Previous Article | Next Article 
Molecular and Cellular Biology, July 1999, p. 4935-4943, Vol. 19, No. 7
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
The Naturally Occurring Mutants of DDB Are Impaired
in Stimulating Nuclear Import of the p125 Subunit and
E2F1-Activated Transcription
Pavel
Shiyanov,1
Steven A.
Hayes,1,
Manjula
Donepudi,1
Anne F.
Nichols,2
Stuart
Linn,2
Betty L.
Slagle,3 and
Pradip
Raychaudhuri1,*
Department of Biochemistry and Molecular
Biology, University of Illinois at Chicago, Chicago, Illinois
606121; Division of Biochemistry and
Molecular Biology, University of California, Berkeley, California
94720-32022; and Division of
Molecular Virology, Baylor College of Medicine, Houston, Texas
770303
Received 25 February 1999/Returned for modification 24 March
1999/Accepted 23 April 1999
 |
ABSTRACT |
The human UV-damaged-DNA binding protein DDB has been linked to the
repair deficiency disease xeroderma pigmentosum group E (XP-E), because
a subset of XP-E patients lack the damaged-DNA binding function of DDB.
Moreover, the microinjection of purified DDB complements the repair
deficiency in XP-E cells lacking DDB. Two naturally occurring XP-E
mutations of DDB, 82TO and 2RO, have been characterized. They have
single amino acid substitutions (K244E and R273H) within the WD motif
of the p48 subunit of DDB, and the mutated proteins lack the
damaged-DNA binding activity. In this report, we describe a new
function of the p48 subunit of DDB, which reveals additional defects in
the function of the XP-E mutants. We show that when the subunits of DDB
were expressed individually, p48 localized in the nucleus and p125
localized in the cytoplasm. The coexpression of p125 with p48 resulted
in an increased accumulation of p125 in the nucleus, indicating that p48 plays a critical role in the nuclear localization of p125. The
mutant forms of p48, 2RO and 82TO, are deficient in stimulating the
nuclear accumulation of the p125 subunit of DDB. In addition, the
mutant 2RO fails to form a stable complex with the p125 subunit of DDB.
Our previous studies indicated that DDB can associate with the
transcription factor E2F1 and can function as a transcriptional partner
of E2F1. Here we show that the two mutants, while they associate with
E2F1 as efficiently as wild-type p48, are severely impaired in
stimulating E2F1-activated transcription. This is consistent with our
observation that both subunits of DDB are required to stimulate
E2F1-activated transcription. The results provide insights into the
functions of the subunits of DDB and suggest a possible link between
the role of DDB in E2F1-activated transcription and the repair
deficiency disease XP-E.
| |
INTRODUCTION |
The human UV-damaged-DNA binding
protein has been linked to the repair deficiency disease xeroderma
pigmentosum group E (XP-E). Cells from about 30% of XP-E patients (6 of 19) were shown to lack the damaged-DNA binding activity of DDB
(3, 27). DDB was originally identified as an activity that
binds to UV-damaged DNA (3). It has high affinities for the
6-4 photoproduct (14, 29, 36). In addition, DDB also binds
to cisplatin-modified DNA (14). It has been proposed that
the damaged-DNA binding activity of DDB is related to a potential DNA
repair function (7, 15, 18, 20, 36). The microinjection of
purified DDB overcomes the repair deficiencies in cells from XP-E
patients lacking the damaged-DNA binding activity of DDB (20,
27). However, purified DDB has no significant effect in
nucleotide excision repair assays in vitro (18). A recent
study proposed that DDB functions as a repair protein in the context of
chromatin structure (27). A repair function of DDB would be
consistent with the fact that the p48 subunit of DDB possesses
extensive sequence homology with the Cockayne syndrome protein CS-A,
which is involved in transcription-coupled DNA repair (2, 9, 12, 19).
DDB also possesses a transcriptional function. The p125 subunit of DDB
possesses homology with CPSF (4) and was implicated in the
transcription of the apolipoprotein B gene. A highly purified preparation of p125 interacted with the apolipoprotein B promoter, and
an antibody against p125 inhibited the transcriptional activity of this
promoter in vitro (22). Hayes et al. (11)
observed that DDB associated with the transcription factor E2F1 and
stimulated E2F1-activated transcription. This study showed that DDB
interacted with the activation domain of E2F1 in experiments that
assayed interactions in vivo and in vitro. Moreover, E2F1 and DDB in
HeLa nuclear extracts cofractionated through several steps of E2F1 purification, including an E2F-specific DNA affinity chromatography. More importantly, the coexpression of DDB with E2F1 resulted in a
cooperative stimulation of transcription from an E2F1-regulated promoter. Using Gal4 fusion constructs, we also showed that a fusion
construct containing the activation domain of E2F1 (residues 363 to
437) but not that of an unrelated transcription factor (hepatocyte
nuclear factor 3), responded to the transcriptional stimulatory
activity of DDB. The transcriptional stimulatory activity required both
subunits (125 and 48 kDa) of DDB (11). These results clearly
suggested a potential role for DDB in E2F1-activated transcription.
DDB is a target of several viral proteins. The large subunit of DDB,
p125, was identified as a cellular target of the hepatitis B virus X
protein (HBx) (23). The HBx protein, which is believed to be
the oncoprotein encoded by hepatitis B virus, was shown to function as
a transcriptional activator protein (35 and
references therein). It was shown that the transcriptionally proficient
HBx proteins were able to bind DDB (35). Moreover, mutants
of HBx that are impaired in binding to p125 were also defective in
their transcription activation function (35). Becker et al.
(1) showed that cells expressing HBx exhibit a reduction in
the DNA repair activity after UV irradiation. Mutational analysis of
HBx also demonstrated a partial correlation between the reduction of
repair activity in cells expressing HBx and the ability of HBx to bind
p125 (1). The p125 subunit of DDB also interacts with the V
proteins of paramyxovirus SV5, mumps virus, human parainfluenza virus, and measles virus (24). It has been postulated that
the interaction between the V proteins and DDB plays a role in the pathogenic function of these viruses (24).
Nichols et al. (26) have characterized two naturally
occurring mutants of DDB from XP-E patients. These are point mutations leading to single amino acid substitutions in the WD motif of the small
subunit (p48) of DDB. These mutations in the small subunit of DDB
correlated with a loss of the damaged-DNA binding activity of DDB.
Hwang et al. (16) demonstrated that these naturally occurring mutants of DDB failed to activate damaged-DNA binding activity when expressed in mammalian cells. These authors also suggested that the two mutant p48 proteins were defective in activating the damaged-DNA binding function of p125.
We have now investigated the naturally occurring mutants of DDB for
their abilities to participate in E2F1-activated transcription. Here,
we show that the p48 subunit stimulated the nuclear localization of the
p125 subunit of DDB. The naturally occurring mutants of p48 were
severely impaired in stimulating the nuclear localization of p125.
These mutant p48 proteins were able to interact with E2F1 but were
unable to stimulate E2F1-activated transcription. The results are
consistent with our previous observation that both subunits of DDB are
required for stimulating E2F1-activated transcription, and they suggest
a possible link between DDB's role in E2F1-activated transcription and
the repair deficiency disease XP-E.
| |
MATERIALS AND METHODS |
Cell culture.
U2OS and C33A cells were grown on
10-cm-diameter dishes in Dulbecco modified Eagle medium supplemented
with 10% fetal bovine serum in 5% CO2.
Mammalian cell expression plasmids.
