ (32,
34). Baumann and West (5) and Hanakahi et al. (21) have
developed an in vitro DNA end-joining assay using human cell
extracts that is dependent upon DNA-PK, Xrcc4, and DNA
ligase IV, indicating that it is a measure of NHEJ. To deter-
mine whether Artemis is involved in NHEJ, we performed this
end-joining assay with HeLa cell extracts, prepared as de-
scribed previously (5), with or without immunodepletion of
Artemis. As shown in Fig. 1A and B, removal of Artemis from
HeLa extracts had little effect on the observed amount of DNA
end joining of a substrate that contained complementary sin-
gle-stranded tails. For controls to ensure that the assay was
dependent upon DNA-PK as previously demonstrated (5), we
performed the end-joining assay in the presence of wortman-
nin (22, 26) or a neutralizing monoclonal antibody to DNA-
PKcs. In both cases, the assay was strongly inhibited, indicating
that it was a measure of the DNA-PK-dependent NHEJ path-
way (Fig. 1B, lanes 6 to 9).
These results suggest that Artemis is not required for NHEJ;
however, this is not a complete test of its possible involvement
in NHEJ, since incompatible DNA termini may require nu-
cleolytic processing that could involve Artemis in a subset of
end-joining reactions. We therefore also examined the repair
of DSBs in vivo after exposure to IR by the method of pulsed-
field gel electrophoresis (10, 11, 29). In Fig. 1C, initial DNA
lesions and DNA retained after 4 h of repair with increasing
doses of radiation are shown. This technique has previously
been used to detect defects in DSB repair after IR exposure in
9208ZHANG ET AL.MOL. CELL. BIOL.
FIG. 1. Artemis is not essential for NHEJ. (A) Immunodepletion of Artemis from HeLa whole-cell extracts as determined by immunoblotting.
HeLa whole-cell extract (lane 1); HeLa whole-cell extract precipitated with protein A-Sepharose beads only (lane 2); whole-cell extract precip-
itated with preimmune serum (Pre.) (lane 3); and Artemis-depleted whole-cell extract (depleted with Artemis antiserum) (lane 4). (B) Immu-
nodepletion of Artemis from HeLa cell extracts does not affect end joining of linearized plasmid DNA. Ethidium bromide-stained agarose gels
show the results of end-joining assays. Plasmid DNA was linearized by digestion with BamHI. The positions of monomer (M) and dimer
(D) plasmids are indicated to the left of the gels. DNA only was used as a control (C) in lane 1. Assays shown in lanes 2 to 4 were performed with
whole-cell extracts treated as described above for panel A. DNA size markers (S) are shown in lane 5. IgG, nonspecific immune serum. Controls
(lanes 6 to 9) show that wortmannin (Wort.) and antibodies to DNA-PKcs inhibit the rejoining reaction. (C) DSB rejoining is normal in
Artemis-deficient cells. P11 cells expressing Artemis, the simian virus 40-transformed human cell line XP2OS (considered the wild type [wt] in
these experiments), and MO59J (DNA-PKcs-deficient) cells were compared as a function of IR dose for DNA rejoining by pulsed-field gel
electrophoresis. (D) DSB rejoining is normal in HEK293 cells depleted of Artemis by siRNA. Depletion of Artemis by siRNA is shown by
immunoblotting. Con., control; Art., Artemis. (E) DSB rejoining examined as a function of time of incubation. The ? indicates the value at the
4-h time point for the MO59J cell line.
VOL. 24, 2004 ARTEMIS IS A CHECKPOINT PROTEIN 9209
Ku80 and DNA-PKcs mutants. As shown, the Artemis-defi-
cient cell line P11 (41) did not exhibit a significant difference in
DSB repair from a similarly simian virus 40-transformed hu-
man fibroblast cell line, whereas a DNA-PKcs-deficient mutant
was clearly defective. To confirm these results, Artemis was
depleted from HEK293 cells by transfection with siRNA, and
pulsed-field gel electrophoresis experiments were repeated.
Compared with cells transfected with a nonspecific (control)
siRNA, Artemis-depleted cells did not exhibit a defect in re-
joining of DSBs after exposure to IR (Fig. 1D). We also ex-
amined the kinetics of DSB repair in Artemis and wild-type
cells and found no significant differences (Fig. 1E). Taken
together with the in vitro results, these findings indicate that
Artemis is not an essential component of NHEJ, although we
cannot rule out the possibility that Artemis may play a minor
role in a subset of end-joining reactions. Nicolas et al. (43) had
previously reached a similar conclusion; they found no defect
in the kinetics of DSB repair in Artemis-deficient cells after
exposure to IR.
