From the Divisions of Developmental Oncology Research
and
Radiation Oncology and the Departments of
¶ Biochemistry and Molecular Biology and
§ Molecular Pharmacology, Mayo Clinic, Rochester, Minnesota
55905
Received for publication, April 3, 2001
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ABSTRACT |
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DNA damage activates cell cycle checkpoint
signaling pathways that coordinate cell cycle arrest and DNA repair.
Three of the proteins involved in checkpoint signaling, Rad1, Hus1, and
Rad9, have been shown to interact by immunoprecipitation and yeast
two-hybrid studies. However, it is not known how these proteins
interact and assemble into a complex. In the present study we
demonstrated that in human cells all the hRad9 and hHus1 and
approximately one-half of the cellular pool of hRad1 interacted as a
stable, biochemically discrete complex, with an apparent molecular mass of 160 kDa. This complex was reconstituted by co-expression of all
three recombinant proteins in a heterologous system, and the reconstituted complex exhibited identical chromatographic behavior as
the endogenous complex. Interaction studies using differentially tagged
proteins demonstrated that the proteins did not self-multimerize. Rather, each protein had a binding site for the other two partners, with the N terminus of hRad9 interacting with hRad1, the N terminus of
hRad1 interacting with hHus1, and the N terminus of hHus1 interacting with the C terminus of hRad9's predicted PCNA-like region.
Collectively, these analyses suggest a model of how these three
proteins assemble to form a functional checkpoint complex, which we
dubbed the 9-1-1 complex.
DNA damage provokes multiple cellular responses, including the
activation of DNA damage checkpoint signaling pathways, which arrest cell cycle progression in G1, S, and
G2/M and possibly coordinate repair of the damage. Loss of
the DNA damage checkpoint response leads to increased sensitivity to
DNA-damaging agents, demonstrating that these pathways are critical for
efficient recovery from DNA damage (reviewed in Refs. 1-6). The
components of the checkpoint signaling pathways were initially
identified by genetic analyses in the yeasts, Schizosaccharomyces
pombe and Saccharomyces cerevisiae, and recent studies
demonstrated that the checkpoint genes are conserved between yeasts and
humans (7-23). Collectively, these studies have given rise to a
conserved model in which DNA damage activates members of the ATM
(ataxia telangiectasia-mutated) family of phosphatidylinositol
3-kinase-related kinases. These protein kinases are then required to
activate the downstream protein kinases Chk1 and Chk2 (20, 22-25),
which regulate cell cycle arrest and possibly other DNA damage-induced responses.
Despite the recent progress in the dissection of the downstream protein
kinase-mediated portions of the checkpoint signaling pathways, yeast
genetic studies demonstrated that additional checkpoint genes also
regulate the DNA damage response (reviewed in Refs. 2-6). For example,
in S. pombe spRad1, spHus1, spRad9, and spRad17 (using
S. pombe nomenclature) are all essential for DNA
damage-induced cell cycle arrest and activation of the spChk1 protein
kinase after DNA damage (4-6). Thus, these results suggest that Rad1, Hus1, Rad9, and Rad17 function upstream of Chk1 activation in the DNA
damage-signaling pathway.
Recently, molecular modeling studies were used to propose functions for
Rad1, Hus1, Rad9, and Rad17 (26-28). In this model Rad9, Hus1, and
Rad1 form a PCNA1 like clamp. PCNA is homotrimeric
doughnut-shaped structure that is loaded onto DNA by the clamp loader,
replication factor C (RFC) (29, 30). Once
loaded, PCNA encircles the DNA as a sliding clamp, tethering
replication proteins, including DNA polymerase In addition to the molecular modeling predictions, limited biochemical
data also support this model. First, the S. cerevisiae homolog of hRad17 interacts with the four small subunits of RFC in a
stable complex, suggesting that the Rad17-RFC complex may indeed be a
clamp loader (32). However, no data currently support this contention.
Second, yeast and mammalian Rad9, Hus1, and Rad1 interact in
immunoprecipitation and yeast two-hybrid analyses (27, 33-35).
However, the nature of the complex has not been explored. Third, hRad1,
hHus1, and hRad9 interact with hRad17 in a manner that requires an
intact hRad17 ATP-binding domain (36). Fourth, following DNA damage,
the Rad9-Hus1-Rad1 complex is converted to an extraction-resistant,
chromatin-bound complex, which may reflect clamping of the complex onto
sites of damage (37). Collectively, these observations suggest a model
in which the Rad17-RFC clamp loader recognizes DNA damage and then
loads a Rad9-Hus1-Rad1 clamp around the DNA. Once loaded around DNA, the Rad9-Hus1-Rad1 complex may tether signaling and repair molecules to
the damaged site.