A mammalian cell
expression vector, pCDNA3, was used to express the DDB subunits and
E2F1 (11). The constructs expressing T7-tagged p48 and p125
were generated by PCR, in which the upstream primers contained
sequences encoding the T7 epitope in frame with p48 or p125 amino acid
sequences, as described previously (11). The PCR primers
(11) used for wild-type T7-p48 were used to obtain mutants
2RO and 82TO in frame with T7. A similar PCR strategy was employed to
generate the plasmids that express hemagglutinin (HA)-tagged p48. The
pGEX-E2F1 construct has been described before (11). The
construct expressing glutathione S-transferase (GST)-E2F4 was a gift from D. M. Livingston (Dana Farber Cancer Institute, Boston, Mass.). The construct expressing HBx was obtained by subcloning the X open reading frame (subtype adw2) from plasmid pSV-X
(1) as a NotI-HindII fragment into
the same restriction sites of the plasmid pRc/CMV (Invitrogen).
Cytosolic and nuclear extracts.
The cytosolic and nuclear
extracts of U2OS cells were prepared following the procedure of Dignam
et al. (6) with the following modifications. Cells in a
hypotonic buffer were lysed by 30 strokes of a Kontes 2-ml tissue
grinder. The nuclei were washed with hypotonic buffer and then
extracted with high-salt buffer (0.5 M KCl). The extracts were not
dialyzed. A second approach was also employed to obtain nuclear and
cytosolic extracts, partly according to the method of Schreiber et al.
(30). In this method cells were lysed in a hypotonic buffer
containing 0.5% Nonidet P-40 (NP-40), the lysates were centrifuged for
30 s to pellet the nuclei, and the supernatant was directly
assayed as cytosol. The nuclei were extracted by a 30-min incubation in
ice with buffer A, which contained 20 mM HEPES (pH 7.9), 400 mM NaCl, 1 mM EDTA, 0.1% NP-40, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl
fluoride, and 20% glycerol. Extracted proteins were separated from the
nuclei by centrifugation at 13,000 × g for 10 min.
p125 antisera and affinity purification.
Based on the
observations made by Lee et al. (23), a chemically
synthesized peptide, with the sequence QYDDGSGMKREATA,
corresponding to p125 (peptide 2 in reference
23) was conjugated to maleimide-activated keyhole
limpet hemocyanin protein (Pierce). The conjugate was used for rabbit
immunizations and antiserum production. For affinity purification, the
same peptide was coupled to cyanogen bromide-activated Sepharose 4B
(Pharmacia), and the peptide-coupled Sepharose beads were used to
purify the antibody according to a previously described procedure
(10).
Immunoprecipitation and Western blotting.
Cells were
harvested after DNA transfection. The harvested cells were washed twice
with phosphate-buffered saline (PBS) and suspended in a lysis buffer
(60 µl for cells from one 100-mm-diameter dish) that contained 20 mM
HEPES (pH 7.9), 400 mM NaCl, 1 mM EDTA, 0.1% NP-40, 1 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 10%
glycerol. After an incubation at 4°C for 30 min, the lysates were
centrifuged at 13,000 × g for 10 min. The supernatants
were used for the immunoprecipitation experiments.
Agarose-linked T7 antibody (Novagen) and protein A-Sepharose-bound HA
antibody (Santa Cruz) were employed. Cell lysates (0.5 mg) were
incubated with beads containing antibodies for 2 h at 4°C. The
beads were then collected by centrifugation. Precipitates were washed
three times with 1 ml of buffer W (20 mM Tris-HCl [pH 7.8], 100 mM
NaCl, 0.1% NP-40, and 1 mM EDTA). The bound proteins were subjected to
Western blot analysis. Western blots were performed by using
anti-rabbit and anti-mouse Fab fragments conjugated to horseradish
peroxidase (Amersham) and Pierce Supersignal detection reagents
according to the manufacturer's instructions.
Immunostaining.
U2OS cells were plated on 10-cm-diameter
dishes containing glass coverslips. After DNA transfection, cells were
fixed with methanol at 
20°C and permeabilized with 0.5% Triton
X-100 in PBS for 10 min. Coverslips were blocked in 3% bovine serum
albumin followed by incubation with anti-T7 tag (1:1,000 dilution) or affinity-purified anti-p125 antibodies (1:100) in PBS containing 3%
bovine serum albumin and 0.1% Triton X-100. Cells were washed four
times with PBS and 0.1% Triton X-100 for 5 min each and incubated with
fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse (1:100
dilution; Gibco BRL) or donkey anti-rabbit immunoglobulin G (1:200
dilution; Jackson Laboratories, Inc.). After being washed, the
coverslips were mounted on glass slides by using 20 µl of a mounting
medium (15% [wt/vol] Vinol 205 polyvinyl alcohol, 33% [vol/vol]
glycerol, 0.1% azide in PBS [pH 8.5]). Cells were visualized, and
images were taken with a CLSM 510 microscope (Zeiss) and a 63×
Achroplan water immersion objective.
DNA transfection and CAT assays.
Transient transfections
were carried out by the calcium phosphate method as previously
described (33, 34). Twenty micrograms of DNA was used per
100-mm-diameter plate. Each experiment was controlled for transfection
efficiency by including 1 µg of pCMV-
-galactosidase in each
transfection and then normalizing for -galactosidase activity.
Chloramphenicol acetyltransferase (CAT) assays were performed by the
xylene extraction method of Seed and Sheen (31).
| |
RESULTS |
P48 stimulates nuclear accumulation of the p125 subunit of
DDB.
We observed that p125, when expressed alone, localizes in the
cytosol, whereas p48 localizes in the nucleus. Because DDB is believed
to possess nuclear functions, we suspected that p48 might play a role
in the nuclear localization of p125. We investigated this possibility
by looking at the distribution of p125 in the nuclear and cytosolic
extracts in the presence and absence of p48 expression. U2OS cells were
transfected with plasmids expressing T7-tagged p125 and p48.
Transfected cells were lysed by using a hypotonic buffer; cytosolic
S100 and high-salt nuclear extracts were prepared following the
procedure of Dignam et al. (6). Because the distribution
between nucleus and cytosol can vary with the level of expression,
three levels of the p125 plasmid were used with the expectation that we
would be able to obtain extracts with comparable levels of p125 in the
presence and absence of p48. The HBx protein was shown to bind p125
(23). A plasmid expressing HBx was used as a control to see
whether any p125-binding protein would have an effect on the
distribution of p125. Cytosolic and nuclear extracts of the transfected
cells were subjected to Western blot assays. The blots were probed with
a T7 antibody that would detect both p125 and p48. As can be seen in
Fig. 1A, the coexpression of p48 resulted
in an increased accumulation of p125 in the nuclear extracts. In the
absence of p48 coexpression there was very little increase in the
nuclear accumulation of p125 with increasing levels of p125 plasmid.
This result is consistent with the notion that p48 plays an important
role in the nuclear localization of p125.
View larger version (96K):
[in this window]
[in a new window]
|
FIG. 1.
P48 stimulates nuclear entry of the p125 subunit of DDB.
(A) Indicated amounts of the plasmid expressing T7-tagged p125 were
transfected into U2OS cells alone or in combination with a plasmid
expressing T7-tagged p48 or HBx. Cytosolic and nuclear extracts were
prepared from the transfected cells according to the method of Dignam
et al. (6). Fifty micrograms of the cytosolic and the
nuclear extracts were subjected to Western blot assays with the T7
antibody. The blots were developed with enhanced chemiluminescence
reagents. The bands corresponding to p125 and p48 are indicated. (B)
Plasmids (1.5 µg) expressing T7-tagged p125 and p48 proteins were
transfected into U2OS cells. Transfected cells were lysed in a
hypotonic buffer containing 0.5% NP-40, and cytosolic and nuclear
extracts were prepared as described in Materials and Methods. Fifty
micrograms of the cytosolic and nuclear extracts were assayed for p125
and p48 with the antibody against the T7 tag. (C) Plasmid (1.5 µg)
expressing T7-tagged p125 was transfected into U2OS cells. The
transfected cells were treated with leptomycin B for the indicated time
periods. The 0-h time point represents no leptomycin B treatment.
