From the ** Howard Hughes Medical Institute and Departments of
Biochemistry and ¶ Medicine-Medical Oncology, Duke
University Medical Center, Durham, North Carolina 27710
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ABSTRACT |
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A human MSH2-human MSH3 (hMSH2·hMSH3) complex
of approximately 1:1 stoichiometry (human MutS (hMutS
)) has been
demonstrated in several human tumor cell lines and purified to near
homogeneity. In vitro, hMutS
supports the efficient
repair of insertion/deletion (I/D) heterologies of 2-8 nucleotides, is
weakly active on a single-nucleotide I/D mispair, and is not detectably
active on the eight base-base mismatches. Human MutS
(hMutS
), a
heterodimer of hMSH2 and hMSH6, efficiently supports the repair of
single-nucleotide I/D mismatches, base-base mispairs, and all
substrates tested that were repaired by hMutS
. Thus, the repair
specificities of hMutS
and hMutS
are redundant with respect to
the repair of I/D heterologies of 2-8 nucleotides. The hMutS
level
in repair-proficient HeLa cells (1.5 µg/mg nuclear extract) is
approximately 10 times that of hMutS
. In HCT-15 colorectal tumor
cells, which do not contain hMSH6 and consequently lack hMutS
, the
hMutS
level is elevated severalfold relative to that in HeLa cells
and is responsible for the repair of I/D mismatches that has been
observed in this cell line. LoVo tumor cells, which are genetically
deficient in hMSH2, lack both hMutS
and hMutS
, and hMSH3 and
hMSH6 levels are less than 4% of those found in repair-proficient
cells. Coupled with previous findings (J. T. Drummond, J. Genschel, E. Wolf, and P. Modrich (1997) Proc. Natl. Acad. Sci.
U. S. A. 94, 10144-10149), these results suggest that hMSH2
partitions between available pools of hMSH3 and hMSH6 and indicate that
hMSH2 positively modulates hMSH6 and hMSH3 levels, perhaps by
stabilization of the polypeptides upon heterodimer formation.
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INTRODUCTION |
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Correction of mismatched base pairs, resulting from replication
errors, contributes significantly to the genetic stability of human
cells (reviewed in Refs. 1-3). Mutations in the gene that encodes the
human MutS homolog MSH2
(hMSH2)1 confer genetic
instability and have been implicated in hereditary non-polyposis colon
cancer and sporadic tumors (4-6). The mutation rate at the
HPRT locus is greatly increased in the
MSH2/
LoVo colorectal tumor cell line. The
majority of the selectable HPRT mutations that occur in LoVo
cells are transitions (80%), with the remainder largely
single-nucleotide frame shifts in mononucleotide repeat sequences (7),
although (CA)n microsatellite repeats are also highly unstable
in this cell line (8). Extracts prepared from LoVo cells are deficient
in repair of heteroduplexes containing either base-base or small
insertion/deletion (I/D) mispairs (8, 9), and a mismatch recognition
activity that restores repair of a G-T base-base mismatch and TG I/D
mispair to LoVo nuclear extracts has been isolated from HeLa cells (9). This activity, designated hMutS
, is a heterodimer of hMSH2 and hMSH6
(also called p160 or GTBP (9, 10)).
Nuclear extracts of the HCT-15 colorectal tumor cell line (11) and the
alkylation-tolerant MT1 lymphoblastoid cell line (12) are also
deficient in mismatch repair due to MutS deficiency (9, 13), but the
MutS
defects in these cell lines are a consequence of MSH6 mutations
(14). Extracts of HCT-15 and MT1 cells are deficient in repair of
base-base mismatches and single-nucleotide I/D mispairs but retain
partial proficiency in the correction of 2-, 3-, and 4-nucleotide I/D
heterologies (9, 13). The HPRT mutation rate is elevated
60-fold in MT1 cells (12), which harbor missense mutations in both
MSH6 alleles (14), and 300-fold in HCT-15 cells (15) in
which both MSH6 alleles have been inactivated by frame shift
mutations. HCT-15 cells also contain a sequence change in a conserved
region of one copy of the gene that encodes DNA polymerase
(11),
but this mutation does not appear to contribute significantly to the
HCT-15 mutator phenotype (16). Although dinucleotide repeats are
relatively stable in these MSH6
/
cell lines,
mononucleotide repeats are prone to mutation (13, 14, 17), but not to
the extent observed with HCT-116 cells (13, 15), which are defective in
base-base and I/D mismatch repair (18, 19) due to mutations in both
alleles of MLH1 (20).
