From the Institute for Molecular and Cellular Biology
and
The Graduate School of Pharmaceutical Sciences, Osaka
University and ¶ CREST, Japan Science and Technology Corp., 1-3 Yamada-oka, Suita, Osaka 565-0871, ** RIKEN, Wako, Saitama 351-0198, and
Yokohama Research Center, Mitsubishi-Tokyo
Pharmaceuticals, Inc., 1000 Kamoshida-cho, Aoba-ku, Yokohama,
Kanagawa 227-8502, Japan
Received for publication, January 30, 2001, and in revised form, February 26, 2001
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ABSTRACT |
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Nucleotide excision repair (NER) is carried out
by xeroderma pigmentosum (XP) factors. Before the excision reaction,
DNA damage is recognized by a complex originally thought to contain the
XP group C responsible gene product (XPC) and the human homologue of
Rad23 B (HR23B). Here, we show that centrin 2/caltractin 1 (CEN2) is
also a component of the XPC repair complex. We demonstrate that nearly
all XPC complexes contain CEN2, that CEN2 interacts directly with XPC,
and that CEN2, in cooperation with HR23B, stabilizes XPC, which
stimulates XPC NER activity in vitro. CEN2 has been shown
to play an important role in centrosome duplication. Thus, those
findings suggest that the XPC-CEN2 interaction may reflect coupling of
cell division and NER.
Centrin (caltractin) found in the centrosomes of a wide variety of
organisms (1) is a member of the highly conserved superfamily of
calcium binding EF-hand proteins. Centrin was first discovered in the
flagellar apparatus of the unicellular green algae Chlamydomonas reinhardii, where it is directly responsible for the contraction of calcium-sensitive structures (2-4). Analysis of a centrin mutant of
green algae suggested that centrin is important for the proper
segregation of the flagellar apparatus during cell division and for
accurate basal body duplication and separation (5). The counterpart of
centrin in Saccharomyces cerevisiae, Cdc31, was shown to be
necessary for the initiation of spindle pole body
(SPB)1 formation (6, 7).
In human, at least three centrin isoforms have been identified: centrin
1/caltractin 2 (CEN1) (8), centrin2/caltractin1 (CEN2) (9), and centrin
3 (CEN3) (10). Among these centrin isoforms, CEN1 displays
tissue-specific expression, whereas CEN2 is expressed
ubiquitously (11). A recent study showed that the expression and
distribution of these three isoforms varies during human ciliated cell
differentiation and proliferation in vitro (12). Moreover,
CEN3, the closest homologue of S. cerevisiae Cdc31, has been
suggested to have a distinct function in centrosome duplication (13).
On the other hand, CEN2 and its isoforms have been detected in the
nuclear fraction, although all centrin isoforms co-exist in the
centrosomes of animal cells (14). The nuclear function of centrins has
not yet been explored.
Xeroderma pigmentosum (XP) is a hereditary disease characterized by
photosensitivity, a high incidence of sunlight-induced skin cancer, and
in some cases, neurological complications. XP patient cells can be
classified into seven different genetic complementation groups (XP-A to
XP-G), all of which are defective in nucleotide excision repair (NER)
(reviewed in Ref. 15), and into one variant group, XP-V, which is
defective in translesion DNA synthesis (16, 17). NER is a versatile and
universal DNA repair pathway that can eliminate most types of the
lesions from DNA. NER reaction consists of four steps: 1) damage
recognition, 2) excision of the damaged DNA by creating incisions on
both sides of the lesion, 3) gap-filling by DNA polymerase activity,
and 4) ligation (18). A group of recent studies revealed that XP gene
products are involved in the early steps of the human NER reaction,
before the gap-filling reaction begins (reviewed in Refs. 19 and 20).
The DNA excision reaction can be reconstituted with six factors: XPA,
the XPC complex, XPF-ERCC1, XPG, general transcription factor
TFIIH, and replication protein A (RPA) on naked DNA (21, 22) or
minichromosomes (23). 24-32 base oligonucleotides containing the
lesion are excised from duplex DNA in the cell-free NER reaction.
The XPC complex acts as a key component of global genome nucleotide
excision repair (GGR), a NER subpathway, by functioning as the initial
damage detector (24). This complex was isolated as a heterodimeric
complex and consists of the XPC gene product (XPC) and the human
homologue of Rad23 B (HR23B) protein (25). Further biochemical analyses
showed that the XPC protein by itself preferentially binds to damaged
DNA and single-stranded DNA (26) and elicits the translocation of
another NER factor, TFIIH, to damaged DNA (27). The second subunit of
the XPC complex, HR23B stimulates the in vitro NER reaction
only in the presence of XPC (28), and this stimulatory activity depends
on the region of HR23B that mediates interaction with XPC (29). Further
analyses showed that the association of HR23B with XPC was important
for its ability to stimulate NER (30). However, the mechanism of NER
stimulation by HR23B is still poorly understood (31).