Cytosolic (C) and nuclear (N) extracts were prepared according to the
procedure of Dignam et al. (6). Twenty micrograms of the
extracts was analyzed by Western blot assays. The blots were probed
with a monoclonal antibody against the T7 epitope (upper panel). The
extracts (20 µg) were also assayed for endogenous cyclin B1 with a
polyclonal antibody (Santa Cruz). The blots were developed with
enhanced chemiluminescence reagents.
|
|
To rule out the possibility that the distribution was an artifact of
the extraction procedure, we employed a different method
to prepare
cytosolic and nuclear extracts. Transfected cells were
lysed in the
presence of 0.5% NP-40, and nuclei and cytosol were
separated
according to the procedure of Schreiber et al. (
30).
Nuclei
were extracted with buffer A (see Materials and Methods).
The nuclear
and cytosolic extracts were assayed for p125. Clearly,
the coexpression
of p48 resulted in a quantitative increase in
the nuclear accumulation
of p125 (Fig.
1B). In these assays, we
always detected a portion of p48
in the cytosolic extracts. We
think that this is a result of leaking of
p48 from nuclei during
the extraction procedure, because in
immunostaining experiments
the majority of p48 was detected in the
nucleus (Fig.
2). The
additional bands in
Fig.
1B detected by the T7 antibody are unrelated
to DDB because they
are also present in lanes corresponding to
the mock-transfected cells.
View larger version (112K):
[in this window]
[in a new window]
|
FIG. 2.
The naturally occurring mutants of p48, like the
wild-type protein, localize in the nucleus. U2OS cells were grown in
plates containing coverslips and transfected with plasmids (5 µg)
expressing T7-tagged p125 or p48 protein. The coverslips containing
transfected cells were fixed and probed with the T7 antibody and the
FITC-labeled secondary antibody as described in Materials and Methods.
The immunofluorescence was detected by using a CLSM 510 microscope.
|
|
It is possible that p125 is capable of nuclear entry, but in the
absence of p48 it is exported out of the nucleus. P125 contains
leucine-rich sequences, which often serve as signals for nuclear
export
(
8a,
9a,
22a). Leucine-rich export signals are recognized
by
the nuclear export receptor CRM1 (
8a,
9a,
22a). Therefore,
we investigated the effect of leptomycin B, an inhibitor of CRM1,
on
the localization of p125. If in the absence of p48 expression
p125 is
exported out of the nucleus by the export receptor CRM1,
leptomycin B
is expected to increase the accumulation of p125
in the nucleus. U2OS
cells were transfected with a plasmid expressing
T7 epitope-tagged
p125. The transfected cells were treated with
leptomycin B for various
lengths of time. Cytosolic and nuclear
extracts were prepared by
following the method of Dignam et al.
(
6). Extracts were
assayed for p125 by Western blotting with
a monoclonal antibody against
the T7 epitope. The Western blots
indicated very little or no
significant effect of leptomycin B
on the localization of p125 (Fig.
1C). The majority of p125 was
detected in the cytosolic extracts after
leptomycin B treatment.
Under these experimental conditions, endogenous
cyclin B1 was
detected mainly in the nuclear extracts (Fig.
1C, lower
panel).
An increase in the band intensity of p125 was detected in the
lane corresponding to 6 h of leptomycin B treatment (Fig.
1C);
however, the nucleus/cytoplasm ratio of p125 levels remained unaltered.
These results are consistent with the notion that p48 is required
for
the nuclear entry of
p125.
The naturally occurring mutants of p48 (2RO and 82TO) are defective
in stimulating nuclear import of p125.
Nichols et al.
(26) characterized two naturally occurring mutants of p48
from cells of XP-E patients. These mutants, 2RO (R273H) and 82TO
(K244E), harbor single amino acid substitutions within the WD motif of
the p48 protein. We compared the mutant forms of p48 with the wild-type
protein for their ability to localize in the nucleus by using an
immunostaining procedure (see Materials and Methods). Cells were grown
in plates containing coverslips and then transfected with plasmids
expressing T7-tagged p48 or the mutants. T7-tagged p125, which
localizes in the cytoplasm, was used as a negative control. Transfected
cells on the coverslips were subjected to immunostaining with a
monoclonal antibody against the T7 tag and an FITC-labeled secondary
antibody. p48-expressing cells were visualized by indirect
immunofluorescence. As can be seen in Fig. 2, the mutant p48 proteins
localized in the nucleus as efficiently as the wild-type p48 protein.
We then investigated the p48 mutants for their ability to import p125
into the nucleus. Under our assay conditions of immunostaining,
p125 is
mainly found in the cytoplasm (Fig.
2). Cells were transfected
with
plasmids expressing p125 and p48 or p48 mutants without any
epitope
tag. The coverslips containing transfected cells were
subjected to
immunostaining with an affinity-purified peptide
antibody specific for
p125 and an FITC-labeled secondary antibody.
In this experimental
setup, the immunostaining would detect only
p125. As can be seen in
Fig.
3, consistent with our results shown
in Fig.
1, a significant amount of fluorescence is detectable
in the
nuclei of cells transfected with p125 and the wild-type
p48 expression
plasmid. Interestingly, the two mutant proteins
were unable to increase
the fluorescence of p125 in the nucleus
(Fig.
3). This would be
consistent with the notion that the two
naturally occurring mutants are
incapable of promoting the nuclear
entry of p125.
View larger version (104K):
[in this window]
[in a new window]
|
FIG. 3.
The naturally occurring mutants of p48 are impaired in
their ability to stimulate nuclear import of p125 as detected by
immunostaining. U2OS cells on coverslips were transfected with a
plasmid (5 µg) expressing p125 alone or in combination with plasmids
expressing the wild-type or mutant p48 proteins. The cells on
coverslips were fixed and probed with an affinity-purified antibody
specific for p125 and then with an FITC-labeled secondary antibody. The
immunofluorescence was detected by using a CLSM 510 microscope.
|
|
The immunostaining results were confirmed by biochemical studies. Cells
were transfected with plasmids expressing T7-tagged
p125 and T7-tagged
p48 or the mutants. The transfected cells were
lysed and fractionated
to obtain cytoplasmic and nuclear extracts
by following the procedure
of Dignam et al. (
6). To determine
the distribution of p125
and p48 between the nucleus and cytoplasm,
the extracts were subjected
to Western blot assays with the T7
antibody. Clearly, p125 was
detectable in the nuclear extracts
of cells cotransfected with the
wild-type p48 expression plasmid
but not in the nuclear extracts of
cells transfected with the
mutant p48 expression plasmids (Fig.
4). Taken together, these
results
indicate that the naturally occurring mutants of p48 (2RO
and 82TO) are
defective in stimulating the nuclear import of p125.
View larger version (42K):
[in this window]
[in a new window]
|
FIG. 4.
The naturally occurring mutants of p48 are unable to
increase the level of p125 in nuclear extracts. U2OS cells were
transfected with a plasmid (5 µg) expressing T7-tagged p125 and
plasmids (5 µg) expressing T7-tagged p48 proteins. Cytosolic and
nuclear extracts of the transfected cells were prepared according to
the procedure of Dignam et al. (6). Fifty micrograms of the
nuclear and the cytosolic extracts was assayed for p125 and p48
proteins with the T7 antibody.
|
|
The p48 mutant 2RO is deficient in its ability to associate with
the p125 subunit of DDB.
We sought to analyze the interaction
between the mutant p48 proteins and p125, because they were unable to
stimulate a nuclear accumulation of p125. Plasmids expressing HA-tagged
p48 were transfected into U2OS cells along with a plasmid expressing
T7-tagged p125 protein. Extracts of the transfected cells were
subjected to immunoprecipitation with HA antibody and then Western
blotting with the T7 antibody. As can be seen in Fig.