Despite the data supporting the clamp model, there are several
experimental inconsistencies with the model. First, studies in S. pombe demonstrated that spHus1 and spRad1 could not interact in
the absence of spRad9 (10, 27), which suggested that spRad9 linked the
two proteins. In contrast, the clamp model predicts that spHus1 and
spRad1 should interact even in the absence of spRad9. Second,
additional studies in S. pombe indicated that spRad9,
spHus1, and spRad1 interacted at very low stoichiometry, calling into
question whether these proteins do indeed form a stable clamp (27).
Third, there are phenotypic differences between sprad1 and
sphus1 null mutants, which would be unexpected if the two
proteins formed a portion of the clamp (38). To address these questions
with the mammalian proteins and to characterize further the biochemical
nature of hRad9, hHus1, and hRad1 complex, we examined the complex in
human cell lysates using size-exclusion chromatography. We also
reconstituted the complex in an insect expression system and mapped the
interaction domains among the three proteins. These studies revealed
that hRad9, hHus1, and hRad1 form a stable protein complex, which we
dubbed the 9-1-1 complex. Our data, which are consistent with a
circular head-to-tail organization for the three-member complex, also
support the proposed sliding clamp model. Additionally, these studies
identified a pool of monomeric hRad1, which may have functions separate
from the 9-1-1 complex.
Cell Culture, Transfections, and Antibodies--
K562 cells were
cultured as previously described (34). All transfections contained 40 µg of DNA, which was adjusted with empty vector if required, and were
performed as described (34). Rabbit antisera against hRad1, hHus1, and
hRad9, and the monoclonal antibody against hRad9 have been described
previously (34, 37). Monoclonal antibodies to Myc, HA, AU1, and AU5
were from Covance. Monoclonal antibodies to FLAG were from Sigma.
Rabbit antisera to green fluorescent protein (GFP) were from Molecular
Probes. S-protein-agarose and S-protein-coupled to horseradish
peroxidase were from Novagen and used according to the manufacturer's recommendations.
Plasmid and Baculovirus Construction--
FLAG-tagged hRad1,
AU1-tagged hRad9, and HA-tagged hHus1 have been described previously
(34). To generate "half-mutants" of hRad1, hHus1, and hRad9, we
examined the molecular modeling structures for these proteins (26-28)
and constructed expression vectors that truncated the proteins in the
putative loop that connects the two lobes. Polymerase chain reaction
(PCR)-based strategies were used to generate the amino-terminal
half-mutants of hRad1, hHus1, and hRad9. The hRad1 amino-terminal
half-mutant contained amino acids 1-146 followed by four copies of the
AU1 epitope tag. The hRad9 amino-terminal half-mutant extended from amino acids 1-129 and tandem HA epitope tags were added to the carboxyl terminus. The hHus1 amino-terminal half-mutant extended from
amino acids 1-148 followed by tandem Myc epitope tags. The hRad9
carboxyl-terminal half-mutant fused S-Tag peptide (MKETAAAKFERNHMDS) to
amino acids 135-275 of hRad9. All epitope-tagged half-mutants were
cloned into pcDNA3, and the cloned constructs were sequenced to
verify fidelity of PCR amplification.
Recombinant baculoviruses encoding His6-tagged hRad9 and
untagged hRad1 and hHus1 were generated using the Bac-to-Bac (Life Technologies, Inc.) expression system. The coding region of hRad9 was
PCR amplified and cloned in-frame with the His6 tag of the pFastBac-HTa baculoviral transfer vector. Similarly, untagged hRad1 and
hHus1 were amplified by PCR and cloned into the pFastBac-1 transfer
vector. All cloned PCR products were sequenced to assure accurate
amplification. Transfer vectors were then transformed into DH10Bac
E. coli and selected for recombination between the transfer
vector and the bacterial bacmid plasmid using an in-plate Coimmunoprecipitation Studies--
Exponentially growing K562
cells (1 × 107 per assay point) were washed in
phosphate-buffered saline. Cells were lysed in lysis buffer (10 mM HEPES, pH 7.4, 150 mM KCl, 10 mM
MgCl2, and 0.1% Triton X-100) supplemented with freshly
added 10 mM Size-exclusion Chromatography--
Exponentially growing K562
cells (2 × 108 per fractionation) were washed in
phosphate-buffered saline and lysed in 0.5 ml of lysis buffer
supplemented with freshly added 10 mM
For analysis of the reconstituted hRad9-hHus1-hRad1 complex, Sf9
cells (4 × 107) were co-infected with baculoviruses
expressing His-tagged hRad9 and untagged hRad1 and untagged hHus1.
