From the Imperial Cancer Research Fund Laboratories, Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DS, United Kingdom
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ABSTRACT |
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Previous reports have indicated that
topoisomerase II (topo II) co-purifies with and is a substrate for
casein kinase II. We have carried out a detailed study of the effect
that purified casein kinase II has on the activity of purified
recombinant human topo II. Co-incubation of topo II
and casein
kinase II led to an apparent activation of the topo II
; however, in
experiments in which topo II
was preincubated at 37 °C with or
without native casein kinase II prior to assaying for decatenation
activity, it emerged that the kinase was exerting its "activating"
function via a decrease in the rate of topo II
enzyme inactivation
during the incubation period. This stabilization of topo II
by
casein kinase II was ATP-independent and was observed in both mutated and truncated derivatives of topo II
lacking the major casein kinase
II phospho-acceptor sites, indicating the lack of a requirement for
phosphorylation. Consistent with a nonenzymatic role for casein kinase
II, stoichiometric quantities of kinase were required for topo II
stabilization. These data indicate that casein kinase II plays a
significant role in regulating human topo II
protein action via
stabilization against thermal inactivation.
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INTRODUCTION |
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DNA topo1 II is a ubiquitous and structurally conserved enzyme required for many different aspects of DNA metabolism (reviewed in Refs. 1 an 2). Studies in bacterial and lower eukaryotic species have revealed that topo II is required for cell viability due to an essential role in the segregation of newly replicated chromosomes during cell division (3-7). During replication, sister DNA molecules become knotted and catenated as a consequence of the difficulties inherent in the unwinding and copying of a double-stranded, helical nucleic acid structure (reviewed in Ref. 8). In eukaryotes, topo II catalyzes the disentanglement of sister chromatids and hence permits their faithful segregation during mitosis. Studies on yeast temperature-sensitive top2 mutants have shown that both chromosome hypercondensation and sister chromatid segregation are dependent upon functional topo II, and that in the absence of topo II chromosomes break or nondisjoin as the mitotic spindle attempts to pull apart the still interlinked DNA molecules (3-8).
In mammalian cells, but not in yeasts, topo II exists as two closely
related isoforms designated (170-kDa form) and
(180-kDa form)
(9-13). The respective roles of the two isoforms are not known
currently, although several differences in their activities/regulation have been noted. For example, the
isoform is a strict proliferation marker in vitro and in vivo and is expressed at a
high level in each cell cycle during the period just prior to cell
division (14-17). This isoform is tightly associated with condensed
chromatin during metaphase. In contrast, topo II
is ubiquitously
expressed in human tissues and is apparently excluded from chromosomes
around the time of mitotic chromosome condensation, although it is
found in the nucleoplasm during interphase (14-18).
Topo II is a phosphoprotein in cells from both higher and lower
eukaryotes (reviewed in Ref. 1). In those species that have been
analyzed, the extent of this phosphorylation is modulated as cells
traverse the different phases of the cell division cycle. In
particular, topo II in mammalian cells is hyperphosphorylated during
mitosis as a result of the actions of at least two different protein
kinases (19-23). However, the precise functional role of these cell
cycle-specific modifications is not clear at this stage.
The kinase most closely associated with the regulation of topo II is
casein kinase II. Topo II from Drosophila, budding and fission yeast, as well as from mammalian cells, is a high affinity substrate for casein kinase II in vitro (24-29). Several
studies have confirmed that this kinase also phosphorylates topo II in intact cells, primarily on sites located within the poorly conserved, noncatalytic C-terminal domain (25, 28-30). The fission yeast topo II
is also phosphorylated on a site in the N-terminal ATPase domain by
casein kinase II (28). To date, three sites have been identified within
the C-terminal domain of human topo II that are phosphorylated by
casein kinase II in vivo (29, 31). None of these sites is
modified differentially during cell cycle transit, and only one, serine
1524 in humans, is highly conserved in all of the mammalian topo II
enzymes. This residue is a major site of phosphorylation in several
different human cell lines studied (29). Of the other two sites, serine
1376 is conserved in mouse and rat topo II
, but its flanking
sequence is not, while threonine 1342 is not conserved, being replaced
by an aspartate in the rodent enzymes (32, 33).
