Molecular basis of the interactions between the p73
N terminus and p300: Effects on transactivation
and modulation by phosphorylation
Sarah Burge, Daniel P. Teufel, Fiona M. Townsley, Stefan M. V. Freund, Mark Bycroft, and Alan R. Fersht1
Medical Research Council Centre for Protein Engineering, Hills Road, Cambridge CB2 0QH, United Kingdom
Contributed by Alan R. Fersht, January 13, 2009 (sent for review October 20, 2008)
can transactivate a number of target genes in common with p53.
Here, we characterized the interaction of the p73 N terminus with
four domains of the transcriptional coactivator p300 and with the
negative regulator Mdm2 by using biophysical and cellular measure-
ments. We found that, like p53, the N terminus of p73 contained two
distinct transactivation subdomains, comprising residues 10–30 and
residues 46–67. The p73 N terminus bound weakly to the Taz1, Kix,
and IBiD domains of p300 but with submicromolar affinity for Taz2,
in contrast to previous reports. We found weaker binding of the p73
N terminus to the p300 domains in vitro correlated with a significant
decrease in transactivation activity in a cell line for the QS and T14A
mutants, and tighter binding of the phosphomimetic T14D in vitro
ylation of T14 increased the affinity of the p73 N terminus for Taz2
10-fold. The phosphomimetic p73? T14D caused increased levels of
p73 ? transcription
consisting of a flexible N-terminal transactivation domain, a DNA-
binding domain, and an oligomerization domain. Both p73 and p63
may exist in one of several C-terminal splice variants; this feature,
combined with the use of two distinct promoters that give rise to
transactivation (TA) and dominant-negative (?N) variants, means
that p73 may be expressed in a wide range of various isoforms. p73
has several functions in common with p53, such as activation of
target gene expression and suppression of cell growth. However,
as a classical tumor suppressor and is rarely mutated in human
cancers (1). Imbalances in the TAp73/?Np73 ratio may be more
important in tumorigenesis and response to chemotherapy than
mutations (2). ?Np73 is preferentially degraded in response to
DNA damage, allowing accumulation of the proapoptotic TAp73
isoform (3). p73, unlike p53, plays an essential role in normal
growth and development, with p73-knockout mice displaying se-
vere developmental defects but no increased susceptibility to spon-
taneous tumorigenesis (4). Despite its important role, relatively
little is known about the various mechanisms by which p73 can
induce apoptosis. p73 can induce G1growth arrest, transactivate
genes such as p21, Mdm2, Bax, and 14-3-3?, and is capable of
inducing apoptosis regardless of p53 status (for reviews see refs. 5
and 6). It is not yet clear whether transactivation of a similar group
of promoters to those of p53 is sufficient for p73 to induce
The transcriptional coactivator p300 is a large multidomain
protein that possesses histone acetyltransferase (HAT) ability (7).
Together with its homolog, CREB-binding protein (CBP), p300
mediates transcription through binding to transcriptional activators
such as JUN, E1A, NF-?B, and to the p53 family (8). Previous
studies have illustrated that the interaction between the p73 N
terminus (p73Nt) and the zinc finger Taz1 domain of p300 is
important for p73 to function as a transactivator (9), whereas the
73 belongs to the p53 family of tumor suppressor proteins. It
shares domain organization similar to that of p53 and p63,
(10). The three members of the p53 family of proteins have distinct
roles during embryogenesis and tumorigenesis; it is interesting to
N-terminal domain, which has the lowest levels of similarity be-
tween family members. Here, we have utilized a combined bio-
physical and cell biology approach to quantify the binding of the
p73Nt to the Taz1 domain and also to three other p300 domains:
p73Nt are involved in binding to the p300 domains Taz1 and Taz2,
whereas only one region is necessary for interaction with the Kix
and IBiD domains. In contrast to previous work showing that the
p73Nt binds only to the Taz1/CH1 domain of p300 (9), we found
from fluorescence anisotropy that the Taz2 domain could bind to
evidence from NMR studies to propose a structural basis for the
observed effects. Further, we found that mutations in the N-
terminal regions that abrogate the interaction with p300 domains
had a significant detrimental impact on the ability of p73? to
function as a transactivator and that phosphorylation of T14 may
play an important regulatory role.