Forty-eight hours after infection, the cells were lysed in lysis buffer
supplemented with freshly added 10 mM Size-exclusion Chromatographic Analysis of hRad9, hHus1, and hRad1
in K562 Cell Lysates--
Previous immunoprecipitation and yeast
two-hybrid analyses indicated that hRad9, hHus1, and hRad1 interacted.
To examine further the interactions among hRad9, hHus1, and hRad1, we
analyzed these proteins by size-exclusion chromatography. K562 cells
were lysed and clarified lysates were fractionated using a Superdex 200 column (Fig. 1A). Lysates and
column fractions were immunoprecipitated and immunoblotted for hRad1.
These studies revealed that hRad1, which has a predicted molecular mass
of 31 kDa, fractionated into two separate pools. One pool eluted with
an apparent molecular mass of ~40 kDa, suggesting that approximately
half of the hRad1 pool exists in monomeric form in cell lysates. The
remaining hRad1 eluted with an apparent molecular mass of 160 kDa,
suggesting that this portion of the hRad1 pool is part of a larger
protein complex.
To determine whether hHus1 and hRad9 exhibited similar elution
profiles, we analyzed the remainder of the column fractions for hRad9
and hHus1. hRad9 has a predicted molecular mass of 43 kDa and hHus1 has
a predicted molecular mass of 31 kDa, and yet both proteins co-eluted
with the fast-eluting form of hRad1, suggesting that they are also part
of larger protein complexes. Unlike hRad1, however, no monomeric hRad9
and hHus1 were present in the cell lysates. To assess whether the
fast-eluting, 160-kDa complex contains interacting hRad1, hHus1, and
hRad9, we immunoblotted the hRad9 immunoprecipitates for hRad1 and
hHus1. Fig. 1B shows that the hRad9 immunoprecipitates
contained both hRad1 and hHus1. Taken together, these results suggest
that hRad9 and hHus1, and much of the hRad1 pool, are part of a larger,
interacting protein complex that elutes from a size-exclusion column
with an apparent molecular mass of 160 kDa. Because the predicted
additive molecular mass of the complex is 105 kDa, this suggests that
the complex is either non-spherical or associates with additional proteins.
hRad9, hHus1, and hRad1 Do Not Self-multimerize--
We then
addressed the possibility that self-multimerization of hRad1, hHus1,
and hRad9 may contribute to complex formation. We co-overexpressed
FLAG-tagged hRad1 with AU5-tagged hRad1, and, as a positive control, we
also co-expressed FLAG-tagged hRad1 with HA-tagged hHus1 (Fig.
2A). Immunoblotting for FLAG
revealed that although FLAG-tagged hRad1 interacted with hHus1 it did
not interact with itself (AU5-tagged hRad1), even when the immunoblot was overexposed. An analysis examining hHus1 self-oligomerization showed that HA-tagged hHus1 interacted with Rad1 but did not associate with itself (Myc-tagged Hus1) (Fig. 2B).
We performed a similar analysis for hRad9. For this analysis, we
generated differentially tagged hRad9 expression vectors that expressed
carboxyl-terminally truncated hRad9 (amino acids 1-270). This
truncation contains the predicted PCNA-like folds (amino acids 1-270)
but removes a carboxyl-terminal extension (amino acids 271-391) of
hRad9 that is not essential for interaction with hRad1 or hHus1 (data
not shown and Fig. 2C). We used this portion of hRad9 for
self-oligomerization analysis because we previously observed that when
overexpressed alone in insect cells and K562 cells, full-length hRad9
elutes as a broad peak (>600 kDa to monomeric size) from Superdex 200. These results suggest that, unlike hRad1 and hHus1, full-length hRad9
aggregates when overexpressed (data not shown). Using the truncated
hRad9 constructs, we found that GFP-tagged hRad9-270 (amino acids
1-270) interacted with hHus1 but did not interact with itself
(AU1-tagged hRad9-270) (Fig. 2C). Taken together, these
studies suggest that self-oligomerization of hRad1, hHus1, and hRad9
does not likely contribute to 9-1-1 complex formation.
The hRad9-hHus1-hRad1 Complex Is Reconstituted in an Insect Cell
Expression System--
To analyze further the hRad9-hHus1-hRad1
complex, we generated recombinant baculovirus expression constructs for
His6-tagged hRad9 and untagged hRad1 and hHus1. hRad9
undergoes extensive phosphorylation in undamaged mammalian cells (basal
phosphorylation), and in response to DNA damage hRad9 is additionally
phosphorylated (hyperphosphorylation) (34). Therefore, we examined
whether hRad9 is basally phosphorylated when expressed in insect cells. Because basal hRad9 phosphorylation dramatically affects protein mobility when analyzed by SDS-PAGE, we examined the mobility of hRad9
in cell lysates derived from baculovirus-infected Sf9 cells. Unmodified hRad9 migrates with an apparent molecular mass of 43 kDa,
equivalent to its predicted molecular mass, whereas fully phosphorylated hRad9 migrates with an apparent molecular mass of 66-68 kDa.