5A, the mutant 2RO failed to
coprecipitate T7-tagged p125, whereas the other mutant (82TO) and
wild-type p48 coprecipitated p125. The mutants and wild-type p48 were
expressed at comparable levels, and no significant difference in the
expression of p125 was detected (Fig. 5A). This observation was
confirmed by another experiment in which the coprecipitation of
endogenous p125 with transfected T7-tagged p48 was assayed. The
endogenous p125 in U2OS cells also failed to coprecipitate with 2RO
(Fig. 5B). Taken together, these results clearly indicate that the main defect in 2RO is its inability to associate with p125. The mutant 82TO
bound p125 but did not stimulate a nuclear accumulation of p125,
suggesting that the interaction with p48 is necessary but not
sufficient for the nuclear accumulation of p125.
View larger version (25K):
[in this window]
[in a new window]
|
FIG. 5.
p48 mutant 2RO is impaired in its ability to associate
with the p125 subunit of DDB. (A) Plasmids (5 µg) expressing the HA
epitope-tagged p48 proteins (wild type and mutants) were transfected
into U2OS cells along with the plasmid (5 µg) expressing T7
epitope-tagged p125. Extracts (50 µg) of the transfected cells were
assayed for the expression of the p48 proteins (upper panel) and p125
(middle panel). The expression of p48 was assayed by using a monoclonal
antibody against the HA epitope, and the expression of p125 was assayed
by using a monoclonal antibody against the T7 epitope in Western blots.
To assay for an interaction between the mutant p48 proteins and p125,
200 µg of the extracts was also subjected to immunoprecipitation (IP)
with a monoclonal antibody against the HA epitope. The
immunoprecipitates were assayed for the presence of p125 by Western
blotting with the antibody against the T7 epitope (lower panel). The
blots were developed with enhanced chemiluminescence reagents by
following a procedure provided by the manufacturer. (B) The
interactions between the mutant p48 proteins and endogenous p125 were
assayed by transfecting U2OS cells with plasmids expressing T7-tagged
p48 or the mutants 2RO and 82TO. Extracts (200 µg) of the transfected
cells were subjected to immunoprecipitation with the T7 antibody. The
immunoprecipitates were analyzed by Western blot assays for the
presence of p125. A peptide antibody specific for p125 was used to
detect p125 in the blot. The Western blot was developed with enhanced
chemiluminescence reagents.
|
|
The mutant p48 proteins are defective in stimulating E2F1-activated
transcription.
We have previously shown that the coexpression of
DDB along with E2F1 resulted in a cooperative stimulation of
transcription from an E2F1-regulated promoter (11). Both
subunits of DDB were needed to observe the transcriptional stimulatory
activity of DDB (11). Because the two mutants 82TO and 2RO
failed to stimulate a nuclear accumulation of p125, we predicted that
these mutants would be impaired in their ability to stimulate
E2F1-activated transcription. The E2F1 gene promoter is a natural
target of E2F1-activated transcription (13, 17) and we
showed that a CAT gene construct containing the E2F1 promoter was
activated by the expression of DDB (11). Moreover, the
coexpression of DDB and E2F1 produced a much greater stimulation of
E2F1-induced transcription from this promoter (15). We
employed this transcription system to compare the activities of the two
p48 mutant proteins, 2R0 (R273H) and 82TO (K244E), with that of
wild-type p48. C33A cells were employed for this analysis because these
cells contain a low endogenous level of the DDB proteins (not shown),
and they exhibited a significant response to DDB expression in
transcription assays (11).
Cells were transfected by using the calcium phosphate precipitation
method. Plasmids expressing E2F1 and p125 were transfected
along with
the mutant or wild-type p48 expression plasmid and
the reporter CAT
gene. A plasmid expressing
-galactosidase was
used to control for
the transfection efficiencies. CAT gene activities
in the extracts were
normalized by using the
-galactosidase values.
An average of fold
stimulation by DDB over E2F1-activated transcription
from three
independent transfection experiments is shown in Fig.
6A. The results of these experiments
clearly indicated that the
two naturally occurring mutants of p48 are
impaired in stimulating
E2F1-activated transcription. Both mutants were
expressed quite
efficiently in these transfection experiments (Fig.
6B), indicating
that the inactivity of the mutants is not due to an
expression
defect.
View larger version (25K):
[in this window]
[in a new window]
|
FIG. 6.
The naturally occurring mutants of p48 are impaired in
stimulating E2F1-activated transcription. (A) Cells were transfected
with the E2F1-CAT reporter gene plasmid (5 µg) along with 0.5 µg of
the E2F1 expression plasmid. Plasmids (5 µg) expressing p125 and T7
epitope-tagged p48 or mutant p48 proteins (2RO and 82TO) were also
included in some of the transfection mixtures. DNA transfection was
carried out as described in Materials and Methods. A plasmid expressing
-galactosidase was included to control for the transfection
efficiencies. Averages of fold stimulation by DDB from three
independent experiments are shown. Error bars indicate standard
deviations. (B) Extracts from one of the above transfection experiments
were assayed for p48 expression. Fifty micrograms of the extract
proteins was separated by SDS-12% PAGE and blotted onto
nitrocellulose membranes. The blots were probed with the T7 antibody
and developed with enhanced chemiluminescence reagents as described
before (11).
|
|
We have previously shown that wild-type p48 could associate with E2F1,
which correlated with the DDB-mediated stimulation
of E2F1-activated
transcription (
11). Therefore, we examined
whether the
mutant p48 proteins were impaired in their abilities
to associate with
E2F1. Plasmids expressing E2F1, p125, and T7-tagged
p48 (wild-type or
mutant form) were transfected into U2OS cells.
Under these conditions
of DNA transfection, the proteins are overexpressed,
and the majority
of E2F1 remains in its free form, as judged by
gel retardation assays
and coimmunoprecipitation experiments with
Rb antibody (data not
shown). Extracts of the transfected cells
were subjected to
immunoprecipitation with a monoclonal antibody
against the T7 epitope.
The immunoprecipitates were subjected
to Western blot assays, which
were probed with a monoclonal antibody
against E2F1. The results of
these experiments clearly indicate
that the mutants, like wild-type
p48, were able to associate with
E2F1, as evidenced by
coimmunoprecipitation (Fig.
7A, left
panel).
In Fig.
7A, 82TO coprecipitated E2F1 at a lower level, the
result
of lower-level expression of 82TO in this experiment (Fig.
7A,
right panel). In other experiments, we did not see any difference
in
the coprecipitation of E2F1 by the p48 mutant forms (data not
shown).
The interactions between the mutant p48 proteins and E2F1
were further
confirmed by additional binding experiments with
a GST pull-down assay.
The p48 proteins were synthesized in reticulocyte
lysates as
[
35S]methionine-labeled proteins. The labeled proteins
were incubated
with GST-E2F1 and subjected to a pull-down assay with
GSH-Sepharose
beads (
11). The bound proteins were analyzed
by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
and autoradiography.
The mutant p48 proteins bound GST-E2F1 as
efficiently as wild-type
p48 (Fig.
7B). We also observed an interaction
between p48 and
GST-E2F4; however, an interaction between these
proteins is yet
to be seen in vivo. These results are consistent with
the notion
that the mutant p48 proteins are impaired in their
transcriptional
function because they are deficient in stimulating a
nuclear accumulation
of p125.
View larger version (29K):
[in this window]
[in a new window]
|
FIG. 7.