The proficiency of MSH6/
cells in the repair
of I/D mismatches suggests the existence of a second mismatch
recognition activity distinct from hMutS
in mammalian cells. Since
hMSH2-deficient cell lines are defective in the repair of both
base-base mispairs and I/D heterologies, this hMSH6-independent
mismatch activity would appear to require hMSH2. There are two
candidates for such an activity: a heterodimeric complex of hMSH2 and
hMSH3, with the locus encoding the latter protein being the first MutS
homolog gene identified in mammalian cells (21, 22), or free hMSH2 in
an unknown oligomeric state. Recombinant hMSH2 has been reported to
bind to I/D mismatches (23), but recent work indicates that this is an
extremely low affinity interaction (24, 25), and free hMSH2 has not
been detected in human cells (26). However, a complex between
recombinant hMSH2 and hMSH3 polypeptides has been demonstrated (24,
25), and a similar complex has been isolated from
methotrexate-resistant human cells in which the DHFR-MSH3
region of chromosome 5 is highly amplified (26).
The hMSH2·hMSH6 complex has been shown to bind specifically to a G-T
mispair and to 1-, 2-, and 3-nucleotide I/D mismatches and to
efficiently restore repair of a base-base and a dinucleotide I/D
mismatch to hMSH2-deficient nuclear extracts (9, 24). By contrast, the
recombinant hMSH2·hMSH3 hMutS complex was shown to bind weakly to
a single-nucleotide insertion/deletion mismatch and with high affinity
to heteroduplexes containing 2, 3, 4, or 10 unpaired nucleotides but
not to the several base-base mismatches tested (24, 25). These in
vitro binding specificities indicate that hMutS
and hMutS
have overlapping specificities for I/D mismatches and that the residual
I/D heteroduplex repair activity observed in extracts of
MSH6
/
cells may be due to hMutS
. We show
here that this is in fact the case and also describe the specificities
of hMutS
and hMutS
in strand-specific mismatch correction.
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EXPERIMENTAL PROCEDURES |
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Cell Lines and Nuclear Extracts-- HeLa S3 cells were obtained from the Lineberger Cancer Center at the University of North Carolina in Chapel Hill. HCT-15 (9, 14), LoVo (8), and HL60-R (21) were cultured as described (9, 26). Nuclear extracts were prepared according to published procedures (9, 27).
Mismatch Repair Assays--
Mismatch repair assays were
performed as described previously (9, 27). Briefly, 50 µg of nuclear
extract was incubated with 100 ng (24 fmol) of a f1-derived
heteroduplex DNA substrate in 10-15 µl at 37 °C for 15 min. The
final salt concentration in the assay was 100-110 mM KCl.
Assays of immunodepleted extracts were performed similarly except that
reactions contained 100 µg of extract protein, and incubation was
extended to 60 min. Complementation of deficient extracts was achieved
by the addition of purified hMutS or hMutS
(9) as indicated. The
hMutS
preparations used for complementation were isolated from
HL60-R cells as described below. Circular phage f1 heteroduplexes were
prepared as described (28, 29) and contained a nick 181 nucleotides 3'
to the mispair (short path) in the case of the 3' substrates or 125 nucleotides 5' to the mispair (short path) in the case of the 5'
substrates. Heteroduplex substrates containing I/D heterologies of
different sizes have the following nonpaired sequences: dA (1 nucleotide), d(CA) (2 nucleotides), d(CTG) (3 nucleotides), d(CTCGA) (5 nucleotides), d(ACACTCGA) (8 nucleotides), d(ACACACACTCGA) (12 nucleotides), d(TTTCTAGACTCGACAGCTGGCTAGCAA) (27 nucleotides). I/D
heteroduplex substrates are sometimes described by the unpaired
sequence that forms the loop, e.g. CA I/D. For 5'-I/D
heteroduplexes the extra nucleotides were in the nicked complementary
DNA strand, except for the A I/D heteroduplex, which contained an
unpaired adenine in a run of six adenines within the continuous viral
strand (9). For the 3'-CA I/D heteroduplex, the two extra nucleotides
were present in the continuous DNA strand.