In this article we report that the XPC complex contains another factor,
CEN2, that is recruited via interaction with XPC. During the analyses
of CEN2, we found that NER stimulatory activity of HR23B is due to
stabilization of XPC protein. We show that CEN2 and HR23B cooperatively
stabilize XPC and thereby stimulate NER in vitro. Thus, we
conclude that CEN2 is a novel NER factor and suspect that the XPC
complex may be involved not only in DNA repair but also in cell division.
Cell Culture and Cell-free Extracts--
Lymphoblastoid cells
(GM2248B, a kind gift of Dr. J. H. J. Hoeijmakers) from an
XPC patient (XP3BE) were cultured at 37 °C with 5% CO2
in RPMI 1640 supplemented with 15% fetal bovine serum. For the
in vitro NER assay, whole cell extracts were prepared as
described previously (32, 33). HeLa cells were cultured in spinner
flasks and harvested for protein purification as described previously
(25).
Purification of XPC-HR23B-CEN2 Complex from HeLa Cells--
The
XPC-HR23B-CEN2 complex was purified from HeLa nuclear extracts as
previously described with minor modifications (25). All procedures were
carried out at 0-4 °C. HeLa nuclear extracts (1.49 g of protein),
obtained from a frozen stock of HeLa cells (236 ml of packed cell
volume), were successively applied to a phosphocellulose column
(Whatman P11 (90 ml)), a single-stranded DNA cellulose column (Sigma (9 ml)), and a CM-cosmogel column (Nacalai Tesque (3.8 ml)). The
fraction (1.2 mg) eluted from the CM-cosmogel column with buffer 1 (20 mM potassium phosphate (pH 7.5), 0.1 mM EDTA,
10% glycerol, 0.01% Triton X-100, 1 mM DTT, 0.25 mM PMSF, 0.2 µg/ml aprotinin, 0.2 µg/ml leupeptin, 0.1 µg/ml antipain, and 50 µM EGTA) containing 0.6 M KCl was dialyzed against buffer 1 containing 0.3 M KCl. The dialysate was adjusted to 0.15 M KCl
and loaded onto a MonoQ PC1.6/5 column (Amersham Pharmacia Biotech (0.1 ml)) equilibrated with buffer 1 containing 0.15 M KCl using
the SMART system (Amersham Pharmacia Biotech). The column was washed
with the equilibration buffer, and adsorbed proteins were eluted with
2.5 ml of a linear gradient of 0.15-0.45 M KCl in buffer
1. XPC-complementing activity was assayed with GM2248B whole cell
extracts. Active fractions containing the HeLa XPC-HR23B-CEN2 complex (150 µg) were frozen at Protein Sequencing--
Protein bands were excised from
the Coomassie Brilliant Blue-stained gel and then digested with
Achromobacter lyticus protease I (35) by an in-gel digestion
method (36). Peptides extracted from the gel were separated by reverse
phase high performance liquid chromatography using a Bakerbond octyl
column (J. T. Baker (4.6 × 250 mm)) with a linear gradient
of 0-60% acetonitrile in 0.1% trifluoroacetic acid for 1 h.
Each peptide fragment was subjected to automated Edman degradation on a
protein sequencer PPSQ-10 (Shimadzu). The amino acid sequences were
ELGENLTDEELQEMIDEADRDGDGEV and QEIREAFDLFDADGTGTIDVK. These two
sequences are identical to deduced amino acid sequences of
CEN2 but distinct from those of CEN1 and
CEN3.
Glycerol Density Gradient Centrifugation--
A portion (40 µl) of the MonoQ fraction 22 was layered onto a 15-40% (v/v)
glycerol gradient (1.2 ml) in buffer 2 (0.3 M KCl, 20 mM potassium phosphate (pH 7.5), 0.1 mM EDTA,
0.01% Triton X-100, 1 mM DTT, 0.25 mM PMSF,
0.2 µg/ml aprotinin, 0.2 µg/ml leupeptin, 0.1 µg/ml antipain, and
50 µM EGTA). Centrifugation was carried out in a Beckman
TLS55 rotor at 194,000 × g for 16 h at 2 °C,
and fractions (60 µl) were collected from the top of the gradient. An
identical gradient containing marker proteins was run at the same time.
The markers were detected by SDS-PAGE followed by staining with
Coomassie Brilliant Blue.