The naturally occurring mutants of p48 are able to
associate with E2F1. (A) Plasmids expressing E2F1 (5 µg) and T7
epitope-tagged wild-type or mutant p48 protein were transfected into
U2OS cells by the Ca phosphate precipitation method (see Materials and
Methods). Thirty-six hours after transfection, cells were harvested and
extracts were prepared with buffer A (see Materials and Methods for
details). Equal amounts (200 µg) of the extract proteins were
subjected to immunoprecipitation with T7 antibody. The
immunoprecipitates were subjected to Western blot assays. The blot was
probed with a monoclonal antibody against E2F1 (left panel). The
migrations of E2F1 and the immunoglobulin G bands are indicated. To
assay for the expression of the p48 proteins in this particular
experiment, the blot was stripped with 2% SDS and 100 mM
2-mercaptoethanol. Following an extensive wash to remove SDS, the blot
was further probed with horseradish peroxidase-linked T7 antibody
(right panel). (B) The p48 and mutant proteins (2RO and 82TO) were
transcribed and translated in vitro by using T7 RNA polymerase and
reticulocyte lysate. The translation was performed in the presence of
[35S]methionine. The translated products (2 µl) were
incubated with 1 µg of the indicated GST fusion proteins.
Glutathione-Sepharose beads (10 µl) were added to each of the
incubation mixtures, followed by incubation on a Nutator for 30 min at
4°C. The beads were collected by centrifugation and extensively
washed with NETN buffer (150 mM NaCl, 20 mM Tris-HCl [pH 7.6], 1 mM
EDTA, 0.5% NP-40). The bound proteins were eluted with SDS gel loading
buffer and were subjected to SDS-10% PAGE followed by
autoradiography. Two microliters of the translated products was
analyzed in the same gel (Load).
|
|
| |
DISCUSSION |
The studies described here are significant with regard to our
understanding of the function of DDB, which has been linked to a rare
inheritable repair deficiency disease, XP-E. We showed that the p48
subunit of DDB, which is found to be mutated in XP-E, plays an
important role in the nuclear accumulation of the p125 subunit of DDB.
This appears to be a specific function of p48, because HBx, which binds
to p125, was unable to stimulate the nuclear accumulation of p125. The
nuclear accumulation of p125 in the presence of p48 most likely
involves an increase in nuclear transport, because the nuclear level of
p125 increased at the expense of its cytosolic level (Fig. 1). However,
we have not ruled out the possibility that p48, in addition to
stimulating nuclear import, also stabilizes p125 in the nucleus. We
analyzed two naturally occurring mutants of p48, 2RO and 82TO. These
mutants harbor single amino acid substitutions in the WD motif of the p48 protein and were isolated from XP-E patients lacking the
damaged-DNA binding activity of DDB. Hwang et al. showed that unlike
wild-type p48, these two naturally occurring mutants of p48 are unable
to stimulate damaged-DNA binding activity when expressed in mammalian cells (16). This is consistent with the notion that p48 is
required for the damaged-DNA binding activity of DDB. It has been
postulated that p48 functions by activating the damaged-DNA binding
activity of the p125 subunit by a "hit and run" mechanism in which
p125 binds damaged DNA and p48 acts to activate the DNA-binding
function of p125 (16). We observed that these naturally
occurring mutants, which failed to bind damaged DNA, were also
defective in stimulating the nuclear entry of the p125 subunit.
The mutant 82TO was isolated from cells of a 41-year-old Japanese
patient. This patient exhibited an acute sun sensitivity but did not
develop skin malignancies (21). The mutant 2RO corresponded to a Dutch patient who developed skin tumors at the age of 14 (5). The difference in severity of the disease phenotype
might be a reflection of how severely the two mutations disrupted the function of p48. This is consistent with our results in that unlike 82TO, 2RO is severely impaired in its ability to associate with the
p125 subunit of DDB. If p125 is an essential functional partner of p48,
we would predict that the mutation in 2RO would severely compromise the
function of p48. The mutation in 2RO (R273H) did not disrupt the
structure of p48 because the mutant protein was able to associate with
E2F1. The result also suggests that the arginine residue at position
273 within the WD motif of p48 is critical for binding to p125. Our
assays of the nuclear localization of p125 and E2F1-activated
transcription failed to distinguish between 82TO and 2RO. This could be
a limitation of the sensitivity of our in vitro assays; however, other
possibilities cannot be ruled out.
The mutant p48 82TO localizes in the nucleus, and it is able to
associate with the p125 subunit. Therefore, it is surprising that this
mutant did not enhance the nuclear import of p125. This would suggest
that binding to p48 is not sufficient for the nuclear accumulation of
p125. It is possible that this mutant is deficient in other
interactions that are critical for the nuclear import or retention of
p125 in the nucleus. p48 has three putative nuclear localization
signals: two overlapping between residues 3 and 7 (PKKRP) and one
between residues 241 and 244 (HKKK). It is important to note that the
nuclear localization signal (HKKK) near the p125-binding site is
mutated in 82TO (K244E). It is possible that this signal plays a role
in carrying p125 into the nucleus. Other possibilities cannot be ruled
out. A modification that is required for the stable nuclear
localization of p125 may be missing in the context of 82TO. Clearly,
further work is required to understand the mechanisms that control the
nuclear localization of the DDB subunits.
We previously observed that DDB could functionally interact with the
transcription factor E2F1, which is involved in the expression of a
variety of cell cycle genes. In this study, we show that the two
mutants of DDB, 82TO and 2RO, are capable of interacting with E2F1 but
failed to functionally cooperate with E2F1 in transcription assays.
This would be consistent with the observation that both subunits of DDB
are necessary to stimulate E2F1-activated transcription because the
mutant p48 proteins (2RO and 82TO) are unable to stimulate the nuclear
accumulation of p125. The results presented here also suggest a link
between the XP-E phenotype and the transcription factor E2F1. However,
it is unclear how a lack of functional interaction between DDB and E2F1
would contribute to the disease phenotype found in XP-E patients.
It was shown that E2F1-deficient mice (E2F1
/) develop
tumors at a high frequency, and it has been suggested that E2F1
possesses tumor suppressor-like activity (8, 38). The tumor
suppressor-like activity of E2F1 might be related to its role in
p53-mediated apoptosis (25, 28, 32, 37). It is possible that
DDB plays a role in activating the E2F1 target genes involved in
apoptosis. However, a role for DDB in the apoptotic function of E2F1 is
yet to be established. In addition, it will be interesting to determine whether the cells from XP-E patients harboring mutations in the DDB
gene are deficient in E2F1-mediated apoptosis.
| |
ACKNOWLEDGMENTS |
We are grateful to Minoru Yoshida (University of Tokyo, Tokyo,
Japan) for the kind gift of leptomycin B. We also thank S. Bagchi, R. Costa, and G. Adami for critically reviewing the manuscript.
The work was supported by grants from the American Cancer Society
(RPG-94-041-04-TBE) and the National Cancer Institute (RO1 CA77637) to
P.R. S.L. is supported by grants (RO1 GM30415 and P30 ES08196)
from the National Institutes of Health.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry and Molecular Biology (M/C 536), University of Illinois at
Chicago, 1819 W. Polk St., Chicago, IL 60612. Phone: (312) 413-0255. Fax: (312) 413-0364. E-mail: Pradip{at}uic.edu.
Present address: Department of Biological Sciences, University of
Nevada, Las Vegas, Las Vegas, NV 89154.
| |
REFERENCES |
| 1.
|
Becker, S. A.,
T.-H. Lee,
J. S. Butel, and B. L. Slagle.
1998.
Hepatitis B virus X protein interferes with cellular DNA repair.
J. Virol.
72:266-272[Abstract/Free Full Text].
|
| 2.
|
Bohr, V. A.,
D. S. Okumoto, and P. C. Hanawalt.
1986.
Survival of UV-irradiated mammalian cells correlates with efficient DNA repair in an essential gene.
Proc. Natl. Acad. Sci. USA
83:3830-3833[Abstract/Free Full Text].
|
| 3.
|
Chu, G., and E. Chang.
1988.
Xeroderma pigmentosum group E cells lack a nuclear factor that binds to damaged DNA.
Science
242:564-567[Abstract/Free Full Text].
|
| 4.
|
Dantonel, J.-C.,
K. G. K. Murthy,
J. L. Manley, and L. Tora.
1997.
Transcription factor TFIID recruits factor CPSF for formation of 3' end of mRNA.
