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INTRODUCTION |
DNA damage triggers a variety of cellular responses in eukaryotic
cells, including the induction of a regulatory signaling network that
activates checkpoint controls (1-3). Checkpoint activation transiently
blocks cell cycle progression by arresting cells in G1 and
G2/M and slowing progression through S phase. Genetic
studies in the yeast Saccharomyces cerevisiae and
Schizosaccharomyces pombe have identified many of the
relevant players in DNA damage-induced checkpoint activation (4, 5). A
common theme that emerges from these studies is that
checkpoint-deficient yeast are dramatically more sensitive to
genotoxins than their wild-type counterparts (1-3), suggesting that
the checkpoint proteins play critical roles in cellular responses to
DNA damage.
Recent studies suggest that key checkpoint regulators may be conserved
between yeast and humans. Cloning of the human gene mutated in AT
(ATM) revealed that the ATM gene exhibited
significant homology with the S. pombe rad3
(sprad3) and S. cerevisiae MEC1 (scMEC1) checkpoint genes (6). The corresponding proteins
scMec1, spRad3, and Atm are protein kinases that share large stretches of homology, including a phosphatidylinositol 3-kinase-related kinase (PIKK)1 domain. The
PIKKs are also functionally conserved as mutation of scMEC1,
sprad3, or ATM disrupts ionizing radiation
(IR)-induced checkpoints and sensitizes the organisms to IR (7).
The PIKKs are integral players in a tentative DNA damage-inducible
signaling pathway that interfaces with the cell cycle machinery (2, 3,
8). In this model, an unidentified sensor recognizes damaged DNA.
Potential sensor candidates include the checkpoint proteins, spRad1,
spRad9, spRad17, and spHus1. The sensor relays a signal to PIKK family
members, which, at least in the case of ATM, are activated by IR (9,
10). In S. pombe, a PIKK-dependent pathway is
required for DNA damage-induced activation of spChk1 (11), a protein
kinase that phosphorylates Cdc25 and blocks its ability to activate
Cdc2, thereby preventing cell cycle transition through the
G2/M boundary.
The similarities between yeast and humans extend beyond these proteins,
as human homologs of sprad1 (12-14), sprad9
(15), sprad17 (16), spchk1 (17), and
sphus1 (18) genes have been identified, fueling speculation
that the checkpoint pathways may be conserved. Despite these advances,
the physical and functional roles of these proteins are largely
unexplored, even in the well studied yeast systems. To gain insight
into the functions of other human checkpoint proteins, we have
undertaken a biochemical and cellular analysis of the novel human
checkpoint proteins hRad1, hHus1, and hRad9. We show that hRad9
undergoes complex post-translational modifications in undamaged cells
and is inducibly phosphorylated in response to DNA damage, suggesting
that this protein participates in a DNA damage-inducible signaling
pathway. We also demonstrate that fully modified hRad9 interacts
selectively with hHus1 and hRad1 in a stable multimolecular complex
that is present even in undamaged cells. Thus, these results
demonstrate that hRad1, hRad9, and hHus1 form a stable radioresponsive
checkpoint complex that actively participates in human cellular
responses to DNA damage.
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EXPERIMENTAL PROCEDURES |
Growth, Transfections, and Irradiation--
K562 cells were
cultured in RPMI 1640 medium supplemented with 10% fetal calf serum
and 2 mM L-glutamine. Cells were transiently transfected by electroporation using a 345-volt, 10-ms pulse delivered with a T 820 electoporator (BTX). All transfections used 40 µg of
plasmid DNA, which included, if required, empty vector added to this
amount. Cells were cultured for 16-24 h after transfection before use
in experiments. Cells were irradiated at a dose rate of 11.4 Gy/min
using a 137Cs
-irradiator.
Cloning and Epitope Tagging of hRad9, hHus1, and hRad1--
All
PCR amplifications were performed with the Expand High Fidelity PCR
System (Boehringer Mannheim). Cloned PCR products were sequenced, and
plasmids without mutations were selected for use.