In this study, we have investigated the role played by casein kinase II
in regulating the activity of human topo II. We show that
co-incubation of topo II
with casein kinase II strongly influences
the ability of topo II
to decatenate kinetoplast DNA. We present
evidence that casein kinase II is able to stabilize topo II
against
inactivation during incubation at 37 °C, and that this effect is not
dependent upon phosphorylation of topo II
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EXPERIMENTAL PROCEDURES |
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Site-directed Mutagenesis--
The topo II cDNA was
mutated using the single-stranded oligonucleotide-directed mutagenesis
method of Kunkel et al. (34), as described in Wells and
Hickson (22). An AvrII/XhoI fragment containing
the 3
end of the topo II
cDNA (12) was subcloned into
pBluescript and a single-stranded uracil-containing template generated
using the Escherichia coli strain CJ236. For the mutation of
serine 1376 to alanine (S1376A) the oligonucleotide
5
-TTCAAGGTCTGCCACGACACTTTT-3
was used; for the mutation of serine
1524 to alanine (S1524A) the oligonucleotide
5
-ACCAGTCTTGGGGCTGGTAAGATA-3
was used; for the truncation at amino
acid 1178 the oligonucleotide 5
-TTAAACAGCCTACAATTCTTCAAT-3
was used.
The double phosphorylation site mutant was generated using the S1376A
and S1524A oligonucleotides simultaneously. Double-stranded mutated
pBluescript constructs were verified by dideoxy DNA sequencing. AvrII/XhoI fragments containing the mutated
phospho-acceptor residues were then subcloned into pYEpWob6 previously
digested with the same enzymes.
Overexpression and Purification of Human Topo II
Enzymes--
The expression construct pYEpWob6 (35) was the basis for
the production of full-length and truncated human topo II
proteins. Full-length, mutated, and truncated proteins were overexpressed in
Saccharomyces cerevisiae and lysates were prepared by
freeze/thawing and shearing cells with glass beads (35). Soluble
proteins were then loaded onto an hydroxyapatite (Ultragel) column, and
the topo II
protein was eluted using a 200-600 mM NaCl
gradient. Peak fractions were pooled and loaded onto a 1-ml HiTrapSP
column (Pharmacia Biotech Inc.), and bound proteins were eluted using a
200 mM to 1 M NaCl gradient. Peak fractions,
containing >95% pure topo II
, as determined by Coomassie Blue
staining of SDS-polyacrylamide gels, were adjusted to 50% glycerol and
stored at
70 °C.
Purification of Casein Kinase II from Rabbit Skeletal
Muscle--
All procedures were carried out at 4 °C. The tissue was
homogenized in 2.5 volumes of 4 mM EDTA, 15 mM
2-mercaptoethanol, 0.1 mM phenylmethylsulfonyl fluoride,
and 1 mM benzamidine at pH 7.0. Following centrifugation at
10,000 rpm for 10 min, the supernatant was collected and adjusted to pH
7.5 with 15 M NH4OH. Solid
(NH4)2SO4 was added slowly with
stirring to 33% saturation. The precipitate was then removed by
centrifugation at 10,000 rpm for 20 min. Further
(NH4)2SO4 was added to the
supernatant to 55% saturation. After precipitation at 10,000 rpm for
10 min, the pellet was resuspended in buffer A (25 mM
Tris-HCl, 1 mM EDTA, 15 mM 2-mercaptoethanol,
pH 7.5) and dialyzed overnight against buffer A. The dialysate was
applied to a phosphocellulose column (Whatman P-11) equilibrated with
buffer A, the column was washed in buffer A containing 400 mM NaCl and eluted with buffer A containing 1 M
NaCl. The eluate was diluted with 4 volumes of buffer A and applied to
a HiTrap Heparin column preequilibrated with buffer A. After washing
with buffer A containing 400 mM NaCl, the bound proteins
were eluted with buffer A containing 1 M NaCl. The casein kinase II-rich fractions were dialyzed against buffer A, before loading
onto a fast protein liquid chromatography Mono Q column equilibrated in
buffer A. The column was washed with buffer A plus 400 mM
NaCl and eluted with a linear gradient of buffer A plus 1 M
NaCl. Casein kinase II activity, as determined by SDS-PAGE and
autophosphorylation assays, eluted from the column at 700 mM NaCl. The active fractions were pooled, dialyzed against
20 mM Tris-HCl, pH 7.5, 350 mM NaCl, 1 mM EDTA, and 15 mM 2-mercaptoethanol and stored
in aliquots at 70 °C. The casein kinase II was judged to be >90%
pure by Coomassie Blue-stained SDS-polyacrylamide gels.