Nature
389:399-402[Medline].
|
| 5.
|
de Weerd-Kastelein, E. A.,
W. Keijzer, and D. Bootsma.
1974.
A third complementation group in xeroderma pigmentosum.
Mutat. Res.
22:87-91[Medline].
|
| 6.
|
Dignam, J. D.,
R. M. Lebovitz, and R. Roeder.
1983.
Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei.
Nucleic Acids Res.
11:1475-1489[Abstract/Free Full Text].
|
| 7.
|
Dualan, R.,
T. Brody,
S. Keeney,
A. F. Nichols,
A. Admon, and S. Linn.
1995.
Chromosomal localization and cDNA cloning of the genes (DDB1 and DDB2) for the p127 and p48 subunits of a human damage-specific DNA binding protein.
Genomics
29:62-69[Medline].
|
| 8.
|
Field, S. J.,
F. Y. Tsai,
F. Kuo,
A. M. Zubiaga,
W. G. Kaelin, Jr.,
D. M. Livingston,
S. H. Orkin, and M. E. Greenberg.
1996.
E2F1 functions in mice to promote apoptosis and suppress proliferation.
Cell
85:549-561[Medline].
|
| 8a.
|
Fornerod, M.,
M. Ohno,
M. Yoshida, and I. W. Mattaj.
1997.
Crm1 is an export receptor for leucine-rich nuclear export signals.
Cell
90:1051-1060[Medline].
|
| 9.
|
Friedberg, E. C.
1996.
Relationships between DNA repair and transcription.
Annu. Rev. Biochem.
65:15-42[Medline].
|
| 9a.
|
Fukuda, M.,
S. Asano,
T. Nakamura,
M. Adachi,
M. Yoshida,
M. Yanagida, and E. Nishida.
1997.
CRM1 is responsible for intracellular transport mediated by the nuclear export signal.
Nature
390:308-311[Medline].
|
| 10.
|
Harlow, E., and D. Lane.
1988.
Antibodies: a laboratory manual.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 11.
|
Hayes, S.,
P. Shiyanov,
X. Chen, and P. Raychaudhuri.
1998.
DDB, a putative DNA repair protein, can function as a transcriptional partner of E2F1.
Mol. Cell. Biol.
18:240-249[Abstract/Free Full Text].
|
| 12.
|
Henning, K. A.,
L. Li,
N. Iyer,
L. D. McDaniel,
M. S. Reagan,
R. Legerski,
R. A. Schultz,
M. Stefanini,
A. R. Lehman,
L. V. Mayne, and E. C. Friedberg.
1995.
The Cockayne syndrome group A gene product encodes a WD repeat protein that interacts with CSB protein and a subunit of RNA polymerase II TFIIH.
Cell
82:555-564[Medline].
|
| 13.
|
Hsiao, K. M.,
S. L. McMahon, and P. J. Farnham.
1994.
Multiple DNA elements are required for the growth regulation of the mouse E2F1 promoter.
Genes Dev.
8:1526-1537[Abstract/Free Full Text].
|
| 14.
|
Hwang, B. J., and G. Chu.
1993.
Purification and characterization of a human protein that binds to damaged DNA.
Biochemistry
32:1657-1666[Medline].
|
| 15.
|
Hwang, B. J.,
J. C. Liao, and G. Chu.
1996.
Isolation of a cDNA encoding a UV-damaged DNA binding factor defective in xeroderma pigmentosum group E cells.
Mutat. Res.
362:105-117[Medline].
|
| 16.
|
Hwang, B. J.,
S. Toering,
U. Francke, and G. Chu.
1998.
p48 activates a UV-damaged-DNA binding factor and is defective in xeroderma pigmentosum group E cells that lack binding activity.
Mol. Cell. Biol.
18:4391-4399[Abstract/Free Full Text].
|
| 17.
|
Johnson, D. G.,
K. Ohtani, and J. R. Nevins.
1994.
Autoregulatory control of E2F1 expression in response to positive and negative regulators of cell cycle progression.
Genes Dev.
8:1514-1525[Abstract/Free Full Text].
|
| 18.
|
Kazantsev, A.,
D. Mu,
A. F. Nichols,
X. Zhao,
S. Linn, and A. Sancar.
1996.
Functional complementation of xeroderma pigmentosum complementation group E by replication protein A in an in vitro system.
Proc. Natl. Acad. Sci. USA
93:5014-5018[Abstract/Free Full Text].
|
| 19.
|
Keeney, S.,
G. J. Chang, and S. Linn.
1993.
Characterization of a human DNA damage binding protein implicated in xeroderma pigmentosum E.
J. Biol. Chem.
268:21293-21300[Abstract/Free Full Text].
|
| 20.
|
Keeney, S.,
A. P. Eker,
T. Brody,
W. Vermeulen,
D. Bootsma,
J. H. Hoeijmakers, and S. Linn.
1994.
Correction of the DNA repair defect in xeroderma pigmentosum group E by injection of a DNA damage-binding protein.
Proc. Natl. Acad. Sci. USA
91:4053-4056[Abstract/Free Full Text].
|
| 21.
|
Kondo, S. J.,
S. Fukuro,
A. Mamada,
A. Kawada, and Y. Satoh.
1988.
Assignment of three patients with xeroderma pigmentosum to complementation group E and their characteristics.
J. Investig. Dermatol.
90:152-157[Medline].
|
| 22.
|
Krishnamoorthy, R. R.,
T.-H. Lee,
J. S. Butel, and H. K. Das.
1997.
Apolipoprotein B gene regulatory factor-2 (BRF-2) is structurally and immunologically highly related to hepatitis B virus S associated protein-1 (XAP-1).
Biochemistry
36:960-969[Medline].
|
| 22a.
|
Kudo, N.,
S. Khochbin,
K. Nishi,
K. Kitano,
M. Yanagida,
M. Yoshida, and S. Horinouchi.
1997.
Molecular cloning and cell cycle dependent expression of mammalian CRM1, a protein involved in nuclear export.
J. Biol. Chem.
272:29742-29751[Abstract/Free Full Text].
|
| 23.
|
Lee, T. H.,
S. J. Elledge, and J. S. Butel.
1995.
Hepatitis B virus X protein interacts with a probable cellular DNA repair protein.
J. Virol.
69:1107-1114[Abstract].
|
| 24.
|
Lin, G. Y.,
R. G. Paterson,
C. D. Richardson, and R. A. Lamb.
1998.
The V protein of the paramyxovirus SV5 interacts with damage-specific DNA binding protein.
Virology
249:189-200[Medline].
|
| 25.
|
Nevins, J. R.
1998.
Towards an understanding of the functional complexity of the E2F and retinoblastoma families.
Cell Growth Differ.
9:585-593[Medline].
|
| 26.
|
Nichols, A. F.,
P. Ong, and S. Linn.
1996.
Mutations specific to the xeroderma pigmentosum group E Ddb-phenotype.
J. Biol. Chem.
271:24317-24320[Abstract/Free Full Text].
|
| 27.
|
Otrin, V. R.,
I. Kuraoka,
T. Nardo,
M. McLenigan,
A. P. M. Eker,
M. Stefanini,
A. S. Levine, and R. D. Wood.
1998.
Relationship of the xeroderma pigmentosum group E DNA repair defect to the chromatin and DNA binding proteins UV-DDB and replication protein A.
Mol. Cell. Biol.
18:3182-3190[Abstract/Free Full Text].
|
| 28.
|
Qin, X. Q.,
D. M. Livingston,
W. Kaelin, Jr., and P. D. Adams.
1994.
Deregulated transcription factor E2F1 expression leads to S phase entry and apoptosis.
Proc. Natl. Acad. Sci. USA
91:10918-10922[Abstract/Free Full Text].
|
| 29.
|
Reardon, J. T.,
A. F. Nichols,
S. Keeney,
C. A. Smith,
J. S. Taylor,
S. Linn, and A. Sancar.
1993.