The hRad9, hHus1, and hRad1 cDNAs were amplified by PCR from a
PCR-ready human testes cDNA library (CLONTECH).
For hRad9, oligonucleotide primers were used that produced an
amino-terminal in-frame fusion with a tandem AU1 epitope tag. The PCR
fragment was cloned into pcDNA3 to yield
pcDNA3-AU12-hRad9. hRad1 was amplified with PCR primers
that appended an amino-terminal tandem FLAG epitope tag fused in-frame
with hRad1. The resulting PCR fragment was cloned into pcDNA3 to
yield pcDNA3-FLAG2-hRad1. The hHus1 expression vector
was prepared by amplifying hHus1 using primers that added a tandem HA
epitope tag to the carboxyl terminus of hHus1. The PCR-derived DNA
fragment was cloned into pEF-BOS-
RI (19) to yield
pEF-BOS-
RI-hHus1-HA2.
Antibodies--
Bacterial expression vectors for
hexahistidine-tagged hRad1, hHus1, and hRad17 were generated by cloning
PCR-amplified DNA fragments into pET24a+ (Novagen, Madison, WI).
Histidine-tagged proteins were induced and purified according to
manufacturer's instructions. The hRad9 cDNA was cloned into
pGEX-KG (20) to generate an in-frame fusion with glutathione
S-transferase. Bacterially produced GST-hRad9 was purified
by affinity chromatography on glutathione agarose. The purified
proteins were used to immunize rabbits with standard procedures. The
anti-HA and anti-AU1 mAbs were from Babco (Berkeley, CA), and the
anti-FLAG mAb was from Eastman Kodak Co.
Coimmunoprecipitation Experiments--
Exponentially growing
K562 cells (1 × 107 per sample) were either
transfected as indicated above or used directly for immunoprecipitation studies. Cells were washed in phosphate-buffered saline, and lysed in
lysis buffer (50 mM HEPES, 1% Triton X-100, 10 mM NaF, 30 mM Na4P207, 150 mM NaCl, 1 mM EDTA, containing freshly added: 10 mM
-glycerophosphate, 1 mM Na3VO4,
20 µg/ml pepstatin A, 10 µg/ml aprotinin, 20 µg/ml leupeptin, 40 µM microcystin-LR). The cell lysates were
immunoprecipitated with the indicated mouse monoclonal antibodies and
protein G-Sepharose (Sigma) or rabbit antisera and protein A-Sepharose
(Sigma) for 1 h. The immunoprecipitates were washed three times
with lysis buffer and fractionated by SDS-PAGE (10% gel). The gels
were transferred to Immobilon-P (Millipore, Bedford, MA) and
immunoblotted. All membranes were developed with SuperSignal
chemiluminescent substrate (Pierce). Membranes that were sequentially
blotted were stripped with two 30-min washes of 8 M
guanidine hydrochloride prior to blocking and immunoblotting.
Mobility Shift Experiments and Phosphatase Treatment--
K562
cells (1 × 107 per assay point) were treated with the
indicated stimuli, and hRad9 was immunoprecipitated. For the
phosphatase experiments, immunoprecipitates were washed three times
with lysis buffer and three times with 50 mM Tris, pH 8.5, 0.1 mM EDTA. Phosphatase-treated samples were incubated
with 0.25 unit of calf intestinal alkaline phosphatase for 30 min at
30 °C, with or without 50 mM
-glycerophosphate, following the manufacturer's directions (Life Technologies, Inc.). The
immunoprecipitates were then washed once with lysis buffer, mixed with
SDS-PAGE sample buffer, and fractionated by SDS-PAGE (10% gel).