To verify that hRad9 phosphorylation patterns were similar when
expressed in either insect or K562 cells, we overexpressed AU1-tagged
hRad9, along with hRad1 (FLAG-tagged) and hHus1 (HA-tagged), in K562
cells (Fig. 3). In parallel, we
co-infected insect cells with His6-tagged hRad9 and
untagged hHus1 and hRad1. Transfected hRad9 (AU1), hHus1 (HA), and
hRad1 (FLAG) were precipitated from K562 cells lysates using the
indicated epitope tags. Lysates from infected Sf9 cells were
immunoprecipitated with anti-hRad9, anti-hHus1, and anti-hRad1. The
precipitates were fractionated by SDS-PAGE in parallel and
immunoblotted for hRad9. As in K562 cells, insect cell-expressed hRad9
migrated as a series of modified forms ranging from partially to fully
modified. Consistent with the results obtained in human cells, hRad1
and hHus1 interacted more efficiently with the highly modified forms of
hRad9. Collectively, these results indicated that hRad9 undergoes
physiologically relevant phosphorylations, and that the complex can be
reconstituted in an insect expression system.
To characterize further the insect cell-expressed hRad9-hHus1-hRad1
complex, we fractionated the complex by size-exclusion chromatography
using a Superdex 200 column (Fig. 4). We
co-infected insect cells with excess hRad1 and hHus1 virus and a
limiting amount of His6-hRad9 virus (data not shown) and
purified the complex using Ni2+-chelate chromatography
prior to size-exclusion chromatography. These experiments revealed that
all three proteins co-eluted as a single peak at precisely the same
position as the peak observed for hRad9, hHus1, and hRad1 in K562
cells. Thus, co-expression of recombinant hRad9, hHus1, and hRad1
reconstituted this checkpoint complex in an insect system. This result
suggests that the slightly anomalous apparent molecular mass of the
complex may be intrinsic to the complex. Alternatively, an insect
cell-derived protein might associate with the complex, which increases
the apparent chromatographic size of the 9-1-1 complex.
hRad9, hHus1, and hRad9 Interact in a Pair-wise Manner--
The
results presented in Figs. 1-4 demonstrated that all three proteins
interacted and formed a trimolecular complex. To begin mapping these
interactions, we examined the association of each protein with the
other two proteins in pair-wise combinations. Insect cells were
infected with the indicated virus combinations (Fig.
5) and immunoprecipitated either for
hRad1, hHus1, or hRad9. The immunoprecipitates were then immunoblotted
for the indicated proteins. As positive controls for the experiments,
we also infected insect cells with all three viruses to generate the
trimolecular complex. These studies revealed that in reciprocal
immunoprecipitates hRad1 interacted with hHus1 in the absence of hRad9
(Fig. 5, A and C, left arrows), and
that hRad1 interacted with hRad9 in the absence of hHus1 (Fig.
5A, right arrow; Fig. 5B, left
arrow). Additional analyses revealed that hRad9 interacted with
hHus1 in the absence of hRad1 (Fig. 5, B and C,
right arrows). Because the proteins are overexpressed in a
heterologous system, these results suggest that the proteins interact
directly in a pair-wise manner; however, it is possible that an insect
homolog may participate in these interactions.
Mapping the Interaction Domains between hRad9, hHus1, and
hRad1--
Molecular modeling studies predict that Rad9, Hus1, and
Rad1 fold into PCNA-like structures (26-28). Each PCNA subunit is
composed of two structurally identical lobes joined by an interdomain
connector loop (39). Using the molecular models, we constructed
expression vectors that divided each protein within the linker domain
connnecting the lobes. We expressed the predicted amino-terminal lobe
of hRad1 (amino acids 1-146) and hHus1 (amino acids 1-148), which we
called amino-terminal half-mutants. Because the predicted PCNA-like
fold of hRad9 extends only to approximately amino acid 270, we called the hRad9 mutant (amino acids 1-129) an amino-terminal half-mutant as
well, even though it only represents one-third of the entire protein.
Given that we were able to detect pair-wise interactions among all
three proteins, we reasoned that these amino-terminal half-mutants
should associate with one binding partner but not the other. We tested
this hypothesis by transiently transfecting K562 cells with
epitope-tagged full-length protein or the corresponding amino-terminal
half-mutants along with epitope-tagged versions of the two potential
binding partners and asked whether the overexpressed amino-terminal
half-mutant associated with one or both partners (Fig.