Artemis is phosphorylated in vivo in response to both IR
and UV. Artemis contains 10 SQ/TQ motifs, nine of which are
conserved in humans and mice (Fig. 2A). These motifs, par-
ticularly when clustered, have been found to be the preferred
sites of phosphorylation by the ATM, ATR, and DNA-PK
PIKKs (28). Artemis has been shown to be a substrate of
DNA-PKcs in vitro (34). To investigate the phosphorylation of
Artemis in vivo, we prepared and affinity purified antibodies
against the nonconserved carboxy-terminal region of the pro-
FIG. 2. Artemis is phosphorylated in vivo after exposure of MCF-7 cells to IR or UV irradiation. (A) Schematic of Artemis indicating locations
of S/TQ motifs. Asterisks above or below the schematic indicate motifs conserved or not conserved in humans and mice, respectively. aa, amino
acids. (B) Immunoblotting shows the absence of Artemis in Artemis-deficient P11 cells. The asterisk indicates a nonspecific loading control.
(C) Treatment with either IR or UV results in phosphorylation of Artemis (Artemis-P). Artemis was immunoprecipitated from extracts of cells
exposed to IR (10 Gy) or UV (20 J/m2) and subsequently incubated for 2 h (lanes 1 and 4). The same immunoprecipitate was incubated with
alkaline phosphatase (lanes 2 and 5) or treated with alkaline phosphatase in the presence of the phosphatase inhibitor Na3VO4(lanes 3 and 6).
The immunoprecipitate from untreated cells (UT) is shown in lane 7. (D) Phosphorylation of Artemis as a function of IR dose examined 2 h after
treatment. (E) Kinetic analysis of the phosphorylation of Artemis after the cells were exposed to IR (10 Gy) for times ranging from 5 min (5m)
to 17 h. (F) Phosphorylation of Artemis as a function of UV dose examined 2 h after treatment. (G) Kinetic analysis of the phosphorylation of
Artemis after exposure of cells to UV (20 J/m2).
9210ZHANG ET AL.MOL. CELL. BIOL.
tein. Immunoblotting indicated that these antibodies recog-
nized a protein migrating at approximately 105 kDa in control
cell lines, but not in the Artemis-deficient cell line P11, which
confirmed their specificity (Fig. 2B). We then examined
whether the gel migration of Artemis was altered in MCF-7
cells by treating cells with IR or UV radiation. Interestingly,
both IR and UV induced a significant shift of Artemis to a
slower-migrating form (Fig. 2C, lanes 1 and 4).
To determine whether this shift was due to phosphorylation,
cellular lysates obtained from cells treated with IR or UV were
incubated with calf intestinal phosphatase (CIP), with or with-
out the addition of a phosphatase inhibitor. Incubation with
CIP resulted in a shift of Artemis to the faster-migrating form
(Fig. 2C, lanes 2 and 5), which did not occur in the presence of
a phosphatase inhibitor (Fig. 2C, lanes 3 and 6). We have also
observed modification of Artemis after exposure of MCF-7
cells to the topoisomerase I inhibitor camptothecin (24) (re-
sults not shown). Essentially similar results of DNA damage-
induced modification of Artemis were obtained with HeLa,
HT-1080, and HEK293 cells (not shown), and taken together,
these results demonstrate that Artemis is phosphorylated in
vivo in response to various types of DNA damage.
We also examined the modification of Artemis as a function
of dose and observed in general that its phosphorylation in-
creased with the level of DNA damage (Fig. 2D and F). Kinetic
studies indicated that the observed phosphorylation of Artemis
could be detected as early as 5 min after exposure to IR and is
sustained for at least 17 h in MCF-7 cells (Fig. 2E). Phosphor-
ylation caused by UV irradiation occurred later and was not
detected until the 2-h time point (Fig. 2G), although in other
experiments, phosphorylation of Artemis after UV can clearly
be seen after 1 h. These findings indicate that Artemis is
phosphorylated in vivo in response to DNA damage induced by
both IR and UV irradiation. In particular, the induction of
phosphorylation by UV was unanticipated and indicates that
Artemis plays a wider role in the cellular response to genotoxic
agents than suggested by previous studies.
Phosphorylation of Artemis is mediated by DNA-PK in vivo.
Artemis has been shown to be phosphorylated by DNA-PKcs
in vitro (34). To determine whether the IR-induced phosphor-
ylation of Artemis observed in vivo (Fig. 2) was DNA-PK
dependent, we examined the DNA-PKcs-deficient cell line
MO59J and the control line MO59K (30). Unlike what we had
observed in MCF-7 or HeLa cells, a constitutive level of phos-
phorylation of Artemis was observed in the absence of treat-
ment in these cell lines, suggesting a DNA-PK-independent
phosphorylation of Artemis (for example, two Artemis bands
can be seen in Fig. 3A, lanes 1 and 4). Treating cells with
caffeine reduced the level of the top band, indicating that this
modification is due to phosphorylation (lane 2). Upon treat-
ment with IR, a decrease in the mobility of Artemis was ob-
served in MO59K cell lysates, but not in MO59J cell lysates
(Fig. 3A, compare lanes 3 and 5). However, the bottom band
in the MO59J lysates disappeared, suggesting that some phos-
phorylation of Artemis did occur in these cells.