Immunological Methods--
Antisera against hMSH3 and hMSH6 were
generated by immunizing rabbits with MAP-conjugated peptides (30).
Peptide sequences were TEIDRRKKRPLENDGPVKKK (21) for hMSH3 and
MQRADEALNKDKIKRLELAV (10) for hMSH6. Blood obtained from rabbits was
clotted overnight at room temperature, the clot and red blood cells
were removed by centrifugation, and the resulting serum was heated for
20 min at 56 °C to inactivate proteases and nucleases. Serum was
stored in aliquots at 20 °C. A monoclonal mouse antibody against
hMSH2 (Ab-1) was purchased from Calbiochem. Secondary antibodies
conjugated with horseradish peroxidase or alkaline phosphatase were
obtained from Sigma.
Purification of hMutS and hMutS
--
hMutS
was purified
to greater than 95% purity from nuclear extracts of HeLa cells as
described (9). Preparations were free of detectable hMutS
as judged
by Western analysis for hMSH3.
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RESULTS |
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Presence of hMutS in Extracts of Human
Cells--
MSH6
/
cell lines like HCT-15 and
MT1 are selectively defective in the repair of base-base mispairs and
single-nucleotide I/D mismatches but are proficient in correction of
I/D heterologies of 2, 3, and 4 nucleotides (9). Since hMSH6 is a
required subunit of hMutS
(9, 10), we reasoned that repair of I/D
heterologies larger than 1 nucleotide in these cell lines must depend
on a second mismatch recognition activity. To isolate this activity, HCT-15 nuclear extract was resolved by chromatography, and fractions were tested for their ability to restore repair of a dinucleotide I/D
heteroduplex to nuclear extract of LoVo colorectal tumor cells, which
are devoid of hMSH2 due to partial deletion of the structural gene (8).
This cell line is also free of detectable hMSH3 and contains only trace
levels of hMSH6 (see below and Ref. 26).
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hMutS Is More Abundant in HCT-15 than in HeLa Cells--
hMSH3
and hMSH6 were quantitated via immunoblotting in nuclear extracts of
HeLa, LoVo, and HCT-15 tumor cell lines (Fig.
2). Neither protein was detectable in
MSH2
/
LoVo cells (<0.03 µg of MSH6
and < 0.005 µg of MSH3 per mg of nuclear extract, Fig. 2),
consistent with an earlier observation with this cell line (26). Since
hMSH3 and hMSH6 were only detected in cell lines expressing hMSH2 and
since both proteins associate tightly with hMSH2 during fractionation
(Fig. 1 and (9, 24, 26), immunological quantitation of hMSH3 and hMSH6
can be used to estimate levels of hMutS
and hMutS
in nuclear
extracts. In HeLa cells, the hMSH3 level is equivalent to about 0.15 µg of hMutS
/mg of nuclear extract protein, while the hMSH6 level
is equivalent to about 1.5 µg of hMutS
/mg. The hMutS
:hMutS
ratio of 10:1 found here for HeLa cells is similar to the value of 6:1 estimated for HL-60 cells based on chromatographic resolution of the
two heterodimers (26). In HCT-15 cells, the hMSH3 level was equivalent
to about 0.5 µg of hMutS
/mg of nuclear extract, but hMSH6 was
undetectable. This confirms the absence of hMutS
in this cell
line.