Comparative analysis of binding of human damaged DNA-binding protein (XPE) and Escherichia coli damage recognition protein (UvrA) to the major ultraviolet photoproducts: T[c,s]T, T[t,s]T, T[6-4]T, and T[Dewar]T.
J. Biol. Chem.
268:21301-21308[Abstract/Free Full Text].
|
| 30.
|
Schreiber, E.,
P. Matthias,
M. M. Muller, and W. Schaffner.
1989.
Rapid detection of octamer binding proteins with mini-extracts, prepared from a small number of cells.
Nucleic Acids Res.
17:6419-6420[Free Full Text].
|
| 31.
|
Seed, B., and J. Y. Sheen.
1988.
A simple phase-extraction assay for chloramphenicol acyltransferase activity.
Gene
67:271-277[Medline].
|
| 32.
|
Shan, B., and W.-H. Lee.
1994.
Deregulated expression of E2F1 induces S phase entry and apoptosis.
Mol. Cell. Biol.
14:8166-8173[Abstract/Free Full Text].
|
| 33.
|
Shiyanov, P.,
S. Bagchi,
G. Adami,
J. Kokontis,
N. Hay,
M. Arroyo,
A. Morozov, and P. Raychaudhuri.
1996.
p21 disrupts the interaction between cdk2 and the E2F-p130 complex.
Mol. Cell. Biol.
16:737-744[Abstract].
|
| 34.
|
Shiyanov, P.,
S. Hayes,
N. Chen,
D. G. Pestov,
L. F. Lau, and P. Raychaudhuri.
1997.
p27Kip1 induces an accumulation of the repressor complexes of E2F and inhibits expression of the E2F-regulated genes.
Mol. Biol. Cell
8:1815-1827[Abstract].
|
| 35.
|
Sitterlin, D.,
T.-H. Lee,
S. Progent,
P. Tiollais,
J. S. Butel, and C. Transy.
1997.
Interaction of the UV-damaged DNA-binding protein with hepatitis B virus X protein is conserved among mammalian hepadnaviruses and restricted to transactivation-proficient X-insertion mutants.
J. Virol.
71:6194-6199[Abstract].
|
| 36.
|
van Assendelft, G. B.,
E. M. Rigney, and I. D. Hickson.
1993.
Purification of a HeLa cell nuclear protein that binds selectively to DNA irradiated with ultra-violet light.
Nucleic Acids Res.
21:3399-3404[Abstract/Free Full Text].
|
| 37.
|
Wu, X., and A. J. Levine.
1994.
p53 and E2F1 cooperate to mediate apoptosis.
Proc. Natl. Acad. Sci. USA
91:3802-3808.
|
| 38.
|
Yamasaki, L.,
T. Jacks,
R. T. Bronson,
E. Harlow, and N. Dyson.
1996.
Tumor induction and tissue atrophy in mice lacking E2F1.
Cell
85:537-548[Medline].
|
Molecular and Cellular Biology, July 1999, p. 4935-4943, Vol. 19, No. 7
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Alekseev, S., Luijsterburg, M. S., Pines, A., Geverts, B., Mari, P.-O., Giglia-Mari, G., Lans, H., Houtsmuller, A. B., Mullenders, L. H. F., Hoeijmakers, J. H. J., Vermeulen, W.
(2008). Cellular Concentrations of DDB2 Regulate Dynamic Binding of DDB1 at UV-Induced DNA Damage. Mol. Cell. Biol.
28: 7402-7413
[Abstract]
[Full Text]
-
Guerrero-Santoro, J., Kapetanaki, M. G., Hsieh, C. L., Gorbachinsky, I., Levine, A. S., Rapic-Otrin, V.
(2008). The Cullin 4B-Based UV-Damaged DNA-Binding Protein Ligase Binds to UV-Damaged Chromatin and Ubiquitinates Histone H2A. Cancer Res.
68: 5014-5022
[Abstract]
[Full Text]
-
Sugasawa, K.
(2008). Xeroderma pigmentosum genes: functions inside and outside DNA repair. Carcinogenesis
29: 455-465
[Abstract]
[Full Text]
-
Stoyanova, T., Yoon, T., Kopanja, D., Mokyr, M. B., Raychaudhuri, P.
(2008). The Xeroderma Pigmentosum Group E Gene Product DDB2 Activates Nucleotide Excision Repair by Regulating the Level of p21Waf1/Cip1. Mol. Cell. Biol.
28: 177-187
[Abstract]
[Full Text]
-
Luijsterburg, M. S., Goedhart, J., Moser, J., Kool, H., Geverts, B., Houtsmuller, A. B., Mullenders, L. H. F., Vermeulen, W., van Driel, R.
(2007). Dynamic in vivo interaction of DDB2 E3 ubiquitin ligase with UV-damaged DNA is independent of damage-recognition protein XPC. J. Cell Sci.
120: 2706-2716
[Abstract]
[Full Text]
-
Al Khateeb, W. M., Schroeder, D. F.
(2007). DDB2, DDB1A and DET1 Exhibit Complex Interactions During Arabidopsis Development. Genetics
176: 231-242
[Abstract]
[Full Text]
-
Li, J., Wang, Q.-E., Zhu, Q., El-Mahdy, M. A., Wani, G., Praetorius-Ibba, M., Wani, A. A.
(2006). DNA Damage Binding Protein Component DDB1 Participates in Nucleotide Excision Repair through DDB2 DNA-binding and Cullin 4A Ubiquitin Ligase Activity.. Cancer Res.
66: 8590-8597
[Abstract]
[Full Text]
-
Hu, Z., Shao, M., Yuan, J., Xu, L., Wang, F., Wang, Y., Yuan, W., Qian, J., Ma, H., Wang, Y., Liu, H., Chen, W., Yang, L., Jin, G., Huo, X., Chen, F., Jin, L., Wei, Q., Huang, W., Lu, D., Wu, T., Shen, H.
(2006). Polymorphisms in DNA damage binding protein 2 (DDB2) and susceptibility of primary lung cancer in the Chinese: a case-control study. Carcinogenesis
27: 1475-1480
[Abstract]
[Full Text]
-
El-Mahdy, M. A., Zhu, Q., Wang, Q.-e., Wani, G., Praetorius-Ibba, M., Wani, A. A.
(2006). Cullin 4A-mediated Proteolysis of DDB2 Protein at DNA Damage Sites Regulates in Vivo Lesion Recognition by XPC. J. Biol. Chem.
281: 13404-13411
[Abstract]
[Full Text]
-
Shimanouchi, K., Takata, K.-i., Yamaguchi, M., Murakami, S., Ishikawa, G., Takeuchi, R., Kanai, Y., Ruike, T., Nakamura, R., Abe, Y., Sakaguchi, K.
(2006). Drosophila Damaged DNA Binding Protein 1 Contributes to Genome Stability in Somatic Cells. J Biochem
139: 51-58
[Abstract]
[Full Text]
-
Kulaksiz, G., Reardon, J. T., Sancar, A.
(2005). Xeroderma Pigmentosum Complementation Group E Protein (XPE/DDB2): Purification of Various Complexes of XPE and Analyses of Their Damaged DNA Binding and Putative DNA Repair Properties. Mol. Cell. Biol.
25: 9784-9792
[Abstract]
[Full Text]
-
Wang, Q.-E., Zhu, Q., Wani, G., El-Mahdy, M. A., Li, J., Wani, A. A.
(2005). DNA repair factor XPC is modified by SUMO-1 and ubiquitin following UV irradiation. Nucleic Acids Res
33: 4023-4034
[Abstract]
[Full Text]
-
Sun, C.-L., Chao, C. C.-K.
(2005). Cross-Resistance to Death Ligand-Induced Apoptosis in Cisplatin-Selected HeLa Cells Associated with Overexpression of DDB2 and Subsequent Induction of cFLIP. Mol. Pharmacol.