The differences in the phosphorylation of Artemis in the
MO59K and MO59J cell lines after exposure to IR suggest that
Artemis is an in vivo substrate of DNA-PKcs, but the MO59J
cell line contains a mutation in ATM and is reported to have
about 33% of normal ATM activity (58). Therefore, to further
examine the role of DNA-PKcs in the modification of Artemis,
we used siRNA to deplete this kinase in HeLa cells (Fig. 3B).
As expected in the absence of treatment, little change in the
migration of Artemis was observed, although there may have
been some increase in the level of constitutive phosphorylation
of Artemis upon depletion of DNA-PKcs (compare lanes 1 and
3), perhaps due to an increase in the amount of unrepaired
DNA damage. Upon treatment with IR, strong phosphoryla-
tion of Artemis is observed even with depletion of DNA-PKcs;
however, this modification is reduced in the presence of caf-
feine (compare lanes 5 and 6), an inhibitor of ATM and ATR
but not DNA-PK (54, 55). These observations clearly indicate
that a caffeine-sensitive kinase(s) is at least partially responsi-
ble for the observed phosphorylation. However, caffeine did
not affect the level of Artemis modification in cells treated with
a control siRNA (compare lanes 7 and 8), indicating that
DNA-PKcs is also involved in the phosphorylation of Artemis
after IR. It should also be noted that there appears to a con-
sistent decrease in the level of Artemis upon depletion of
DNA-PKcs, suggesting that this kinase may be required for
Artemis stability. Additionally, phosphorylation of Artemis af-
ter IR exposure was also inhibited in MCF-7 cells in the pres-
ence of wortmannin, which is an inhibitor of ATM, ATR, and
DNA-PK (22, 26), but not in the presence of the Chk1 inhib-
itor UCN-01 (20) (Fig. 3C).
We also examined the interaction between Artemis and
DNA-PKcs by coimmunoprecipitation from HCT116 cells
(Fig. 3D). In untreated cells, Artemis antibodies coprecipi-
tated DNA-PKcs; however, upon exposure to IR, the interac-
tion between Artemis and DNA-PKcs increased and was max-
imal between 8 and 16 h after exposure to IR. For a further
control for the interaction of Artemis with DNA-PKcs, we
stably expressed a GST-Artemis fusion protein in HEK293
cells. Upon exposure to IR, approximately half of the overex-
pressed fusion protein was phosphorylated (Fig. 3E). Immu-
noprecipitation with DNA-PKcs antibodies showed that both
phosphorylated and unphosphorylated forms of Artemis coim-
munoprecipitated with DNA-PKcs. Taken together and com-
bined with the results of previous in vitro studies (34), these
results indicate that Artemis is a likely phosphorylation sub-
strate of DNA-PK and that this modification is induced by IR
in vivo. However, these findings also suggest that other kinases
are involved in the phosphorylation of Artemis after DNA
Artemis interacts with checkpoint proteins and is phosphor-
ylated by ATM and ATR in vitro. We have shown above that
Artemis interacts with DNA-PK and is a likely phosphoryla-
tion substrate of this kinase in vivo. However, as indicated
above, these findings also suggest that phosphorylation of Ar-
temis may also be mediated by other kinases. ATM is a central
kinase involved in the cellular response to IR-induced DNA
damage (1, 16, 56). To determine whether Artemis interacts
with ATM, we performed an immunoblot analysis for ATM
after an immunoprecipitation from HeLa extracts with Arte-
mis affinity-purified antibodies. As shown in Fig. 4A (top gel),
this experiment indicated that ATM coimmunoprecipitated
with Artemis. ATM has previously been shown to be a com-
ponent of a large complex termed BASC (61). In addition to
ATM and BRCA1, this complex includes Mre11, Rad50, and
VOL. 24, 2004 ARTEMIS IS A CHECKPOINT PROTEIN9211
9212 ZHANG ET AL.MOL. CELL. BIOL.
Nbs1 (MRN), as well as other proteins involved in DNA re-
pair, checkpoint signaling, and chromatin remodeling (6, 19).
To determine whether Artemis interacts with other compo-
nents of BASC, we immunoblotted for BRCA1 and MRN and
found that all were specifically coimmunoprecipitated from
HeLa cell extracts by Artemis antibodies (Fig. 4A, top blot).