Phosphorylation of Topo II--
Phosphorylation of topo II
was typically carried out at 30 °C for 15 min using 2 µg of topo
II
and 0.4 µg of casein kinase II in a final volume of 20 µl of
kinase buffer (20 mM Tris-HCl, pH 7.5, 50 mM
KCl, 10 mM MgCl2) supplemented with either 1 mM ATP or 110 µM [
-32P]ATP
(final specific activity >23 TBq/mmol). PKI, the peptide inhibitor of
protein kinase A, and H-7 were obtained from Sigma.
Determination of the Stoichiometry of Phosphorylation--
12
µg of wild-type topo II were incubated with 2.4 µg of casein
kinase II and 110 µM [
-32P]ATP in a
total volume of 140 µl of kinase buffer at 30 °C. 20-µl aliquots
were taken at 0, 2, 5, 10, 20, and 30 min, and the reaction was stopped
by the addition of 10 µl of SDS gel loading buffer. The samples were
separated by SDS-PAGE, and the amount of phosphate incorporated into
the topo II band on the dried gel was quantitated using a Molecular
Dynamics PhosphoImager model 425.
Phosphopeptide Mapping of Topo II--
This procedure was
carried out as described by Wells et al. (29).
DNA Topo II Assays-- DNA topo II activity was assayed by measuring the decatenation of kDNA (Topogen). The standard mix for a 20-µl reaction consisted of 50 mM Tris-HCl, pH 7.9, 85 mM KCl, 10 mM MgCl2, 0.5 mM EDTA, 10 mM ATP, 1 mM dithiothreitol, 20 µg/ml bovine serum albumin, and 240 ng of kDNA. The reaction mixture was incubated at 30 °C, and at timed intervals the reaction was stopped by the addition of 5 µl of stop solution (5% SDS, 25% Ficoll, and 0.05% bromphenol blue). The samples were loaded onto a 1% agarose gel in Tris borate EDTA buffer. After electrophoresis, the gel was stained with ethidium bromide and photographed under UV illumination.
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RESULTS |
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Co-purification of a Kinase with Topo II from HeLa Cell Nuclear
Extracts--
Our initial premise was that because casein kinase II
phosphorylates human topo II
in vivo, it was very likely
to play a role in the regulation of topo II
function. Moreover, a
kinase with properties very similar to those of casein kinase II has been reported to co-purify with topo II in extracts from
Drosophila, mouse and yeast cells (26, 27, 36). To address
whether this was also the case for human topo II
, we tested the
highly purified topo II
preparations from HeLa cell nuclei that we
have described previously (23) for kinase activity. Fig.
1 shows that a kinase capable of
phosphorylating topo II
(as well as casein; data not shown)
co-purifies with topo II
from HeLa nuclei. Next, we addressed whether this copurifying kinase displayed two of the features diagnostic of casein kinase II; sensitivity to heparin and an ability
to utilize GTP, as well as ATP, as a phosphate donor (reviewed in Ref.