6). The tagged half-mutant or
corresponding full-length proteins were then immunoprecipitated and the
immunoprecipitates were immunoblotted for the co-transfected binding
partners. These studies verified that full-length hRad9 interacted with
both hRad1 and with hHus1. In contrast, the amino-terminal hRad9
half-mutant interacted with hRad1 but not with hHus1 (Fig.
6A). Thus, we concluded that the amino-terminal portion of
hRad9 associates with hRad1. We repeated the analogous interaction
studies with the hHus1 and hRad1 amino-terminal half-mutants, which
revealed that the amino terminus of hHus1 interacted with hRad9 (Fig.
6B) and that the amino terminus of hRad1 associated with
hHus1 (Fig. 6C).
To further map these interactions, we also generated expression vectors
for the carboxyl-terminal, PCNA-like lobes of hHus1 (amino acids
135-280) and hRad1 (amino acids 135-282). Despite generating several
different constructs with a variety of tags (including large tags such
as glutathione S-transferase), the carboxyl-terminal hHus1
and hRad1 fragments were expressed very poorly compared with other
half-mutants, suggesting that they did not fold appropriately.
Consistent with these observations, these carboxyl-terminal
half-mutants were unable to interact with any other members of the
9-1-1 complex (data not shown). In contrast, an expression vector for
the carboxyl-terminal, PCNA-like lobe of hRad9 (amino acids 135-270)
was expressed well. We then asked whether the Rad9 carboxyl-terminal
half-mutant could interact selectively with either the amino-terminal
hHus1 or hRad1 half-mutants. These studies revealed that the
carboxyl-terminal hRad9 (R9-C) interacted exclusively with the
amino-terminal hHus1 half-mutant (Fig.
7A) but not with the
amino-terminal Rad1 half-mutant (Fig. 7B). Taken together
with the results in Fig. 6, these results suggest that the PCNA-like
portion of hRad9 (amino acids 1-270) is flanked on its amino terminus
by hRad1 and on its carboxyl terminus by the amino terminus of
hHus1.
Studies in the yeast S. pombe demonstrated that Rad1,
Hus1, and Rad9 are required for early events in the activation of the DNA damage checkpoint-signaling pathway. Initially, there were few
clues regarding their functions, although the proteins had been shown
to interact. To further our understanding of how hRad1, hHus1, and
hRad9 interact to form a checkpoint complex, we examined the endogenous
complex, reconstituted the complex in a heterologous expression system,
and mapped interactions among the subunits. In this report we show that
the majority of human hRad9, hHus1, and hRad1 exist in a heterotrimeric
complex. We also found that the PCNA-like portions of hHus1
(full-length), hRad1 (full-length), and hRad9 (amino acids 1-270) do
not self-multimerize. Additionally, we reconstituted the 9-1-1 complex
in an insect cell expression system and demonstrated that it had
identical chromatographic behavior. Finally, our mapping studies
demonstrated that each member of the complex interacted in a pair-wise
fashion, and these studies also mapped the interactions among the three
9-1-1 complex members. Collectively, these results provide novel
insights into the assembly, structure, and potential functions of the
9-1-1 DNA damage-responsive checkpoint complex.
The self-oligomerization analysis suggests that hRad9, hHus1, and hRad1
do not homomultimerize. This is in contrast to a study by Hang and
Lieberman (35), which demonstrated that hHus1 and hRad9
self-oligomerize. One possible explanation for this discrepancy stems
from our observation that full-length hRad9 forms large, higher-order
aggregates when overexpressed without its partners hRad1 and
hHus1.2 These higher-order
aggregates may be physiologically relevant. However, analysis of
endogenous hRad9, hHus1, and hRad1 (Fig. 1) did not reveal such
complexes. Thus, we propose that hRad9, hHus1, and hRad1 form a
heterotrimeric 9-1-1 complex that does not include self-multimers of
any individual component.
Examination of the interactions within the 9-1-1 complex revealed that
each member interacted in a pair-wise manner. We also mapped the
pair-wise interactions using amino- and carboxyl-terminal expression
constructs. These results showed that: 1) the amino terminus of hRad9
interacted selectively with hRad1, 2) the amino terminus of hRad1
interacted with hHus1, and 3) the amino terminus of hHus1 interacted
with hRad9. Further mapping, using the carboxyl terminus of the hRad9
predicted PCNA fold, demonstrated that this region associated with the
amino terminus of hHus1. Thus, the PCNA-like portion of hRad9 is
flanked by hRad1 on its amino terminus and by hHus1 on its carboxyl
terminus. Although we were unable to fully map the interactions among
the subunits, our studies support a PCNA-like clamp model (Fig.