We were also able to demonstrate a reciprocal coimmunopre-
cipitation of exogenously expressed GST-Artemis by antibod-
ies to ATM (Fig. 4A). This interaction was not mediated by
DNA, since prior incubation of the extracts with DNase I did
not prevent the coimmunoprecipitation. For a control to dem-
onstrate the specificity of the Artemis antiserum, we used
siRNA to deplete HeLa cells of Artemis and then repeated the
coimmunoprecipitation experiment. As shown in Fig. 4B), Ar-
temis siRNA effectively depleted HeLa cells of Artemis but did
not affect Rad50 levels. In the absence of Artemis, coimmu-
noprecipitation of Rad50 with Artemis antibodies was greatly
reduced, demonstrating that the results shown in Fig. 4A are
not due to nonspecific interactions of the Artemis antibody.
Since Artemis interacts with ATM, we examined whether
Artemis is a substrate of ATM or ATR by performing an
immunoprecipitation kinase assay (3, 39). As shown in Fig. 4C,
Artemis was phosphorylated by both ATM and ATR in vitro
but not by kinase-dead variants of these enzymes. These find-
ings, taken together with the interactions with known check-
point proteins, suggest that Artemis has a potential role in cell
signaling pathways induced by DNA damage.
Phosphorylation of Artemis in vivo in response to IR is ATM
dependent. We have shown above that Artemis interacts with
ATM in vivo and is phosphorylated by this kinase in vitro. To
determine whether ATM plays a role in the IR-induced phos-
phorylation of Artemis in vivo, we first examined ATM-defi-
cient cells and observed altered migration of Artemis as seen in
other cell lines (Fig. 5A). This result is consistent with a major
role for DNA-PK in Artemis phosphorylation after exposure to
IR irradiation, as discussed above, but does not rule out a role
for ATM. To determine whether ATM is involved, we used
transfection of siRNA oligonucleotides to eliminate expression
of ATM in the DNA-PKcs-deficient cell line MO59J (Fig. 5B,
top gels) and subsequently examined the phosphorylation of
Artemis after IR or UV radiation (Fig. 5B, bottom gel). In cells
transfected with a control siRNA, phosphorylation of Artemis
due to IR or UV treatment was observed. However, in the
ATM siRNA-transfected cells, phosphorylation of Artemis was
reduced after IR, but not after UV (Fig. 5B, bottom gel,
compare lanes 2 and 5 and lanes 3 and 6), irradiation. Taken
together with our in vitro results, these findings demonstrate
that ATM is involved in the modification of Artemis that
occurs in response to IR and are consistent with previous
studies indicating that ATM does not play a significant role in
the response to UV irradiation. Thus, Artemis phosphoryla-
tion after IR exposure is dependent upon both ATM and
Phosphorylation of Artemis in vivo in response to UV is
ATR dependent. As shown above (Fig. 2), Artemis is phos-
phorylated in response to both IR and UV. The major PIKK
that mediates the cellular response to UV is ATR (1, 16, 56,
63). To assess the role of ATR in UV-induced phosphorylation
of Artemis, we used a recently developed HCT116 conditional
null cell line (ATRflox/?) in which one allele of ATR is dis-
rupted and the other contains loxP sites flanking exon 2 (12).
Infection of the ATRflox/?cell line with an adenovirus encod-
ing Cre recombinase reduced expression of ATR to low levels
(Fig. 5C, top gels). We then examined the phosphorylation of
Artemis after exposure to UV or IR in cells with or without
expression of Cre recombinase. Eliminating the expression of
ATR did not affect the phosphorylation of Artemis induced by
IR (Fig. 5C, bottom gel, compare lanes 3 and 6) but did reduce
the phosphorylation of Artemis after UV treatment (Fig. 5E,
bottom gel, compare lanes 2 and 5). Also note that the phos-
phorylation of Artemis after UV treatment is inhibited in the
presence of caffeine (Fig. 3B, lane 5). Thus, the ATR-mediated
modification of Artemis induced by UV radiation is consistent
with the observed in vitro phosphorylation of Artemis shown
Artemis is involved in a G2/M DNA damage checkpoint.
Artemis-deficient cells are hypersensitive to IR (9, 42, 44), but
as shown above (Fig. 1), this sensitivity does not appear to be
due to a significant defect in NHEJ. Another possible expla-
nation for this phenotype is loss of checkpoint function, par-
ticularly since both IR and UV irradiation induce phosphory-
lation of Artemis (Fig. 2). IR induces an ATM-dependent
G2/M checkpoint arrest in mammalian cells. To assess this
checkpoint, we used siRNA to deplete HEK293 cells of Arte-
mis. Cells depleted of Artemis by transfection with siRNA and
control transfected cells were subsequently irradiated with IR
(6 Gy), and DNA content and level of phospho-histone H3
(P-H3) were measured as a function of time (Fig. 6A). Trans-
fection with Artemis siRNA resulted in a slight accumulation
in the G1population in untreated cells compared to transfec-
tion with the control siRNA (see the 0-h time points in Fig.