37). As shown in Fig. 1, the co-purifying kinase was inhibited by
heparin, but not by a specific peptide inhibitor of protein kinase A,
or by H-7, the inhibitor of protein kinase C, cyclic
nucleotide-dependent kinases, and myosin light chain kinase. Moreover, this kinase could use GTP as a phosphate donor (data
not shown), suggesting that it was either casein kinase II or a very
closely related enzyme. These data, together with those of previous
studies, suggest that topo II
may be an important target for casein
kinase II in human cells.
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Purification of Recombinant Topo II Protein--
Using this
information, we set out to analyze the role of casein kinase II in
regulating topo II
enzyme activity in vitro. However,
using human nuclear extracts as a source of enzyme, it proved very
difficult to separate topo II
from the co-purifying kinase. As a
consequence, we adopted an alternative approach exploiting the recent
description of an overexpression system for recombinant human topo
II
using S. cerevisiae as a host (35). With this system,
we prepared homogeneous human topo II
from yeast extracts (see Fig.
6 below) using a new purification protocol (see "Experimental Procedures"). In addition, we purified casein kinase II to apparent homogeneity from rabbit skeletal muscle using an established protocol. We first confirmed that the recombinant topo II
preparation lacked intrinsic kinase activity and that the topo II
was a substrate in vitro for the purified casein kinase II (Fig.
2a). Analysis of this reaction
showed that the stoichiometry of phosphorylation was approximately 2 moles of phosphate per mole of topo II monomer, even after extended
incubation periods (Fig. 2b). Thus, we conclude that this
yeast-based expression system provides a means to analyze the effects
of casein kinase II on topo II
activity without the compounding
influence of a co-purifying kinase.
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Effect of Casein Kinase II on the Activity of Recombinant Topo
II Protein--
We next tested whether the purified rabbit casein
kinase II could influence the activity of the recombinant topo II
protein. Fig. 3 shows that the purified
human topo II
displayed significantly higher activity in a
decatenation assay following preincubation with casein kinase II than
it did following an otherwise identical incubation in buffer containing
either a control protein (BSA) or heat-inactivated casein kinase II.
Thus, native casein kinase II is required for this activation of topo
II
. As a control, we confirmed that the casein kinase II preparation
contained no contaminating topoisomerase activities (data not
shown).
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Stabilization of Topo II by Casein Kinase II Is Independent of
Phosphorylation and Requires Stoichiometric Amounts of
Kinase--
Omission of ATP from the preincubation buffer had no
effect on the ability of casein kinase II to stabilize topo II
,
suggesting a phosphorylation-independent mode of action (data not
shown). Nevertheless, it was formally possible that the enzyme
preparations themselves provided sufficient ATP to permit
phosphorylation of topo II
to take place during the preincubation
period prior to performing the decatenation assay. However, to exclude
a role for phosphorylation by casein kinase II, we studied mutated and truncated derivatives of recombinant topo II
. We have shown
previously that casein kinase II phosphorylates topo II
in
vitro primarily on two sites in the C-terminal domain (29). These
sites, serine 1376 and serine 1524, are also phosphorylated in
vivo (29). In addition, Ishida et al. (31) have shown
recently that threonine 1342 is phosphorylated in human cell lines.
Recombinant topo II
proteins containing mutation of serine 1376 to
alanine (S1376A), serine 1524 to alanine (S1524A), or a double mutant
lacking both phospho-acceptor serine residues (designated topo
II
-SDM), were overexpressed in yeast and purified to apparent
homogeneity (Fig. 6 and data not shown).
In addition, a truncated derivative comprising residues 1-1178, which
was designated topo II
-CTT, was purified in a similar manner (Fig.
6). This truncated derivative lacks residues 1179-1530, which
encompass the entire regulatory C-terminal domain of topo II
and the
three previously mapped phospho-acceptor sites discussed above.