Interaction of the p73Nt with the p300 Domains. To determine the
extent of the p73Nt-binding site, we monitored shifts in the1H15N
heteronuclear single quantum correlation (HSQC) spectra of
p73Nt constructs upon the addition of various p300 domains.
Two-dimensional1H15N HSQC spectra for free p73 1–67 and p73
bound to the four p300 domains and Mdm2 are shown in Fig. 1 A
and B. Residues of the p73Nt that shift upon addition of p300
domains are detailed in Fig. 1C. Some peaks in the bound spectra
were not assignable because of line-broadening effects and disap-
Upon addition of the zinc finger domains of p300 (Taz1 and
Taz2), we saw changes in chemical shift for residues in two distinct
regions of the p73Nt. It clearly consisted of two separate subdo-
from residues 46–67, unlike p53, where TAD1 and TAD2 corre-
spond to residues 1–40 and 41–60, respectively (11). There is a
linker between TAD1 and TAD2, broadly corresponding to Q33–
G45. In this region, few changes in chemical shift were observed
upon addition of p300 domains, highlighting the distinct nature of
the subdomains. There were no further changes in chemical shifts
on addition of Taz2 to greater concentrations than p73Nt, indicat-
ing formation of a 1:1 complex.
1D) whereas the TAD2s vary more. The changes in chemical shifts
in the HSQC spectrum show that Taz1, Taz2, and Mdm2 bind to
Author contributions: S.B., D.P.T., F.M.T., S.M.V.F., M.B., and A.R.F. designed research; S.B.,
F.M.T., and S.M.V.F. performed research; D.P.T. contributed new reagents/analytic tools;
S.B., F.M.T., S.M.V.F., and A.R.F. analyzed data; and S.B. and A.R.F. wrote the paper.
The authors declare no conflict of interest.
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
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no. 9 www.pnas.org?cgi?doi?10.1073?pnas.0900383106
the p73Nt in a fashion similar to the p53Nt, involving both
transactivation subdomains. But, in contrast to p53, binding of the
p300 domains Kix and IBiD results in few chemical shift changes in
The Transactivation Domain of p73 Bound More Weakly to the p300
Domains Than Does p53. In all cases, the full-length peptide bound
Table 1). Dissociation constants ranged from 0.89 ?M for Taz2 to
25.5 ?M for IBiD. There were significant increases in affinity
between the shorter peptides and the full-length peptides for the
Taz2 domain. This finding is consistent with the NMR data and
suggests that both subdomains are required for maximum affinity.
Interestingly, this was also the case for Mdm2, which has a Kdvalue
comparable with that of Taz2 for binding to the full-length peptide,
unlike with p53. Although NMR data for Taz1 and Mdm2 binding
to p73 confirmed that both subdomains were involved in binding,
Kd values obtained via fluorescence anisotropy suggest that, by
itself, TAD2 contributes weakly to binding. All four domains of
p300 studied here interacted with p53 1–57 with binding constants
of ?10 ?M (11). In contrast, the p73Nt displays a greater range of
affinities. Taz2 and Mdm2 had the smallest dissociation constants
consistent with their binding to only the first transactivation do-
main. With the exception of the Taz2 domain, we were unable to
Mdm2 by using fluorescence anisotropy. Although NMR data
IBiD binding to the p73Nt, Kdvalues were obtained by using only
the full-length peptide. In general, although extending the peptides
to include the second transactivation domain resulted in a notice-
able enhancement of avidity, the effect is less marked than for p53.
The Mutant L18QW19S Bound Weakly to the p300 Domains. The QS1
(L22Q/W23S) and QS2 (W53Q/F54S) mutants of p53 have im-
paired transcriptional activity because of their greatly reduced
affinity for p300 (12–14). Although there is no direct equivalent of
the hydrophobic residues V51 F52 or V61 M62 may fulfill a role
presence of an excess of Taz1 (blue), Taz2 (red), and Mdm2 (green). (B) HSQC spectra of free15N p73 1–57 in the presence of an excess of Kix (blue) and IBiD (red). (C)
Chemical shift map of p73Nt 1–67 in the presence of p300 domains and Mdm2. Peaks that disappear upon binding are shown in red bold; peaks that shift ?0.01ppm
for Biotechnology Information accession numbers are as follows: p53 from H. sapiens, gi:120407068 and p73 from H. sapiens, gi:4885645.