6A). This accumulation, which was consistently observed in all
cell lines examined, could be due to a fraction of arrested cells
caused by increased spontaneous chromosomal damage in the
absence of Artemis. Rooney et al. (51) observed significantly
higher levels of chromosomal aberrations in Artemis-deficient
mouse embryo fibroblasts compared with wild-type cells. One
hour after IR irradiation, both Artemis-depleted and control
FIG. 3. Artemis is phosphorylated in vivo by DNA-PKcs after IR. (A) Phosphorylation of Artemis in vivo after exposure of cells to IR is
deficient in MO59J cells. MO59J and MO59K cells were treated with IR (10 Gy), incubated for 1 h prior to extract preparation, and compared
with untreated cells (UT). Caff., caffeine. (B) Depletion of DNA-PKcs by siRNA shows that DNA-PKcs and a caffeine-sensitive kinase(s)
participate in the phosphorylation of Artemis after IR. LC, loading control. The presence (?) or absence (?) of control and DNA-PKcs siRNA,
caffeine, and IR irradiation are indicated above the blots. (C) Phosphorylation of Artemis after IR treatment is inhibited by wortmannin (Wor.)
in MCF-7 cells, but not by the Chk1 inhibitor UCN-01 (UCN.). (D) Artemis antibodies coimmunoprecipitate DNA-PKcs with and without
exposure to IR in HCT116 cells. (E) DNA-PKcs interacts with phosphorylated and unphosphorylated forms of Artemis as determined by
coimmunoprecipitation (IP) with DNA-PKcs antibodies. HEK293 cells stably transfected with pDEST27-Artemis (expressing GST-Artemis) were
exposed to IR and incubated for 1 h. Protein A-Sepharose beads were used as a control.
VOL. 24, 2004 ARTEMIS IS A CHECKPOINT PROTEIN9213
FIG. 4. Artemis interacts with checkpoint proteins and is phosphorylated by ATM and ATR. (A, top blot) Artemis antibodies coimmunopre-
cipitate ATM, BRCA1, Rad50, Nbs1, and Mre11 from HeLa extracts. Protein A-Sepharose beads were used as a control. IP, immunoprecipitation;
IgG, nonspecific immune serum. Immunoblotting (IB) was performed using antibodies to the indicated proteins. (A, lower blot) Reciprocal
coimmunoprecipitation of Artemis by ATM antibodies. ATM?I, incubation of the extracts with DNase I prior to coimmunoprecipitation. (B)
Depletion of Artemis in HeLa cells by transfection of siRNA eliminates the coimmunoprecipitation of Rad50 by Artemis antibodies. ?-Artemis
IP, immunoprecipitation with anti-Artemis antibodies; Pre., preimmune serum; Imm., Artemis antiserum. (C) Immunoprecipitation (IP) kinase
assay shows that Artemis was phosphorylated by both ATM and ATR but by kinase-dead (kd) variants of the enzymes. The top shows an
autoradiogram of Artemis phosphorylation by ATM or ATR. The bottom shows immunoblots of immunoprecipitated kinases used in the assays.
cells exhibited a dramatic reduction in P-H3 staining; however,
at later time points, Artemis-depleted cells exhibited a higher
level of P-H3 staining than did the control cells, suggesting a
failure of the former cells to fully arrest or to maintain an
arrest in G2/M (Fig. 6A and B). This trend was also reflected
in the cell cycle data, where 16 h after IR irradiation, 51.6% of
the Artemis-depleted cells were in G2/M compared with 72.5%
of the control cells.
To ensure that the increased levels of P-H3 staining ob-
served in Artemis-depleted cells was not due to a failure to exit
mitosis with normal kinetics, we performed the experiment in
the presence of nocodazole 16 h after IR irradiation. As shown
in Fig. 6C, the increase in P-H3 staining in Artemis-depleted
cells also occurred in the presence of nocodazole, indicating
that it is due to premature transition from G2to M and not to
a differential exit from mitosis. Finally, these results have been
confirmed by depletion of Artemis in HEK293 cells with a
second siRNA and by similar experiments conducted with
HCT116 and HeLa cells (not shown) and indicate that Artemis
is required for a normal G2/M cell cycle delay that occurs after
To further distinguish the roles of Artemis and DNA-PKcs,
we depleted expression of each of these proteins separately by
siRNA transfection and compared the fraction of G2/M cells
16 h after IR irradiation. As expected, Artemis-depleted cells
exhibited an incomplete arrest, whereas cells transfected with
a control or DNA-PKcs siRNA showed normal and slightly
enhanced enforcement of the checkpoint, respectively (Fig.