8) and define the orientation of the
three proteins in the complex. It is important to bear in mind,
however, that confirmation of the structure awaits biophysical analysis
of purified 9-1-1 complex.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, to the replicating
DNA. RFC is composed of four small subunits (p36, p37, p38, and p40)
and one large subunit (p140), and all the subunits contain consensus
nucleotide-binding Walker A and B motifs (31). RFC binds to
primer-template junctions that are generated during replication, opens
the PCNA clamp, and loads the clamp around the DNA in an
ATP-dependent manner. Thus, the molecular modeling studies
suggest that Rad9, Hus1, Rad1, and Rad17 may have functions analogous
to the functions of PCNA and RFC.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase assay. Recombined bacmids were transfected into Sf9 cells using CellFectin reagent (Life Technologies, Inc.) and viral supernatants were harvested after 3 days. hRad9, hHus1, and hRad1
protein expression was verified by immunoblotting cell lysates from
infected Sf9 cells.
-glycerophosphate, 1 mM
Na3VO4, 20 µg/ml pepstatin A, 10 µg/ml
aprotinin, 20 µg/ml leupeptin, and 20 ng/ml microcystin-LR for 10 min
on ice. Cleared lysates (21,000 × g for 5 min) were
immunoprecipitated with the indicated mouse monoclonal antibodies and
protein G-Sepharose (Sigma) or rabbit antisera and protein A-Sepharose
(Sigma) at 4 °C for 1 h. Immunoprecipitates were washed three
times with lysis buffer, solubilized in SDS-polyacrylamide gel
electrophoresis (PAGE) buffer, and fractionated by SDS-PAGE. Proteins
were transferred to Immobilon P membrane and immunoblotted as described
(34).
-glycerophosphate, 1 mM Na3VO4, 80 µg/ml pepstatin
A, 40 µg/ml aprotinin, 80 µg/ml leupeptin, and 80 ng/ml
microcystin-LR and briefly sonicated. Cell lysates were cleared by
ultracentrifugation (290,000 × g) for 15 min and then
filtered through a 0.2-µm filter prior to injection of 300 µl of
lysate onto a Superdex 200 HR 10/30 (Amersham Pharmacia Biotech)
equilibrated in lysis buffer. The column was run at a flow-rate of 0.3 ml/min, and 0.5 ml fractions were collected. hRad1, hHus1, and hRad9
were immunoprecipitated from the total cell lysates and column
fractions. The immunoprecipitates were immunoblotted as described (34).
The column was calibrated by separating protein standards
(thyroglobulin, 660 kDa; ferritin, 440 kDa; catalase, 232 kDa;
aldolase, 159 kDa; ovalbumin, 43 kDa) under identical conditions.
-glycerophosphate,
1 mM Na3VO4, 20 µg/ml pepstatin
A, 10 µg/ml aprotinin, 20 µg/ml leupeptin, and 20 ng/ml microcystin-LR for 10 min on ice. Clarified lysates (21,000 × g) were then incubated with 0.5 ml of
Ni2+-charged His-Bind Resin (Novagen) for 1 h at
4 °C. The resin was washed with lysis buffer containing 5 mM imidazole. The complex was eluted in lysis buffer
containing 300 mM imidazole, pH 7.4, and filtered through a
0.2-µm filter. The filtered sample was analyzed on a Superdex 200 HR10/30 column as described above.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Size-exclusion chromatographic analysis
of endogenous hRad9, hHus1, and hRad1 in K562 cell lysates.
A, exponentially growing K562 cells (2 × 108) were lysed, and the lysate (300 µl) was fractionated
on a Superdex 200 HR 10/30 size-exclusion column equilibrated in lysis
buffer, and 500 µl fractions were collected. Portions of the cell
lysate and each fraction were immunoprecipitated (IP) with
the indicated rabbit antisera (hRad1 and hHus1) or monoclonal antibody
(hRad9). The precipitates were separated by SDS-PAGE and immunoblotted
as indicated. The column was calibrated with protein standards, and the
elution position of each standard is indicated in the figure.
B, the hRad9 immunoprecipitates from A were
immunoblotted sequentially for hRad1 and hHus1.
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Fig. 2.
hRad9, hHus1, and hRad1 do not
self-multimerize. A, K562 cells (1 × 107 per sample) were transfected as indicated, and
harvested 20 h after transfection, and detergent-soluble lysates
were prepared. A, lysates were immunoprecipitated
(IP)with either AU5 or HA mAb. Immunoprecipitates were
separated by SDS-PAGE (10% gel) and blotted for FLAG followed by AU5.