Interactions of the p73 N terminus with p300 domains as determined by NMR spectroscopy. (A) NMR HSQC spectra of free15N p73 1–67 (black) and in the
Burge et al.
March 3, 2009 ?
vol. 106 ?
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also abrogate binding to the p300 domains. We were unable to
detect any binding of the double-point mutation peptide, L18Q/
W19S, to any of the p300 domains by using fluorescent anisotropy
p73Nt Binds to Taz2 in a Fashion Similar to That of p53. The binding
from the p300 homolog CBP has been reported (15). Shifts in the
HSQC spectra of p73 1–67 (Fig. 1 A and B) upon binding to the
p300 domains and Mdm2 were indicative of helix formation by the
transactivation domains. Formation of small helical regions is
common among transcriptional activators; for example, p53 forms
a 13-residue helix upon binding Mdm2 (16), and the c-Jun N
terminus also forms helical regions upon binding to Kix (17). It is
to be expected that the first transactivation domain of p73 behaves
in a similar fashion.
The HSQC spectrum of15N-labeled Taz2 alone and in complex
in the 2D HSQC of the complex were assignable because of
line-broadening effects and disappearing peaks. Reassignment of
the bound state was challenging because of the low concentration
of labeled protein and exchange broadening, despite attempting
triple resonance experiments. The structure of Taz2 from the p300
homolog CBP consists of four helices with three zinc-binding
motifs, and the binding surface of a peptide representing residues
14–28 of p53 has been mapped (15). Peaks corresponding to Taz2
residues that shifted significantly upon binding (or disappearing/
unassignable peaks) to the p73Nt coincided with those that are
reported to shift upon binding of the p53 N-terminal peptide. In
particular, residues L69–L72, which showed large shifts upon
binding to the p53 (14–28) peptide, showed large shifts or were not
assignable when in complex with the p73Nt. Given the homology
between the p53 (14–28) peptide and the equivalent region of the
area on the Taz2 surface consisting primarily of residues L69–L72,
which form part of the third helix. The abrogation of binding
obtained by substituting nonpolar residues L18W19 for Q18S19
suggests that binding between the Taz2 and p73Nt is primarily
driven by the interaction of two hydrophobic surfaces. In addition
to shifts described by Wright and coworkers (15), we also observed
N83-C90, and we propose that the second transactivation domain
Interaction Between the TAp73 N Terminus and p300 Was Essential for
TAp73 to Function as a Transactivator of Bax. The Bax protein
(Bcl-2-associated protein X) was the first identified proapoptotic
member of the Bcl-2 family of proteins (18). p73 has a 2-fold effect
on Bax: first, it increases steady-state levels of Bax protein through
direct transactivation; and second, it promotes translocation of Bax
from the cytosol to the mitochondrial membranes through an
p73?L18Q/W19S to transactivate the bax promoter in H1299 cells.
Because we were unable to find binding of the QS peptide to any
p300 domain by using fluorescence anisotropy, we hypothesized
that p73?QS would be severely weakened in its ability to transac-
tivate Bax expression. To test this hypothesis, we used a bax-
show a marked reduction in bax reporter activity (Fig. 4A); residual
transactivation ability may be caused by binding between the
transactivation and p300 domains that is too weak to detect by
fluorescence anisotropy. To eliminate the possibility of Bax trans-
the TAp73–p300 interaction, we used siRNA to achieve a 3-fold
reduction in p300 levels and then repeated the luciferase assay. In
cells where p73?WT was cotransfected with p300 siRNA, we saw
activity (Fig. 4B), demonstrating that the ability of p73? to trans-
a weakening of the p300–p73 interaction through mutation and a
decrease in the levels of p300 obtained through siRNA knockdown
resulted in a decrease in bax transactivation ability.
0 10 20 30 40 50 60 70 80 90 100
QS p73 10-40
0 10 20 30 4050 60 70
WT p73 10-40
0 10 20 30 40 50 60 70 80 90 100
WT p73 10-70
0 10 2030 4050 6070
WT p73 41-70
p300 domains Taz1, Taz2, Kix and IBiD, and Mdm2.