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hMutS Is Responsible for I/D Mismatch Repair in HCT-15
Extracts--
Nuclear extracts of HCT-15 cells were immunodepleted by
adsorption to an anti-MSH3 support (see "Experimental Procedures"). As shown in Fig. 3, this resulted in loss
of repair activity on a CA I/D heteroduplex, but activity on the
dinucleotide I/D heteroduplex substrate was restored upon the addition
of purified hMutS
. By contrast, a G-T heteroduplex was not repaired
by either the depleted or the mock-depleted extract, even upon the
addition of hMutS
. As expected, the addition of hMutS
to
immunodepleted extracts restored repair of both the base-base and the
I/D heteroduplexes. The addition of hMutS
also restored G-T
heteroduplex repair activity to the mock-depleted extract, but repair
of the CA I/D substrate was not significantly increased above
endogenous levels. hMutS
therefore has a unique role in G-T
heteroduplex repair, but hMutS
and hMutS
are redundant with
respect to correction of the dinucleotide I/D substrate. The finding
that immunodepletion of HeLa nuclear extracts with the hMSH3 antiserum
did not affect repair of either heteroduplex (data not shown) is also
consistent with this view.
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Mismatch Specificities of hMutS and hMutS
in Strand-specific
Repair--
Inasmuch as MSH2
/
LoVo cells
are also phenotypically deficient in hMSH3 and hMSH6 (see Fig. 2 and
Ref. 26), extracts of this cell line can be used to establish the
mismatch repair specificities of hMutS
and hMutS
, since the
potential for subunit exchange is precluded. As noted above (see
"Experimental Procedures"), the hMutS
and hMutS
preparations
used in this study are free of detectable cross-contamination. As shown
in Fig. 4, hMutS
restored near normal
levels of repair for each of the eight base-base mismatches to LoVo
extract, but comparable amounts of hMutS
did not detectably increase
repair above base line in any case. The amounts of hMutS
and
hMutS
used in these experiments was 0.8 µg/mg LoVo extract,
corresponding to about 50 and 500% of the levels of hMutS
and
hMutS
in HeLa nuclear extract, respectively (see above).
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Like hMutS, hMutS
Supports Bidirectional Mismatch
Repair--
Human strand-specific mismatch repair can be directed by a
single-strand break located either 5' or 3' to the mispair on the incised strand (29). The experiments summarized in Figs. 4 and 5 used
5'-heteroduplexes. In similar experiments, supplementation of LoVo
extract with either hMutS
or hMutS
restored repair of a 3'-CA I/D
heteroduplex (3.9 fmol/15 min for hMutS
and 4.2 fmol/15 min for
hMutS
). However, repair of a 3' G-T heteroduplex was restored only
upon the addition of hMutS
(4.9 fmol/15 min for hMutS
and <0.3
fmol/15 min for hMutS
). Like hMutS
(9), hMutS
therefore
supports bidirectional repair, and its mismatch specificity is
evidently orientation-independent.
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DISCUSSION |
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Two phenotypes have been described for mismatch repair-deficient
human cell lines with mutations in genes that encode MutS homologs.
MSH2 mutations confer hypermutability at selectable loci and
destabilize simple repeats such as (A)n and (CA)n (5,
14). MSH6/
mutants, on the other hand, show
an increased HPRT mutation rate and instability of
single-nucleotide repeat sequences, but mutations in dinucleotide
repeats are rare (13-15, 17). hMSH3 deficiency has been reported in
hematological malignancies (33) and sporadic cancers (34, 44), but the
effect of hMSH3 deficiency on genetic stability has not been carefully
evaluated.
The differential nature of the mutation spectra of
MSH2/
and MSH6
/
human cells can be understood in terms of the repair specificities of
hMutS
and hMutS
that we describe here. We have shown that hMutS
supports repair of all eight base-base mismatches, as well as
each I/D mispair tested ranging from 1 to 8 unpaired nucleotides. By
contrast, hMutS
is inactive in base-base mismatch repair and is
weakly active on the single-nucleotide I/D mismatch tested but supports
efficient repair of I/D mismatches of 2 to ~8 unpaired nucleotides.