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DISCUSSION |
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We have shown that casein kinase II co-purifies with and strongly
influences the activity of human topo II. This activation of topo
II
is apparently independent of any phosphorylation and does not
require sequences present in the regulatory C-terminal domain of the
enzyme. Although casein kinase II treatment increases the activity of
topo II
to a level above that of mock-treated enzyme, this does not
represent true activation, in that the role of the kinase appears to be
to stabilize topo II
and hence prevent a decline in activity that
normally occurs during a period of incubation at 37 °C. We would
suggest that this effect on enzyme activity might be widespread among
nuclear enzymes and therefore important for chromosome function, since
casein kinase II has been found in association with a wide variety of
other enzymes important for different aspects of DNA metabolism (see
below). Of possible significance in this regard is the observation that that the
subunit of casein kinase II confers thermal stability upon
casein kinase II
subunits, in which the catalytic activity resides
(38).
How might casein kinase II effect its role as an enzyme stabilizer? The
most obvious mechanism is via direct association with topo II. Our
data showing the co-purification of topo II
with casein kinase II
and the requirement for stoichiometric amounts of casein kinase II to
effect stabilization of topo II
enzyme activity, are consistent with
this suggestion. Moreover, Bojanowski et al. (39) have shown
that the
subunit of casein kinase II interacts with topo II from
budding yeast. However, despite extensive efforts, utilizing a variety
of different methodologies including immunoprecipitation and chemical
cross-linking, we have been unable to "trap" a stable complex
containing pure human topo II
with casein kinase II. This does not
rule out the possibility that such an association occurs under certain
conditions, or that the association is transient in nature. Casein
kinase II is known to associate with a number of its other
substrates, including topo I, p53, and protein
phosphatase 2A (40-42). In addition, casein kinase II
has been shown to stimulate topo I activity via a direct physical
association (40).
Recently, Kimura et al. (43) reported that casein kinase II
has no effect on the activity of mouse topo II. Further, they concluded that their previously reported results (27) showing a
stimulation of topo II
activity by casein kinase II could not be
attributed to the kinase, but to the composition of the incubation buffer. Specifically, incubation in buffers containing low, but not
high, concentrations of glycerol was stimulatory, and this effect was
dependent upon topo II
being present at a high concentration. Our
results are partially in agreement with those of Kimura et al. (43) in that we could not identify a role for phosphorylation per se in the regulation of topo II
activity by casein
kinase II. However, our findings that native casein kinase II is a
necessary component of the buffer, and that denatured casein kinase II
is not active in this regard, are not consistent with their
conclusions. Moreover, we have been unable to reproduce the stimulatory
effects of manipulating the glycerol concentration alone, reported by Kimura et al. (43). The reason(s) for these discrepancies is not apparent at this stage.
We have shown that the C-terminal domain of topo II is not important
for catalytic activity in vitro, and that phosphorylation of
target residues within this domain is not required for the regulation
of that activity by casein kinase II. It would be predicted, therefore,
that a failure to phosphorylate these residues would not alter the
known ability of the topo II
cDNA to rescue
temperature-sensitive yeast top2-4 mutants at the
restrictive temperature (35, 44). Consistent with this prediction, we
have shown that cDNAs carrying mutations of serine 1524 and/or
serine 1376 to alanine will fully complement top2-4
strains.2 It is conceivable
that the role of phosphorylation of these residues is normally not to
regulate catalytic activity, but instead to influence subcellular or
subnuclear localization in human cells, as has been shown for fission
yeast topo II (28). Our data showing that residues 1179-1530 are
dispensible for the catalytic activity of human topo II
in
vitro are consistent with previous analyses on truncated versions
of the S. cerevisiae and Drosophila topo II
proteins (45, 46). The possibility that phosphorylation of sites
outside the C-terminal domain may influence the stability of the enzyme
can be discounted, since the truncated protein, which was shown to
incorporate only 0.02 mol of phosphate/mol of topo II
monomer, is
stabilized by casein kinase II in a fashion similar to that of the
wild-type enzyme.