Fluorescence anisotropy titrations of N-terminal peptides of p73 to the
Table 1. Dissociation constants for binding of various p300 domains and Mdm2 to p73
N-terminal peptides as determined by fluorescence anisotropy
Taz1 Taz2 KixIBiD Mdm2
39.0 ? 1.94.5 ? 0.5
28.0 ? 2.6
0.89 ? 0.25
0.47 ? 0.05
1.9 ? 0.1
10.3 ? 2.5
4.62 ? 0.09
19.5 ? 1.6
15.0 ? 3.0
25.5 ? 1.8
23.5 ? 3.1
0.87 ? 0.13
0.74 ? 0.16
119 ? 25
126 ? 34
1.13 ? 0.2
52 ? 4
4.1 ? 0.1
0.027 ? 0.01
110 ? 30
43 ? 4
3.0 ? 0.4
0.6 ? 0.1
121 ? 37
7.9 ? 1.60.16 ? 0.04
Values for equivalent p53 N-terminal peptides are included for comparison and are taken from ref. 11.
www.pnas.org?cgi?doi?10.1073?pnas.0900383106Burge et al.
Phosphorylation of the p73Nt at T14 Increased Its Avidity for the p300
Domains. Phosphorylation of the p53Nt is known to inhibit the
p53–Mdm2 interaction. We examined the binding of a p53 T18P
analog, p73 10–40 T14P, to the p300 domains and Mdm2 (Fig. 5
and Table 1). This phosphorylated peptide bound with ?10-fold
greater affinity for the p300 domains Taz1 and Taz2, whereas only
a 2-fold increase was seen for Mdm2. The phosphorylation of T14
also increased the affinity of the p73Nt to Kix and IBiD; however,
a direct comparison between the phosphorylated and unmodified
for the unmodified peptide for Kix and IBiD. To examine the
bax-luciferase assays with the mutants TAp73?T14A and
reduced bax transactivation ability whereas the T14D phosphomi-
metic mutant would show increased bax transactivation. Indeed,
this was found to be the case, with the T14D resulting in almost
twice the transactivation ability of wild-type p73 (Fig. 6A). We
further confirmed this with a dose–response luciferase assay with
T14D (Fig. 6B). We expect, therefore, the full T14P phosphoryla-
of T14 is part of an important mechanism that controls p73
behavior in vivo.
p73 is thought to be a key determinant of cellular sensitivity to
anticancer therapeutics (20), particularly in tumors lacking p53.
p73? is, to a greater extent than p73?, widely induced by chemo-
and the transcription machinery (for a review see ref. 7). Using
combined biophysical and cell-based methods, we have quantified
the binding of the p73Nt to various domains of p300 and directly
correlated binding strength to cellular activity. In contrast to
previous studies that suggested that the p73Nt bound only to the
Taz1/CH1 domain of p300 (9), we have found that Taz2 bound to
the p73Nt and did so with a higher affinity than did Taz1. Further,
the p73Nt could bind to several domains of p300. Binding of the
N-terminal regions of p53 family members via the Taz2 domain is
concurrent with p300 behaving as a general coactivator for a wide
range of transcription factors. Despite previous GST pulldown
assays not showing an interaction between the Taz2 domain and
the high affinity of the p73Nt for Taz2 and its structurally similar
mode of binding to p53, it is clear that Taz2-p53 family N terminus
between p53 family-specific target genes is achieved by other
Role of p300 Domains. Although we have quantified binding of the
p73Nt to four separate domains of p300, it is not yet clear whether
all domains are involved in effecting p73 transcriptional activity.
The possibility of four p300 domains binding simultaneously to a
p73 tetramer cannot be discounted; however, given the weaker
binding of the remaining domains, it is difficult to postulate a
biological reason for such a mode of binding.
Binding of p73 to Mdm2. The E3 ubiquitin ligase Mdm2 is a crucial
negative regulator of p53. Overexpression of Mdm2 is associated
with transformation of NIH 3T3 and Rat2 cell lines; it is believed
of p53 occurs via two mechanisms: first by inhibition of p53
Mdm2 can alleviate p73-induced apoptosis (25). But, the role of
Mdm2 in modulating p73 activity is more limited because the
diversity in the C terminus of p73 precludes ubiquitination occur-
1–67 (blue). For clarity, not all peaks are labeled.