6D). This conclusion was also confirmed by examination of
cyclin B levels 16 h after IR irradiation. With control siRNA,
cyclin B levels were higher in IR-treated cells than in untreated
cells. This effect was even more pronounced in cells depleted
of DNA-PKcs, whereas Artemis-depleted cells showed little
increase in cyclin B levels after IR irradiation (Fig. 6D), indi-
cating a failure to delay in G2/M for the latter cells. Taken
together, these findings indicate that Artemis is involved in a
G2/M checkpoint and are consistent with a requirement for
ATM and ATR in the DNA damage-induced phosphorylation
A host of factors have been identified in the mediation of
DNA damage-induced G2/M checkpoint responses in mamma-
lian cells (13, 52, 63). Two kinases that act downstream of
ATM and ATR in checkpoint responses are Chk1 and Chk2.
Since Artemis is rapidly phosphorylated after IR irradiation
(Fig. 2), we examined whether the phosphorylation of either
Chk1 or Chk2 was affected in Artemis-depleted cells. As shown
in Fig. 7A, no differences in the phosphorylation of these
proteins were observed after exposure to IR or UV compared
with control cells or cells depleted of DNA-PKcs, indicating
that Artemis does not act upstream of these checkpoint ki-
nases. H2AX is a variant histone that is rapidly phosphorylated
after DNA damage (?-H2AX) and is required for the forma-
tion of foci by other checkpoints proteins, such as Nbs1,
53BP1, and BRCA1 (48, 49, 50, 59). Examination of the phos-
phorylation of H2AX showed that it occurred normally in both
Artemis-depleted and control cells (Fig. 7B).
Both Rad17, a component of the checkpoint clamp-loading
complex, and 53BP1, a BRCT repeat-containing protein, are
rapidly phosphorylated after DNA damage and have been
shown to be required for G2/M checkpoint responses (4, 14, 18,
59). To determine whether the phosphorylation of Artemis
depends upon either of these proteins, Rad17 was depleted
from HCT116 RAD17flox/?cells by introduction of Cre recom-
binase (60), and 53BP1 was depleted from HEK293 cells by
siRNA transfection (59). As shown in Fig. 7C and D, the
phosphorylation of Artemis after IR or UV exposure was not
FIG. 5. Phosphorylation of Artemis is partially dependent upon
ATM after IR irradiation and upon ATR after UV irradiation. (A) Ar-
temis is phosphorylated in ATM-deficient cells (GM9607) after IR
irradiation. UT, untreated cells. (B, top blot) Depletion of ATM ex-
pression in MO59J cells by siRNA. LC, loading control. (B, bottom
blot) Phosphorylation of Artemis in MO59J cells depleted of ATM is
affected after IR irradiation, but not after UV irradiation. (C, top blot)
Elimination of ATR expression by infection of ATRflox/?cells with
adenovirus expressing Cre recombinase. (Bottom blot) Phosphoryla-
tion of Artemis in cells depleted of ATR is affected after UV irradi-
ation, but not after IR irradiation.
VOL. 24, 2004 ARTEMIS IS A CHECKPOINT PROTEIN 9215
9216 ZHANG ET AL.MOL. CELL. BIOL.
affected by the depletion of these checkpoint proteins. Thus,
while phosphorylation of Artemis after DNA damage is de-
pendent upon ATM and ATR, it does not appear that it is a
component of the well-established G2/M checkpoint pathway
that functions downstream of these PIKKs (13, 52, 63).
Previous studies of Artemis have shown that it plays a crucial
role in V(D)J recombination because in its functional absence,
T and B lymphocytes fail to mature, leading to SCID syndrome
(9, 41, 42, 44). The role of Artemis in V(D)J recombination has
been defined as an endonuclease that cleaves the hairpins at
coding joints, producing unmistakable DSBs that are subse-
quently repaired by the NHEJ pathway (34). Additionally,
Artemis-expressing cells are radiosensitive, suggesting the at-
tractive concept that Artemis also plays a role in rejoining
IR-induced DSBs via NHEJ (34). This hypothesis, although
seemingly widely accepted in the literature, has, to our knowl-
edge, never been directly demonstrated; in fact, Artemis-defi-
cient cells have been reported to have normal repair kinetics of
DSBs in vivo (43). Consistent with these earlier findings, we
have shown here that Artemis is not an essential element of
DSB rejoining in vitro and that Artemis-deficient cells are not
significantly defective in vivo in restoration of DSBs after IR
treatment, although we cannot completely rule out the possi-
bility that Artemis may be involved in a minor subset of reac-
tions in this pathway. Nevertheless, these findings indicate that
the significant radiosensitivity of Artemis-deficient cells is un-
likely to be due to defects in NHEJ. In addition to our findings,
several other lines of evidence also support the conclusion that
Artemis does not play a major role in NHEJ. First, Artemis-
deficient cells are not defective in signal joint rejoining, al-
though DNA-PKcs-deficient cells are defective in the fidelity of
this process (41, 51). Second, inactivating mutations in other
components of NHEJ, including Ku70, Ku80, DNA-PKcs,
Xrcc4, and DNA ligase 4, have not been observed in human
immunodeficiencies, presumably due to the essential nature of
NHEJ in human cells, whereas Artemis is clearly involved in
human SCID (41). Third, a recent report on the transposition
of the Sleeping Beauty element showed that NHEJ, but not
Artemis, is required for this recombination pathway (25).