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RESULTS |
hRad1, hRad9, and hHus1 Associate in a Checkpoint
Complex--
Comparisons of the predicted human and yeast protein
sequences indicate that the human proteins are 25-30% identical and
53-57% similar to their respective homologs, with homologies
extending over extensive portions of each protein (12-18). In S. pombe, spRad1 and spHus1 associate in wild-type, but not
rad9, mutant yeast. One interpretation of this result is
that spRad9 may physically link spRad1 to spHus1, although this
hypothesis has not been validated experimentally (18). To address
whether conservation extends to a functional level, we examined the
ability of the human homologs to form biochemical complexes similar to
those reported in yeast.
hHus1 immunoprecipitates (Fig.
1A, upper left
panel) contained a 34-kDa protein, which is similar in size to
hHus1's predicted mass of 32 kDa. The hHus1 immunoprecipitates also
contained an anti-hRad1-reactive 33-kDa band (Fig. 1A,
middle left panel), which comigrated with immunoprecipitated
hRad1 (Fig. 1A, middle right panel),
demonstrating that hRad1 associated with hHus1. Immunoblotting of the
hHus1 immunoprecipitate with an anti-hRad9 antiserum revealed a 70-kDa
band (Fig. 1A, bottom left panel) that comigrated
with immunoprecipitated hRad9 (Fig. 1A, lower center
panel). This was surprising, as hRad9 has a predicted molecular mass of 45 kDa, which is much smaller than the 70-kDa band we observed.
However, as we show later, hRad9 undergoes extensive modification (see
Figs. 2 and
3), which likely accounts for the discrepancy between the apparent and predicted molecular masses.

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Fig. 1.
hRad9, hHus1, and hRad1 associate in a stable
complex. A, K562 cell lysates were immunoprecipitated
with preimmune (PI) antisera, anti-hRad9, anti-hRad1, or
anti-hHus1 rabbit antisera. Immunoprecipitates were immunoblotted with
hRad9, hHus1, or hRad1. B, K562 cell lysates were
immunoprecipitated with anti-hHus1 or anti-hRad17 (left
panel) or anti-hRad1 or anti hRad17 (right panel).
Immunoprecipitates were immunoblotted with anti-hRad17 (upper
panels) or anti-hHus1 (lower left panel) or anti-hRad1
(lower right panel).
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Fig. 2.
Transiently overexpressed hRad9 undergoes
complex modifications. K562 cells were transiently transfected
with 40 µg of pcDNA3 empty vector only (lanes 1,
2, and 4), with 5 µg of
pEF-BOS- RI-hHus1-HA2, and 5 µg
pcDNA3-FLAG2-hRad1 expression vectors (lane
3), with 20 µg AU1-hRad9 alone (lane 5) or with these
amounts of all three checkpoint expression vectors (lane 6).
The following day, cell lysates were immunoprecipitated with either
preimmune serum (PI, lane 1), anti-hRad9
(lanes 2 and 3), or anti-AU1 mAb (lanes
4-6). Immunoprecipitations (IP) were
immunoblotted with anti-hRad9. The arrows indicate apparent
molecular masses of AU1-hRad9 calculated using commercially available
protein standards.
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Fig. 3.
Transiently expressed, epitope-tagged hRad9,
hHus1, and hRad1 interact. Cells were transfected with 40 µg of
pcDNA3 (lanes 1, 5, 9,
13), 30 µg of pcDNA3-AU12-hRad9
(lanes 3, 4, 7, 8,
11, 12, 15, 16), 5 µg of
pEF-BOS- RI-hHus1-HA2 (lanes 2, 4,
6, 8, 10, 12,
14, 16), or 5 µg of
pCDNA3-FLAG2-hRad1 expression vectors. Twenty hours
after transfection, the cells were lysed, and an aliquot of each lysate
was prepared for electrophoresis (Lysate). The lysates were
immunoprecipitated with anti-FLAG, anti-HA, or anti-AU1 mAb and
lysates, and the precipitates were immunoblotted sequentially with
anti-hRad9 (C), anti-FLAG (A), followed by
anti-HA (B). The apparent molecular mass of AU1-hRad9 was
estimated by comparison with protein standards.