67: 1307-1314
[Abstract]
[Full Text]
-
Takata, K.-i., Yoshida, H., Yamaguchi, M., Sakaguchi, K.
(2004). Drosophila Damaged DNA-Binding Protein 1 Is an Essential Factor for Development. Genetics
168: 855-865
[Abstract]
[Full Text]
-
Yanagawa, Y., Sullivan, J. A., Komatsu, S., Gusmaroli, G., Suzuki, G., Yin, J., Ishibashi, T., Saijo, Y., Rubio, V., Kimura, S., Wang, J., Deng, X. W.
(2004). Arabidopsis COP10 forms a complex with DDB1 and DET1 in vivo and enhances the activity of ubiquitin conjugating enzymes. Genes Dev.
18: 2172-2181
[Abstract]
[Full Text]
-
Obuse, C., Yang, H., Nozaki, N., Goto, S., Okazaki, T., Yoda, K.
(2004). Proteomics analysis of the centromere complex from HeLa interphase cells: UV-damaged DNA binding protein 1 (DDB-1) is a component of the CEN-complex, while BMI-1 is transiently co-localized with the centromeric region in interphase. GENES CELLS
9: 105-120
[Abstract]
[Full Text]
-
Itoh, T., O'Shea, C., Linn, S.
(2003). Impaired Regulation of Tumor Suppressor p53 Caused by Mutations in the Xeroderma Pigmentosum DDB2 Gene: Mutual Regulatory Interactions between p48DDB2 and p53. Mol. Cell. Biol.
23: 7540-7553
[Abstract]
[Full Text]
-
Bondar, T., Mirkin, E. V., Ucker, D. S., Walden, W. E., Mirkin, S. M., Raychaudhuri, P.
(2003). Schizosaccharomyces pombe Ddb1 Is functionally Linked to the Replication Checkpoint Pathway. J. Biol. Chem.
278: 37006-37014
[Abstract]
[Full Text]
-
Rapic-Otrin, V., Navazza, V., Nardo, T., Botta, E., McLenigan, M., Bisi, D. C., Levine, A. S., Stefanini, M.
(2003). True XP group E patients have a defective UV-damaged DNA binding protein complex and mutations in DDB2 which reveal the functional domains of its p48 product. Hum Mol Genet
12: 1507-1522
[Abstract]
[Full Text]
-
Leupin, O., Bontron, S., Strubin, M.
(2003). Hepatitis B Virus X Protein and Simian Virus 5 V Protein Exhibit Similar UV-DDB1 Binding Properties To Mediate Distinct Activities. J. Virol.
77: 6274-6283
[Abstract]
[Full Text]
-
Fitch, M. E., Cross, I. V., Ford, J. M.
(2003). p53 responsive nucleotide excision repair gene products p48 and XPC, but not p53, localize to sites of UV-irradiation-induced DNA damage, in vivo. Carcinogenesis
24: 843-850
[Abstract]
[Full Text]
-
Zolezzi, F., Fuss, J., Uzawa, S., Linn, S.
(2002). Characterization of a Schizosaccharomyces pombe Strain Deleted for a Sequence Homologue of the Human Damaged DNA Binding 1 (DDB1) Gene. J. Biol. Chem.
277: 41183-41191
[Abstract]
[Full Text]
-
Andrejeva, J., Poole, E., Young, D. F., Goodbourn, S., Randall, R. E.
(2002). The p127 Subunit (DDB1) of the UV-DNA Damage Repair Binding Protein Is Essential for the Targeted Degradation of STAT1 by the V Protein of the Paramyxovirus Simian Virus 5. J. Virol.
76: 11379-11386
[Abstract]
[Full Text]
-
Bontron, S., Lin-Marq, N., Strubin, M.
(2002). Hepatitis B Virus X Protein Associated with UV-DDB1 Induces Cell Death in the Nucleus and Is Functionally Antagonized by UV-DDB2. J. Biol. Chem.
277: 38847-38854
[Abstract]
[Full Text]
-
Bergametti, F., Sitterlin, D., Transy, C.
(2002). Turnover of Hepatitis B Virus X Protein Is Regulated by Damaged DNA-Binding Complex. J. Virol.
76: 6495-6501
[Abstract]
[Full Text]
-
Rapic-Otrin, V., McLenigan, M. P., Bisi, D. C., Gonzalez, M., Levine, A. S.
(2002). Sequential binding of UV DNA damage binding factor and degradation of the p48 subunit as early events after UV irradiation. Nucleic Acids Res
30: 2588-2598
[Abstract]
[Full Text]
-
Chen, X., Zhang, Y., Douglas, L., Zhou, P.
(2001). UV-damaged DNA-binding Proteins Are Targets of CUL-4A-mediated Ubiquitination and Degradation. J. Biol. Chem.
276: 48175-48182
[Abstract]
[Full Text]
-
Nag, A., Datta, A., Yoo, K., Bhattacharyya, D., Chakrabortty, A., Wang, X., Slagle, B. L., Costa, R. H., Raychaudhuri, P.
(2001). DDB2 Induces Nuclear Accumulation of the Hepatitis B Virus X Protein Independently of Binding to DDB1. J. Virol.
75: 10383-10392
[Abstract]
[Full Text]
-
Nag, A., Bondar, T., Shiv, S., Raychaudhuri, P.
(2001). The Xeroderma Pigmentosum Group E Gene Product DDB2 Is a Specific Target of Cullin 4A in Mammalian Cells. Mol. Cell. Biol.
21: 6738-6747
[Abstract]
[Full Text]
-
Hara, R., Mo, J., Sancar, A.
(2000). DNA Damage in the Nucleosome Core Is Refractory to Repair by Human Excision Nuclease. Mol. Cell. Biol.
20: 9173-9181
[Abstract]
[Full Text]
-
Lin, G. Y., Lamb, R. A.
(2000). The Paramyxovirus Simian Virus 5 V Protein Slows Progression of the Cell Cycle. J. Virol.
74: 9152-9166
[Abstract]
[Full Text]
-
Gomez, C. A., Ptaszek, L. M., Villa, A., Bozzi, F., Sobacchi, C., Brooks, E. G., Notarangelo, L. D., Spanopoulou, E., Pan, Z. Q., Vezzoni, P., Cortes, P., Santagata, S.
(2000). Mutations in Conserved Regions of the Predicted RAG2 Kelch Repeats Block Initiation of V(D)J Recombination and Result in Primary Immunodeficiencies. Mol. Cell. Biol.
20: 5653-5664
[Abstract]
[Full Text]
-
Shiyanov, P., Nag, A., Raychaudhuri, P.
(1999). Cullin 4A Associates with the UV-damaged DNA-binding Protein DDB. J. Biol. Chem.
274: 35309-35312
[Abstract]
[Full Text]
-
Wakasugi, M., Kawashima, A., Morioka, H., Linn, S., Sancar, A., Mori, T., Nikaido, O., Matsunaga, T.
(2002). DDB Accumulates at DNA Damage Sites Immediately after UV Irradiation and Directly Stimulates Nucleotide Excision Repair. J. Biol. Chem.
277: 1637-1640
[Abstract]
[Full Text]
-
Nichols, A. F., Itoh, T., Graham, J. A., Liu, W., Yamaizumi, M., Linn, S.
(2000). Human Damage-specific DNA-binding Protein p48. CHARACTERIZATION OF XPE MUTATIONS AND REGULATION FOLLOWING UV IRRADIATION. J. Biol. Chem.
275: 21422-21428
[Abstract]
[Full Text]
-
Liu, W., Nichols, A. F., Graham, J. A., Dualan, R., Abbas, A., Linn, S.
(2000). Nuclear Transport of Human DDB Protein Induced by Ultraviolet Light. J. Biol. Chem.
275: 21429-21434
[Abstract]
[Full Text]
Top
Abstract
Introduction