Phosphorylation of Artemis in response to genotoxic stress
is mediated by ATM, ATR, and DNA-PK. Artemis has been
shown to be a phosphorylation substrate of DNA-PKcs in vitro
(34). We have extended these studies to show that phosphor-
ylation of Artemis in vivo after IR treatment is mediated by
DNA-PK. However, these experiments also indicated that an
additional kinase or kinases were involved in the phosphory-
lation of Artemis after IR irradiation and that Artemis was also
phosphorylated after UV irradiation. We also showed that
Artemis interacts with some components of BASC, a multifac-
torial complex that plays a central role in the mediation of both
cell cycle checkpoints and DNA repair pathways (61) and that
Artemis is an in vitro substrate of ATM and ATR. Consistent
with the defined roles of these two PIKKs in the cellular re-
sponse to DNA damage, in vivo phosphorylation of Artemis
mediated by ATM and ATR was shown to be induced by IR
and UV treatment, respectively. These findings suggest a wider
role for Artemis in the cellular response to DNA damage than
has been previously appreciated. It is unlikely that this role
involves a direct participation in DNA repair pathways, be-
cause DNA damage induced by IR or UV radiation results in
lesions of distinctly different types, and their repair is mediated
by disparate pathways that have little overlap in mechanism or
components. Thus, a direct role for Artemis in multiple path-
ways of DNA repair appears less plausible than a role in the
mediation of cell cycle checkpoints in response to various types
of DNA damage. Consistent with this interpretation, we
showed that Artemis-deficient cells are defective in a G2/M cell
cycle checkpoint induced by IR.
The observation of ATM/ATR-mediated phosphorylation of
Artemis induced by genotoxic stress is consistent with a defec-
tive checkpoint response that we have described here; how-
ever, the phosphorylation of Artemis after IR irradiation by
DNA-PK remains unexplained. Our findings and those re-
ported previously by others (2) indicate that DNA-PKcs is not
required for a G2/M DNA damage checkpoint induced by IR.
In fact, in the absence of DNA-PKcs, the checkpoint appears
to be enhanced, presumably due to a decreased level of repair
of DSBs. DNA-PKcs interacts with both the unphosphorylated
and phosphorylated forms of Artemis, and the association be-
tween the two proteins appears to increase until it reaches a
maximum 8 to 16 h after IR irradiation. This result suggests the
possibility that DNA-PK is required to maintain the phosphor-
ylation of Artemis after DNA damage.
Artemis is involved in cell cycle checkpoint pathways. The
conclusion that Artemis functions in cell cycle checkpoint
pathways is based on the following observations. (i) Both IR
and UV induce phosphorylation of Artemis. (ii) Artemis in-
teracts with checkpoint proteins and is phosphorylated by the
checkpoint kinases ATM and ATR in vitro. (iii) Phosphoryla-
tion of Artemis in vivo after genotoxic stress requires ATM
and ATR as well as DNA-PK. (iv) Artemis is required for a
normal G2/M arrest after IR irradiation. The mechanism of the
Artemis-mediated checkpoint arrest does not appear to in-
volve the well-established pathways of G2/M delay that func-
FIG. 6. Artemis is required for a normal DNA damage-induced G2/M cell cycle checkpoint. (A) Analysis of the G2/M checkpoint after IR.
Fluorescence-activated cell sorting profile of Artemis-depleted and control HEK293 cells stained with propidium iodide and anti-phospho-histone
H3 (P-H3) at the indicated times after IR irradiation (6 Gy). The percentages of cells in the G1/S and G2/M phases are shown. (B) Graphical
analysis of P-H3 staining indicates a defect in the G2/M checkpoint in Artemis-depleted cells. The fraction of P-H3-positive cells is expressed as
a percentage of that measured at the 0-h time point. (C) P-H3 accumulation is not due to differential exit from mitosis. Values of P-H3 levels for
the untreated sample (UT) were set at 1.0, and results for the other samples are shown as a percentage of this value. Nocodazole was added 30
min after IR treatment, and data were collected after 16 h. (D) Comparison of the cell cycle fractions of Artemis- and DNA-PKcs-depleted cells
16 h after IR (graphs at the top). The levels of cyclin B in the same cells are shown (middle blot). Immunoblotting showing depletion of DNA-PKcs
by siRNA in HEK293 cells is shown (bottom blot). LC, loading control.