HSQC of15N Taz2 free (red) and in a 1:1.2 complex with unlabeled p73
encoding p300. p300 and pcDNA 3.1(?) were used as controls. The QS mutant
displays much weaker binding to p300 domains and displays greatly reduced
transactivation ability. (B) p73 bax transactivation depends on p300. H1299 cells
p73? QS, bax-Luc, and p300 siRNA or control siRNA. A reduction in p300 levels is
concurrent with a decrease in bax transactivation.
0 25 50 75 100 125 150 175 200 225
p73 10-40 T14P
10–40 T14P to the p300 domains Taz1, Taz2, Kix and IBiD, and Mdm2.
Fluorescence anisotropy titrations of the phosphorylated peptide p73
Burge et al.
March 3, 2009 ?
vol. 106 ?
no. 9 ?
ring in a fashion similar to p53. Mdm2 is, therefore, only able to
Zeng et al. (9) demonstrated that Mdm2 competes with p300 for
binding to the p73Nt but not to p53 and hypothesized that binding
between p73Nt and p300 occurred via the Taz1 domain. However,
our results clearly show that Taz2 is capable of binding the p73
N-terminal domain and does so with an affinity comparable with
that of Mdm2 and in excess of that of Taz1. Our data, therefore,
support a p73 control mechanism that involves competition be-
tween Mdm2 and p300. But, the high affinity of p73Nt for the Taz2
domain implies that it is Taz2 that is involved in p300–p73 binding.
Control of p73 transactivation may, therefore, be achieved through
the relative levels of p300 and Mdm2 within the cell in concert with
selected posttranslational modifications.
Modification of Binding by Phosphorylation. p53 behavior is modu-
lated extensively through the use of posttranslational modifications
(for a review see refs. 26 and 27. In contrast, posttranslational
modifications of p73 have not yet been investigated as comprehen-
sively. Hck and c-Src phosphorylate Y28 (28); JNK may phosphor-
ylate S8 (among other sites not located in the N-terminal region)
(29) and catalytic subunit ? of protein kinase A has been shown to
phosphorylate the N terminus at an as yet unidentified site. Other
posttranslational modifications have been reported: acetylation of
lysines 321, 327, and 331 by p300 activate the apoptotic functions of
p73 (30); phosphorylation of tyrosine 86 by various cyclins results
in a decrease in p73 transcriptional activity (31, 32) whereas
phosphorylation by c-abl (33, 34) has the opposite effect, resulting
in an increase in apoptotis-inducing behavior. Phosphorylation of
the N terminus is an obvious candidate for altering p73 transcrip-
tional activity; modifications may increase the affinity of the N
terminus for p300 with a subsequent increase in transactivation.
Alternatively, Mdm2-mediated modifications of the N terminus
may decrease the transactivation ability of TAp73, as is the case for
Mdm2-mediated NEDDylation (35). Here, we investigated the
impact of phosphorylation of T14, equivalent to the p53 phosphor-
as a recognition site for CK1, and so phosphorylation in vivo may
be achieved by an unidentified kinase. We found that phosphory-
lation of p73 10–40 at T14 resulted in a 10-fold increase in affinity
for the Taz2 domain; if this increase in avidity is extended to the
full-length peptide, we would expect the phosphorylated 10–70
region to bind with a Kdof ?90 nM. Further, the phosphomimetic
expect the T14 phosphorylation to have even greater transactiva-
tion ability. Given the homology and functional overlap between
the p53Nt and p73Nt, we expect these data to be validated by
further in vivo work to identify a kinase responsible for T14
phosphorylation. Interestingly, although the T18 phosphorylation
of p53 resulted in a decrease in affinity for Mdm2, we found that
the equivalent phosphorylation in p73, T14P, results in a 2-fold
increase in affinity. Recent work has demonstrated that TAp73? is
able to enhance p53 stability by two mechanisms: through antag-
onizing p53 at the Mdm2 promoter level and by competing for
concomitantly with p53 phosphorylation in response to stress
factors, p73–Mdm2 binding will be favored at the expense of
phosphorylated p53, thereby enhancing p53 stability at a time of
Plasmid Construction. p73 (residues 1–57 and 1–67) coding sequences were
amplified from I.M.A.G.E Clone 40125802 (Geneservice Ltd.). Expression vectors
into the pRSETHisLipoTev vector (39).