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To further verify that these proteins associate, we immunoprecipitated
hRad9. In these precipitates we readily observed a hHus1-reactive band
(Fig. 1A, upper center panel) that comigrated with immunoprecipitated hHus1 (Fig. 1A, upper left
panel). We also immunoprecipitated hRad1 and demonstrated that
hHus1 (Fig. 1A, upper right panel) and hRad9
(Fig. 1A, lower right panel) were present in
anti-hRad1 immunoprecipitates. However, we did not find hRad1 in
anti-hRad9 immunoprecipitates (Fig. 1A, middle center
panel), even though we did observe hRad9 in anti-hRad1 immunoprecipitates (Fig. 1A, lower right panel).
One explanation for these discrepant results is that hRad1 and the
immunodominant anti-hRad9 antibodies share an overlapping binding site,
which precludes simultaneous interaction. In support of this Fig. 3 demonstrates that anti-epitope immunoprecipitates of AU1-tagged hRad9
contain hRad1. These results suggest that this group of human
checkpoint proteins assembles into a multimolecular complex even in the
absence of genotoxic stimuli.
hRad1 and hHus1 Do Not Interact with hRad17--
Epistasis studies
identify genetic interactions and are frequently indicative of
biochemical interactions as well. Studies in S. cerevisiae
have shown genetic and biochemical interactions among the members of
the scRAD24 epistasis group (21, 22), which includes
S. cerevisiae homologs for HRAD1
(scRAD17), HRAD17 (scRAD24), and
HRAD9 (partially homologous to scDDC1).
Additionally, previous work demonstrated that hRad17 and hRad1
interacted in a two-hybrid system (16). Therefore, we addressed the
possibility that hRad1 and hHus1 might interact with the checkpoint
protein hRad17. We immunoprecipitated hHus1 and hRad17 (Fig.
1B, left panel) and hRad1 and hRad17 (Fig.
1B, right panel). The immunoprecipitates were
immunoblotted first with anti-hHus1 (Fig. 1B, lower
left panel) or anti-hRad1 (Fig. 1B, lower right
panel), followed by anti-hRad17 (Fig. 1B, upper
panels). We found no interactions between hRad17 and hRad1 or
hHus1. Thus, these results suggest that the human checkpoint complexes
may be differentially assembled in undamaged cells. Alternatively, the
interactions may be transient and not detectable under our experimental conditions.
hRad9 Undergoes Complex Post-Translational Modifications--
To
generate a system amenable to further biochemical analysis, we prepared
epitope-tagged expression vectors for hRad1, hHus1, and hRad9. When
analyzed by SDS-PAGE (Fig. 1), hRad9 migrated with an apparent
molecular mass (70 kDa) that was much larger than predicted (45 kDa),
suggesting that the cellular pool of hRad9 may undergo extensive
post-translational modifications (Fig. 2, lanes 2 and
3). Consistent with this observation, overexpression of
AU1-tagged hRad9 revealed multiple species when resolved by SDS-PAGE
(Fig. 2, lane 5). The major detectable band had an apparent molecular mass of 55 kDa. This is significantly smaller than the 70-kDa
endogenous hRad9 (Fig. 2, lanes 2 and 3), but
still larger than the predicted molecular mass (45 kDa), even when the
2-kDa epitope tag is taken into account. Thus, the major 55-kDa may be
either an unmodified form that migrates anomalously or a partially modified version. In addition to the 55-kDa form, multiple slower migrating bands were present above this band, suggesting several steps
of post-translational modification (Fig. 2, lane 5).
Remarkably, coexpression of hRad9 with hHus1 and hRad1 increased the
amount of a highly modified, 72-kDa form of hRad9 (Fig. 2, lane
6). This slow-migrating form of hRad9 had an apparent molecular
mass slightly greater than endogenous hRad9, which is due to the
addition of the tandem AU1 tag on hRad9.