VOL. 24, 2004 ARTEMIS IS A CHECKPOINT PROTEIN9217
tion downstream of ATM and ATR (13, 52, 63). Although
Artemis is rapidly phosphorylated after IR irradiation, it is not
required for the damage-induced phosphorylation of Chk1,
Chk2, or H2AX. In addition, the phosphorylation of Artemis is
not dependent upon Rad17 or 53BP1 and was not inhibited by
the Chk1 inhibitor UCN-01. Rad17 has recently been shown to
be important for ATR-dependent but not ATM-dependent
checkpoint signaling through Chk1 (60) and may facilitate the
phosphorylation of other targets downstream of ATR (64).
Thus, our findings suggest that Artemis does not act down-
FIG. 7. Artemis is not involved in established G2/M checkpoint pathways. Where not indicated, the IR dose was 20 Gy and the UV dose was
50 J/m2. All experiments were performed 2 h after irradiation. (A) Phosphorylation of Chk1 (phospho-Ser345) and Chk2 (phospho-Thr68) are
unaffected in Artemis-depleted cells after IR or UV irradiation. UT, untreated cells; LC, loading control. (B) Phosphorylation of H2AX is
unaffected in Artemis-depleted cells. Numbers shown after the indicated treatment specify the dosage in grays (IR) or joules per square meter
(UV). (C) Phosphorylation of Artemis is unaffected in Rad17-depleted cells. Depletion of Rad17 in RAD17flox/?cells is shown after infection with
adenovirus expressing Cre recombinase. (D) Phosphorylation of Artemis is unaffected in 53BP1-depleted cells.
9218 ZHANG ET AL.MOL. CELL. BIOL.
stream of Chk1. There is, however, precedence for Rad17-
independent checkpoint signaling mediated by ATR homo-
logues in both budding and fission yeasts (37, 38). In addition,
the checkpoint phenotype of ATR-deficient cells is more se-
vere than that of Rad17-deficient cells, indicating that a
Rad17-independent pathway(s) exists in mammalian cells (60).
53BP1 is a mediator of DNA damage-induced checkpoints that
acts downstream of ATM and ATR and is required for phos-
phorylation of BRCA1 and Chk2 (14, 18, 59). Our results
indicate that Artemis does not function downstream of 53BP1
and thereby suggest that it does not act downstream of Chk2.
Artemis has been shown to possess both endonuclease and
exonuclease functions that act on DNA hairpins and broken
ends (34). It is possible that the checkpoint function of Artemis
could be mediated through its nucleolytic activity. Recently, it
has been shown that single-stranded DNA is the activating
signal for the ATR- and possibly ATM-mediated checkpoint
responses (45, 65). Also, there is precedence for a factor which
contains a nuclease function and which is also involved in
checkpoint mechanisms. The MRN complex possesses exonu-
clease and hairpin-opening activities (46, 47) and has been
shown to function in a DNA damage-induced checkpoint in S
phase (17, 33, 62). Alternatively, since Artemis does not ap-
pear to act through the canonical pathways of G2/M check-
point arrest that are highly conserved between yeast and mam-
mals, it is possible that Artemis may be in a pathway involved
in the maintenance of checkpoint arrest, rather than in its
initiation. Studies of yeast have shown that initiation and main-
tenance of checkpoint signaling are functionally separable
(13). This hypothesis is supported by our finding that Artemis-
deficient cells are not defective in the initial imposition of cell
cycle arrest that occurs within 1 h after IR irradiation but
rather appear unable to maintain the arrest at later time
points. Such a pathway would likely monitor DNA repair to
ensure its completion before releasing the checkpoint. Main-
tenance pathways are not well understood, but conceivably the
interaction between Artemis and DNA-PKcs, which is maximal
at these later time points, could be part of a mechanism to
monitor completion of DSB repair and provide a signal to
relieve the arrest and allow cells to proceed through the cell
cycle. Obviously, an important element of defining the role of
Artemis in cell cycle responses to genotoxic agents will be to
correlate the phosphorylation of Artemis mediated by ATM
and ATR to the observed failure to properly arrest after DNA
We thank the following investigators for gifts of reagents. The ATR
and ATM expression constructs were provided by Karlene Cimprich
and Michael Kastan, respectively. P11 and P14 cell lines were provided
by J. P. de Villartay. The ATRflox/?cells were provided by Stephen
Elledge, and the RAD17flox/?cells were provided by Lei Li. We thank
Lei Li for comments on the manuscript.
This work was supported in part by grants from the NIH (CA52461,
CA96574, CA90270, and ES07784).
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