It is noteworthy that hMutS
supported the repair of each I/D
mismatch that was also corrected in a hMutS
-dependent reaction. This indicates extensive overlap in I/D mismatch specificity for the two activities, although we cannot exclude the possibility that
hMutS
and hMutS
may differentially respond to other I/D mispairs
depending on sequence context or the nature of the unpaired nucleotides
(24).
hMutS and hMutS
repair spectra are also in general accord with
chromosome transfer experiments in which chromosome 5 bearing MSH3+ or chromosome 2 bearing
MSH2+ and MSH6+ genes was
introduced into the MSH3
/
MSH6
/
HUAA double mutant cell line. Mono-, di-,
tri-, and tetranucleotide repeats are unstable in HUAA cells (34).
Introduction of MSH3 on chromosome 5 stabilized a
tetranucleotide repeat and dinucleotide repeats, but failed to
stabilize a d(A)n repeat (34). HUAA extracts are defective in
repair of base-base and I/D mispairs, but extracts prepared from HUAA
cells containing wild type MSH2 and MSH6 genes
repair base-base mismatches as well as 1-, 2-, and 4-nucleotide I/D
mismatches (35). Extracts derived from HUAA cells harboring a wild type
copy of MSH3 were inactive on base-base mismatches but were
found to repair mononucleotide and tetranucleotide I/D mispairs,
provided that the unpaired nucleotides were in the incised DNA strand
(34). These findings differ somewhat from our results with hMutS
. We
have found that a 3'- CA I/D heteroduplex containing the unpaired
dinucleotide within the continuous strand is a good substrate for
repair mediated by hMutS
(above) and have previously shown that di-,
tri-, and tetranucleotide I/D heteroduplexes are good substrates for
nick-directed correction in MSH6-deficient HCT-15 and MT1 nuclear
extracts without regard to location of the unpaired nucleotides in the
incised or continuous strand (9). The latter experiments also
demonstrated that an unpaired d(T) in the nicked heteroduplex strand is
a poor substrate for hMutS
in HCT-15 and MT1 cell extracts. It is
possible that some of these discrepancies are due to differences in
assay conditions or to the effect of sequence context, since we have
observed weak but significant hMutS
-directed repair of an A I/D
mismatch under conditions of reduced ionic strength. Furthermore, while
mononucleotide repeats are highly unstable in hMSH6-deficient human
cells (13, 14, 17), the degree of destabilization of such sequences is not as great as that observed with MLH1
/
cells (13, 15), suggesting that some processing of mononucleotide mismatches by hMutS
does occur. Interestingly, mononucleotide repeats appear to be relatively stable in
MSH6
/
murine cells (35).
While the mismatch repair specificities of hMutS and hMutS
described here are consistent with mutation spectra and in
vitro assay of hMSH6-deficient and hMSH3-deficient cells, they
differ significantly from hMutS
specificity deduced from gel shift
assay using a heterodimer produced by in vitro
transcription/translation (25). These experiments failed to detect
interaction of hMutS
with I/D heteroduplexes of 2 or 10 unpaired
nucleotides, whereas hMutS
produced in a similar manner was found to
bind to both. Since other studies have shown that hMutS
efficiently
binds I/D heterologies with one, two, and three unpaired nucleotides
(9, 24), it is possible that hMutS
produced by in vitro
transcription/translation is not fully active. In addition, since other
activities are involved in mismatch rectification, mismatch binding may
not provide a completely accurate indicator of the specificities of
hMutS
and hMutS
in the overall repair reaction. Repair, on the
other hand, is expected to reflect the influence of these other
activities.