Protein Expression and Purification. Escherichia coli C41 cells containing the
expression plasmids for p73 1–57 and 1–67 were grown at 37 °C to log phase in
2? TY medium, at which point 0.3 mM isopropyl 1-thio-?-D-galactopyranoside
was added. Expression was conducted at 20 °C for 16 h. The N-terminal domains
affinity column that was preequilibrated with binding buffer and eluted with
elution buffer [50 mM potassium phosphate (pH 8.0), 400 mM NaCl, 2 mM
2-mercaptoethanol, and 250 mM imidazole]. Fusion proteins with TEV protease
added were dialyzed overnight into reloading buffer [50 mM potassium phos-
phate (pH 8.0), 100 mM NaCl, and 2 mM 2-mercaptoethanol]; the TEV protease
cleaved the His-lipoyl tag. The unwanted tag was separated from the protein of
interest by binding to a nickel affinity column; the protein of interest was
collected in the flow-through. p73 1–57 and 1–67 were then subjected to HPLC
purification on a C18 column and eluted by using a water:acetonitrile gradient.
were combined, lyophilized, and resuspended in appropriate buffer. p300 do-
proteins were expressed in M9 minimal medium with 1.1g/L15NH4Cl as the sole
for double-labeled proteins) and vitamin mix. Isotopically labeled proteins were
purified as above.
NMR.1H15N heteronuclear single quantum correlation experiments were con-
ducted on a Bruker DRX-600 or 500 spectrometer equipped with CryoProbes.
mM NaCl, 5 mM DTT. The concentration of labeled p73 was ?100 ?M; p300
Backbone assignments were obtained by using standard HNCA, HNCACB,
HN(CO)CA, CBCA(CO)NH, and HN(CA)CO triple-resonance experiments. Spec-
tra were assigned by using Sparky (University of California, San Francisco);
chemical shift differences were calculated in accordance with the methodol-
ogy presented by Hajduk et al. (40).
Fluorescence Anisotropy. Peptides were synthesized as described in ref. 11.
equipped with a Hamilton Microlab M dispenser. Protein awaiting titration was
393 nm, respectively. Typically, 250 ?L of protein at various concentrations was
titrated into 1 mL of 0.5 ?M peptide. Buffer conditions were identical to those
Cell Biology. The coding sequence for WT TAp73? was obtained via PCR from
bility. (A) H1299 cells were transiently cotransfected with p73? WT, p73? T14D,
p73? T14A, and bax-Luc; pcDNA 3.1(?) was used as a negative control. The
phosphomimetic displays greater transactivation ability than wild-type, consis-
mutant. Increased amounts of transfected DNA correspond to increased lucif-
Effects of T14D phosphomimetic mutant on p73? transactivation capa-
www.pnas.org?cgi?doi?10.1073?pnas.0900383106 Burge et al.
tech). The coding sequence for the QS mutant (L18Q /W19S) was obtained by
using site-directed mutagenesis; all constructed plasmids were verified by se-
Upstate Bioscience (now Millipore).
confluence to a 1:6 dilution into 6-well plates. Transfection was performed with
Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.
by using RIPA buffer to which Complete protease inhibitor (Roche) had been
the following primary antibodies: mouse anti-p73? (ab19941; AbCam) and
mouse anti-p300 (ab3164; AbCam).
Luciferase Assays. H1299 cells were grown and transfected as detailed above.
Each well received 0.1 ?g of pCMV-Renilla (Promega), 1.0 ?g of pGL3-Bax-
or 0.5 ?g of pCMV?-p300. Cells were harvested 48 h after transfection, and
luciferase assays were performed with the Dual-Luciferase Reporter Assay sys-
prepared in triplicate, and error bars represent 1 SD.
Transfections with siRNA. In addition to the p73?, pCMV-Renilla, and Bax-
luciferase plasmids, cells were transfected with 100 pmol of p300 SMARTpool
siRNA transfection was repeated 24 h later. After 48 h, the cells were harvested,
levels were confirmed via Western blotting.
ACKNOWLEDGMENT. S.B. is supported by a Medical Research Council Career
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