Our work and that of Kimura et al. (43) on mouse topo II
have shown no direct effect on topo II
activity of phosphorylation by casein kinase II. In addition, in both studies stoichiometric quantities of kinase were necessary to give efficient phosphorylation. This is in contrast to reports of the effect of casein kinase II
phosphorylation on topo II activity from lower eukaryotes. Topo II from
Drosophila is a high affinity substrate for casein kinase II
both in vitro and in vivo and is fully
phosphorylated using substoichiometric quantities of kinase (24, 30).
The phosphorylated enzyme has an enhanced rate of ATP hydrolysis and a
3-fold higher plasmid relaxation activity compared with that of the
dephosphorylated enzyme, effects that are fully reversed by
dephosphorylation of the enzyme (24, 30, 47). Similarly, S. cerevisiae topo II is readily phosphorylated by casein kinase II,
resulting in a greater than 10-fold stimulation of decatenation activity (36). In Drosophila and yeast there is a single
gene encoding topo II, whereas in mammalian cells there are two closely related, but genetically distinct, isoforms. Thus it is possible that
higher eukaryotes have evolved a mechanism for differentially regulating the two topo II isoforms.
Gasser and co-workers (36, 48) have reported that casein kinase II not
only reactivates dephosphorylated yeast topo II, but also influences
the extent of enzyme multimerization. Although it remains to be
confirmed that higher order (greater than dimeric) forms of topo II
have any functional significance in eukaryotic cells, it is formally
possible that the regulatory phenomenon that we have observed reflects
an ability of casein kinase II to prevent topo II
from adopting a
subunit composition that exhibits little or no catalytic activity.
Further work is required to ascertain whether or not this suggestion is
true.
The primary role of topo II in human cells is almost certainly to
act during mitosis. It would appear that the level and activity of this
enzyme is very tightly controlled by a combination of features acting
at different levels. Thus, the mRNA is virtually absent from
G0/G1 cells and only accumulates to high levels
during late S phase (14-16, 49). The topo II
protein peaks later,
in G2/M, and is phosphorylated by at least two different
kinases during mitosis (14, 22, 23). Indeed, it would appear that topo
II
activity is strongly activated only during a narrow time window
when its decatenase function is required for mitotic chromosome condensation/segregation. Casein kinase II might contribute to this
tight regulation at (at least) two levels; either via modulation of
intrinsic topo II
enzyme stability, or via its known ability to
modulate the action of other kinases/phosphatases required for mitosis,
including protein phosphatase 2A and the universal mitotic controller,
p34cdc2 (42) (reviewed in Ref. 37). It might be significant
that both casein kinase II and topo II
are among the proteins that are phosphorylated during mitosis, leading to the generation of the
phosphoepitope recognized by the MPM-2 antibody (50, 51).
In summary, we have shown that the activity of topo II is maintained
during an incubation at 37 °C by the action of casein kinase II in a
manner that is independent of phosphorylation. The challenge is now to
delineate the functional significance of this regulation in
vivo and to address whether casein kinase II plays a widespread
role in regulating the stability of other important nuclear
enzymes.
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ACKNOWLEDGEMENTS |
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We thank members of the ICRF Molecular Oncology Laboratories for helpful discussions, Dr. J. C. Wang for pYEpWob6, and Dr. C. Norbury for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by the Imperial Cancer Research Fund.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.
Current address: Molecular Biology and Virology Laboratory, The
Salk Institute, San Diego, CA 92138.
§ Current address: Dept. of Molecular Biology, University of Geneva, CH-1211 Geneva 4, Switzerland.
¶ To whom correspondence should be addressed. Tel.: 44 1865 222417; Fax: 44 1865 222431; E-mail: hickson{at}icrf.icnet.uk.
1 The abbreviations used are: topo, topoisomerase; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin.
2 C. Redwood, S. L. Davies, N. J. Wells, A. M. Fry, and I. D. Hickson, unpublished data.
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REFERENCES |
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