Epitope-tagged Checkpoint Proteins Associate in a
Modification-dependent Manner--
To test whether the
epitope-tagged checkpoint proteins recapitulate complex formation, K562
cells were transfected with empty vector, a combination of hHus1 and
hRad1, hRad9 alone, or expression vectors for all three proteins. The
anti-hRad9 immunoblots of cell lysates (Fig. 3, lanes 1-4)
revealed that epitope-tagged hRad9 was highly overexpressed compared
with the 70-kDa endogenous hRad9 (Fig. 3C, lanes
3 and 4), which was not visible on this exposure.
Consistent with the results presented in Fig. 2, there were multiple
forms of AU1-tagged hRad9, and coexpression of hRad1 and hHus1 enhanced
the accumulation of the highly modified form (Fig. 3C,
lane 15 versus lane 16).
To assess associations among the transfected proteins, we
immunoprecipitated hRad1 (Fig. 3, lanes 5-8) and found
epitope-tagged hRad1 (Fig. 3A), hHus1 (Fig. 3B),
and both endogenous hRad9 (Fig. 3C, lane 6) and
epitope-tagged hRad9 (Fig. 3C, lane 8) in these immunoprecipitates. We also immunoprecipitated with anti-HA (Fig. 3,
lanes 9-12) and looked for associated hRad1 (Fig.
3A), hHus1 (Fig. 3B), and hRad9 (Fig.
3C). Again, as in the hRad1 (anti-FLAG) immunoprecipitates,
endogenous and AU1-tagged hRad9 were present in the complex.
Strikingly, in both the hRad1 and hHus1 precipitations, only the most
highly modified form of transfected hRad9 associated with these
proteins, suggesting that the modification is essential for interaction.
In the reciprocal experiment (Fig. 3, lanes 13-16) we
observed that hRad1 (Fig. 3A) and hHus1 (Fig. 3B)
coprecipitated with hRad9. Unlike our observations with the endogenous
proteins, hRad1 (Fig. 3A) was detected readily in the
anti-AU1-hRad9 immunoprecipitations, thus suggesting strongly that the
rabbit anti-hRad9 antiserum indeed masks or disrupts the hRad1
interaction (see Fig. 1). Taken together, these results revealed that
the human checkpoint proteins hRad1 and hHus1 associated selectively
with modified hRad9 in a protein assembly that mimics the endogenous complex.
hRad9 Is Phosphorylated in Response to DNA Damage--
scDdc1 is a
putative S. cerevisiae homolog of spRad9 and hRad9. Because
scDdc1 is phosphorylated in response to DNA damage (22), we explored
the possibility that hRad9 may also be phosphorylated in response to
genotoxins. It is important to note that endogenous hRad9 (although
highly modified) migrates as a single band when isolated from undamaged
cells. However, we noticed that the single endogenous hRad9 band
(70-kDa form) exhibited a progressively greater reduction in
electrophoretic mobility when isolated from cells irradiated with
increasing doses of IR (Fig.
4A). We also explored the time
course of the mobility shift, which showed that hRad9 was modified
within 30 min, was maximal at 2 h, and persisted for at least
6 h (Fig. 4B). To determine whether the mobility shift
reflected DNA damage-induced phosphorylation, we treated hRad9
immunoprecipitates isolated from irradiated cells with calf intestinal
phosphatase (Fig. 4C). As expected for phosphorylation, the
DNA damage-induced mobility shift was readily reversed by treatment
with the phosphatase. Moreover, the phosphatase inhibitor
-glycerophosphate blocked the effects of the phosphatase, suggesting that effects of the phosphatase preparation are not the result of
contaminating activities.

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Fig. 4.
hRad9 is phosphorylated in response to DNA
damage. A, K562 cells were treated with nothing ( ) or
5, 10, 20, or 50 Gy IR and cultured for 5 h after irradiation.