B, lysates were immunoprecipitated with Myc or FLAG mAb and
immunoblotted sequentially for HA and Myc. C, lysates were
immunoprecipitated with AU1 or HA mAb and immunoblotted for GFP
followed by AU1.
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Fig. 3.
Reconstitution of the hRad9-hHus1-hRad1
complex in an insect cell expression system. K562 cells were
transfected with AU1-hRad9, HA-tagged hHus1, and FLAG-tagged hRad1.
Sf9 cells were co-infected with His6-tagged hRad9
and untagged hHus1 and untagged hRad1. K562 cells were harvested
20 h after transfection, and Sf9 cells were harvested 3 days after infection. For the K562 samples, cell lysates were
precipitated with anti-AU1 for hRad9, anti-HA for hHus1, or anti-FLAG
for hRad1. For the Sf9 lysates, cell lysates were precipitated
with monoclonal anti-hRad9 or anti-hHus1, or polyclonal anti-hRad1.
After fractionation by SDS-PAGE, membranes were then immunoblotted for
hRad9. The faint shadow present in the hRad1 immunoprecipitate
(right-hand lane) is from the immunoglobulin heavy chain
used to immunoprecipitate hRad1.
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Fig. 4.
Size-exclusion analysis of the
hRad9-hHus1-hRad1 complex purified from insect cells. Sf9
cells (5 × 107) were co-infected with baculoviruses
expressing His6-hRad9 and untagged hHus1 and hRad1. The
infected cells were harvested and lysed 48 h later. The complex
was purified on Ni2+-charged His Bind Resin (Novagen) and
fractionated on a Superdex 200 HR 10/30 column as described in Fig. 1.
Portions of each cell lysate and column fractions were separated by
SDS-PAGE and immunoblotted for the indicated proteins.
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Fig. 5.
Analysis of pair-wise interactions among
hRad9, hHus1, and hRad1. Sf9 cells were co-infected with
His6-hRad9, untagged hHus1, or untagged hRad1 as indicated.
Cells were harvested and lysed 48 h later. The lysates were
immunoprecipitated with the indicated rabbit antisera (hRad1) or
monoclonal antibody (hRad9 and hHus1). The immunoprecipitates, along
with a portion of each cell lysate, were separated by SDS-PAGE and
immunoblotted as indicated. A, the indicated
immunoprecipitates were immunoblotted for hRad1. B, the
indicated immunoprecipitates were immunoblotted for hRad9.
C, the indicated immunoprecipitates were immunoblotted for
hHus1. The faint shadows present in the hRad1 immunoprecipitates on the
hRad9 immunoblot (B) are due to rabbit immunoglobulin heavy chain used
for the immunoprecipitations. Arrows mark the lanes
demonstrating the interactions that are depicted above the
arrows.
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Fig. 6.
Mapping the interaction domains of hRad9,
hHus1, and hRad1 amino termini. K562 cells (1 × 107) were transfected with the indicated full-length or
half-mutant constructs. Cells were cultured for 24 h, harvested,
and lysed. The lysates were immunoprecipitated with antibodies to the
indicated tags. The washed immunoprecipitates were separated by
SDS-PAGE and immunoblotted as shown. A, analysis of the
amino-terminal hRad9 half-mutant (R9-N). B,
analysis of the amino-terminal hHus1 half-mutant (H1-N).
C, analysis of the amino-terminal hRad1 half-mutant
(R1-N).
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Fig. 7.
The carboxyl terminus of the predicted hRad9
PCNA domain interacts with the amino terminus of hHus1. K562 cells
(1 × 107 per sample) were transfected with S-tagged
full-length (R9 (S)) or carboxyl terminus half-mutant
(R9-C(S), amino acids 135-270) and Myc-tagged
amino-terminal hHus1 (1). Transfected cells were harvested 20 h after transfection, and lysed. A, detergent-soluble
lysates were precipitated (IP) with S-protein-agarose and
blotted for Myc followed by blotting with S-protein-horseradish
peroxidase. B, K562 cells were transfected with S-tagged
full-length (R9(S)) or the carboxyl terminus half-mutant
(R9-C(S), amino acids 135-270) and AU1-tagged
amino-terminal hRad1-(1-146). Detergent-soluble lysates were
precipitated with S-protein-agarose or HA mAb and blotted for AU1
followed by S-protein-horseradish peroxidase.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 8.
Model for the 9-1-1 clamp complex. This
model demonstrates the interactions that were defined by this study,
showing that the amino terminus of hRad9 interacts with hRad1, the
amino terminus of Rad1 interacts with hHus1, and the amino terminus of
hHus1 interacts with Rad9. In each case, the interaction of the amino
terminus is predicted to occur with the carboxyl terminus of the
binding partner.