Our findings and the chromosome transfer experiments discussed above
also suggest that significant differences exist between the
specificities of hMutS and hMutS
and their Saccharomyces cerevisiae counterparts. As in the case of hMutS
, genetic
evidence and gel shift data support a role for yeast MSH2·MSH6 in the
repair of base-base mispairs and single-nucleotide and dinucleotide I/D mismatches (36-38). Yeast MSH2·MSH3 is also able to support the repair of single-nucleotide and dinucleotide I/D mismatches, but only this complex appears to be involved in the repair of larger I/D
heterologies (39-41).
Heterodimer formation between hMSH2 and hMSH6 or between hMSH2 and hMSH3 results in two activities with distinct, partially overlapping specificities that are highly active in mismatch binding and repair (9, 10, 24, 25). What determines the mispair binding of these two complexes? An intriguing possibility is that both subunits of the heterodimer contribute elements to the mismatch binding site. In this model, the mismatch binding site created in the hMSH2·hMSH6 complex would be able to accommodate both I/D and base-base heterologies, whereas only I/D heterologies would fit well into the binding site created by hMSH2·hMSH3 heterodimerization. Alternatively, the mismatch binding site may reside within one subunit, with the other subunit serving to activate this binding center.
This and other studies suggest that hMutS and hMutS
participate
in a common mismatch repair pathway. We previously demonstrated that
hMutS
is present in a 6-fold molar excess over hMutS
in exponentially growing HL-60 cells (26) and have shown here that in
mitotically active HeLa cells, 90% of the nuclear hMSH2 is present in
the hMutS
heterodimer. The partitioning of hMSH2 between these two
complexes appears to be important for genetic stabilization, since
overproduction of hMSH3, which increases the hMutS
pool at the
expense of hMutS
, is associated with a large increase in mutation
rate (26). As shown here, the hMutS
pool is also elevated in HCT-15
cells, which do not produce hMutS
due to genetic inactivation of
MSH6. Several other lines of evidence indicate regulation of
hMutS
and hMutS
pools in human cells.
MSH2
/
LoVo cells, which lack detectable
levels of the hMSH2 polypeptide, are phenotypically deficient in hMSH3
and hMSH6 proteins (Fig. 2 and Ref. 26). The distribution of the common
hMSH2 subunit between hMutS
and hMutS
may be a consequence of
mass action, with hMSH2 partitioning between hMSH3 and hMSH6 according
to pool size and heterodimer formation stabilizing the hMSH3 and hMSH6 subunits. However, other forms of regulation have not been ruled out.
The relative abundance of hMutS and its broad mismatch specificity
suggest that the hMSH2·hMSH6 heterodimer is the primary mismatch
recognition activity for correction of DNA biosynthetic errors. While
hMutS
may complement the specificity of hMutS
for I/D
heterologies, depending on sequence context of the mismatch and the
nature of the unpaired nucleotides, it is also possible that the former
activity may have distinct functions in DNA metabolism. For example,
Haber and colleagues (42, 43) have shown that S. cerevisiae
MSH2 and MSH3, but not MSH6, are involved in the processing of
nonhomologous ends during recombinational double-strand break
repair.
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ACKNOWLEDGEMENTS |
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We thank E. Penland and S. Larson for culturing cell lines, and M. McAdams for the synthesis of peptides.
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FOOTNOTES |
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* 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.
This work was supported in part by National Institutes of Health Grants GM45190 (to P. M.) and K08 CA71554 (to S. J. L.) and a fellowship from the Deutsche Forschungsgemeinschaft (to J. G.).
§ These authors contributed equally to this work.
Present address: Dept. of Biology, Indiana University,
Bloomington, IN 47405.
To whom correspondence should be addressed. Tel.: 919-684-2775;
Fax: 919-681-7874; E-mail: modrich{at}biochem.duke.edu.
1
The abbreviations used are: hMSH2, -3, and -6, human MSH2, -3, and -6, respectively; hMutS and hMutS
, human
MutS
and -
, respectively; DTT, dithiothreitol; I/D,
insertion/deletion.
2 S. J. Littman and J. Genschel, unpublished results.
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REFERENCES |
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