Lysates were immunoprecipitated with anti-hRad9 and immunoblotted with
anti-hRad9. B, K562 cells were treated with nothing ( ) or
50 Gy IR and cultured for 0.5, 1, 2, 4.5, or 6.5 h. Samples were
processed as in A. C, K562 cells were
treated with nothing or 50 Gy IR and cultured for 5 h. hRad9
immunoprecipitates were washed with lysis buffer, followed by
phosphatase buffer, and treated with nothing or with 0.25 unit of calf
intestinal alkaline phosphatase (CIAP), in the absence and
presence of 50 mM -glycerophosphate ( -GP).
The immunoprecipitates were washed once with lysis buffer and
immunoblotted with anti-hRad9.
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DISCUSSION |
The present results demonstrate that hRad9 undergoes extensive and
quantitative modification even in the absence of exogenous genotoxic
stimuli. This modification is significant, because it is crucial for
hRad9 to interact with the checkpoint proteins hRad1 and hHus1.
However, there is an intricate interplay between hRad9 modification and
complex formation. Overexpression of hRad1 and hHus1 promote the
appearance of fully modified hRad9. One possible explanation for this
result is that hRad1 and hHus1 are required for hRad9 modification.
Another, perhaps more plausible explanation is that hRad9 is not stable
when overexpressed singly, but once modified it associates with hRad1
and hHus1 and forms a stable multimolecular complex.
In addition to the modification required for hHus1 and hRad1
interactions, hRad9 is phosphorylated in response to DNA damage, like
its S. cerevisiae homolog scDdc1 (22). Phosphorylation of
scDdc1 requires the Atm homolog scMec1 (23). However, in SV40-transformed AT fibroblasts, we observed DNA damage-induced hRad9
phosphorylation (data not shown), suggesting that other PIKKs, possibly
Atr, may be key mediators of this signaling pathway. Although we do not
yet know the significance of hRad9 phosphorylation, it has no effect on
association with hRad1 or hHus1 (data not shown), suggesting that
phosphorylation regulates interactions with other members of the
checkpoint signaling cascade.
There is a precedent for other checkpoint proteins undergoing extensive
modifications that dramatically alter their apparent molecular mass and
regulate their interactions with other proteins. The unrelated scRad9
is phosphorylated in response to DNA damage, and this modification is
essential for scRad9's interaction with the checkpoint protein kinase
scRad53 (24). There are several potential molecular modifications that
may contribute to hRad9's mobility shift, including phosphorylation
that is resistant to phosphatase treatment, possibly due to protection
by hHus1 and hRad1 interaction. Although the nature of the hRad9
modification is currently unknown, we are intensively investigating
these and other possible molecular alterations of the protein.
Previous studies in yeast demonstrated that hRad1 and hHus1 genetically
and biochemically interact and are required for activation of
checkpoints in response to DNA damage and replication inhibitors (18).
Although much genetic evidence attests to their importance in cell
cycle arrest and survival, even in the well studied yeast models little
is known about their functions. To add further complexity, spRad9,
hRad9, spHus1, and hHus1 have no signature sequences or homologies with
other proteins that yield clues to their functions. However, spRad1 and
hRad1 have significant homology with Ustilago maydis Rec1
exonuclease (12-14, 25). Additionally, hRad1 may possess 3'
5'
exonuclease activity (13), although other groups could not confirm this
hypothesis (12). The presence of a putative DNA-metabolizing protein in
the multimolecular checkpoint complex, coupled with genetic data that
place spRad1, spHus1, and spRad9, and their S. cerevisiae
counterparts, early in the response pathway (2, 3, 21), suggests that
the complex may function as a sensor that scans the genome for damaged
DNA. Once damaged DNA is detected, this complex may initiate
endonucleolytic processing of the lesions and trigger interactions with
downstream signaling elements. Alternatively, the checkpoint complex
may link unknown damage recognition components to downstream
signal-transducing pathways that include ATM and hChk1, both of which
are implicated in actively enforcing cell cycle arrest after DNA
damage. Thus, the present data provide the first identification of a
DNA damage-responsive human checkpoint complex that is fundamentally
conserved between yeast and humans.