Studies in S. pombe demonstrated that Myc-tagged spHus1 eluted as a complex with an apparent molecular mass of 450 kDa (27). Moreover, only very small fractions of the total spRad9 and spRad1 pools were associated with the tagged spHus1. These results suggest that either the complex is very unstable or that the proteins do not form a clamp-like structure. In contrast, we found that all the hHus1 and hRad9, and about one-half of the hRad1 were assembled into a stable, discrete complex in human cell lysates, indicating that the complex was the favored form in cell lysates and presumably in intact cells. Such results suggest that the complexes may vary significantly between yeast and mammals. Alternatively, the addition of 13 Myc tags to spHus1, even though the tagged protein complemented a sphus1 null mutant, may dramatically affect the stability of protein-protein interactions with spHus1.
The present studies also differ from the studies in yeast with respect to interactions between individual complex members. Two studies have shown that S. pombe spHus1 and spRad1 do not co-immunoprecipitate in the absence of spRad9 (6, 10), suggesting that spRad9 links spRad1 and spHus1. This result differs from our data and is difficult to reconcile with the clamp model. We found that each protein interacted efficiently in pair-wise reconstitution experiments, which is consistent with a clamp model. The reasons for the discrepancies between the yeast and human studies are not known. The two organisms may differentially regulate complex assembly. Alternatively, the discrepancy may stem from dissimilar technical approaches used to study yeast and mammalian cells. For example, the harsher conditions required to disrupt yeast cells may also destabilize the interactions among the 9-1-1 complex members, especially if one complex member is missing as is the case in the studies with the spRad9 mutant.
The results presented here may also provide insight into a disparity between the clamp model and genetic studies in S. pombe. Although the PCNA-like clamp model for the 9-1-1 complex is compelling, it does not account for all the available data. A previous analysis of telomere maintenance in S. pombe revealed that sprad1 null mutants had telomere-shortening defects, whereas sphus1 null and sprad9-192 (null for all tested phenotypes) mutants had normal telomeric lengths (38). These observations are not compatible with a clamp composed of three different subunits; if any one subunit were missing, the clamp would no longer be able to encircle DNA. Thus, null mutants of sprad1 and sphus1 should have identical phenotypes. A possible explanation for this discordance is that the functions of these proteins may have diverged. Evidence for this possibility includes the observation that cells derived from mHus1 knockout mice are sensitive to ultraviolet but not ionizing radiation (40). In contrast, sphus1 null S. pombe are sensitive to both forms of radiation, suggesting that mammals may have evolved additional mechanisms to respond to DNA damage. An alternative explanation is that the monomeric hRad1 that we observed in human cell lysates might have functions independent of a 9-1-1 clamp. For instance, spRad1 and the related Ustilago maydis Rec1 checkpoint protein possess 3'-5' exonuclease activity (11, 41). Thus, hRad1 may have dual biochemical functions; in a complex with hRad9 and hHus1 it might be part of a clamp, and as a monomer it may operate as an exonuclease.
On the basis of the genetic, biochemical, and structural prediction
data available for Rad9, Hus1, Rad1, and Rad17, the following model has
arisen. Rad17, in complex with the small RFC subunits, may recognize
directly or indirectly DNA damage. The Rad17-RFC clamp loader will then
open and load the 9-1-1 clamp onto the DNA. Once loaded, the 9-1-1 clamp becomes resistant to extraction, and by analogy with PCNA,
tethers proteins to sites of DNA damage. Once clamped onto the DNA, the
function of the 9-1-1 clamp may be to "mark" DNA lesions, thereby
recruiting repair machinery and downstream signaling components that
activate the checkpoint signal. The studies presented here provide
compelling evidence that hRad9, hHus1, and hRad1 form a clamp-like
structure, providing further impetus to test the predicted clamp model.
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ACKNOWLEDGEMENTS |
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We thank Scott Kaufmann, Junjie Chen, and Jann Sarkaria for thoughtful discussions and critical evaluations of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant CA-84321 and the Mayo Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: Mayo Foundation, Oncology Research, Guggenheim 13, Rochester, MN 55905. Tel.: 507-284-3124; Fax: 507-284-3906; E-mail: karnitz.larry@mayo.edu.
Published, JBC Papers in Press, May 4, 2001, DOI 10.1074/jbc.M102946200
2 M. A. Burtelow and L. M. Karnitz, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: PCNA, proliferating cell nuclear antigen RFC, replication factor C; HA, hemagglutinin; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; GFP, green fluorescent protein.
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