Co-immunoprecipitation and in Vitro Binding
Assay--
Whole cell extracts (200 µg) were mixed gently at 4 °C
for 16 h with 5 µl of protein A-Sepharose 4FF (Amersham
Pharmacia Biotech) beads covalently conjugated to affinity-purified
anti-XPC, anti-HR23B, or anti-CSA in buffer 3 (0.15 M KCl, 20 mM Tris-HCl (pH 8.0), 0.2 mM EDTA, 10% glycerol, 0.01% Nonidet P-40, and 0.2 mg/ml
bovine serum albumin). The beads were washed three times at 4 °C
with 100 µl of buffer 3, suspended in SDS-PAGE sample buffer (50 mM Tris-HCl (pH 6.8), 2% SDS, and 10% glycerol), boiled
for 2 min, directly subjected to SDS-PAGE, and analyzed by
immunoblotting. Binding assays using nickel-chelating Sepharose
(Amersham Pharmacia Biotech) were carried out in binding buffer (20 mM sodium phosphate (pH 6.8), 0.3 M NaCl, 20 mM imidazole, 0.1% Triton X-100, and 0.2 mg/ml bovine
serum albumin) as described previously (29). The amounts of protein
used in the binding assays are indicated in the figure legend.
Construction of Plasmids for Protein Expression in Escherichia
coli--
The human CEN2-coding sequence was obtained by
PCR with first-strand cDNA synthesized using HeLa poly(A)+ RNA as
template. The sequences of the oligonucleotides used were
5'-GTACACGTCGGTTGCCTAAC-3' and 5'-TTCTTCACGCTTGTGTGCTC-3'. The PCR
products were further amplified by a second PCR using oligonucleotides
5'-CCTTTGACCATGGCCTCCAAC-3' and 5'-AGAGAATTCTGATCTTAATAGAGGC-3', and
then the second PCR products were cloned into pGEM-T Easy vector
(Promega) to generate pGEM-CEN2. As a result of the second PCR, an
NcoI site was introduced into the CEN2 cDNA
that allowed the isolation of CEN2 cDNA as a 550-base pair fragment after NcoI digestion of pGEM-CEN2. The
fragment was inserted into the NcoI site of E. coli expression vector, pET24d (Novagen), to generate pET24-CEN2.
The stop codon in pET24-CEN2 was converted to an XhoI site
by site-directed mutagenesis using the oligonucleotide
5'-GAGAATTCTGCTCGAGATAGAGGCTGGTC-3'. This was performed with the
reagent Mutan-K (Takara Shuzo). The resulting plasmid was digested by
XhoI and self-ligated to fuse the C terminus of CEN2
in-frame with eight amino acids (LEHHHHHH). This construct was
designated pET24-CEN2H. All constructs were verified by DNA sequencing
using an ALF-Red DNA sequencer (Amersham Pharmacia Biotech).
Expression and Purification of Recombinant CEN2
Proteins--
Recombinant nontagged CEN2 protein was expressed and
purified essentially according to the previous methods of Baron
et al. (37) and Wiech et al. (38). Ten
milliliters of a fresh, full-grown culture of E. coli BL21
(DE3) carrying pET24-CEN2 was inoculated into 1 liter of Super
Broth medium (5 g of NaCl, 32 g of Bacto-trypton, and
20 g of yeast extract/liter (pH 7.0)) containing 50 µg/ml of
kanamycin. Cells were cultured at 30 °C with vigorous shaking to an
absorbance at 600 nm of 0.4, and then synthesis of the recombinant protein was induced by further incubation in the presence of 0.5 mM isopropylthioglucoside for 2 h at 30 °C. The
cell pellet was washed with ice-cold 10% glycerol, suspended in lysis
buffer (50 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 2 mM DTT, 0.5 mM PMSF, 0.4 µg/ml
aprotinin, 0.4 µg/ml leupeptin, 0.2 µg/ml antipain 0.1 mM EGTA, and 10 µg/ml lysozyme), and disrupted by
freezing and thawing. The supernatant (129 mg of protein), obtained by
centrifugation, was loaded onto a HiTrap DEAE column (Amersham
Pharmacia Biotech (5 ml)) that was equilibrated with buffer 4 (50 mM Tris, 34 mM HCl, and 1 mM DTT).
The column was washed with the equilibration buffer, and the adsorbed
proteins were eluted with 30 ml of a linear salt gradient of 0-0.2
M NaCl in buffer 4. CEN2 eluted around 0.2 M
NaCl was collected and dialyzed against buffer 4 containing 1.5 M NaCl, 5 mM CaCl2, and 10%
glycerol. The dialysate was incubated in a water bath at 80 °C for
10 min and chilled on ice. After centrifugation to remove denatured
proteins, the supernatant (25.2 mg) was loaded onto a phenyl-Superose
HR5/5 column (Amersham Pharmacia Biotech (1 ml)) equilibrated with the same buffer used for denaturation. The column was washed with the
equilibration buffer, and the adsorbed proteins were eluted with buffer
5 (10 mM Tris, 6.8 mM HCl, 5 mM
EGTA, 1 mM DTT, and 10% glycerol). Because a portion of
CEN2 was recovered in the flow-through fraction, the fraction was
applied to and eluted from the column three times. The eluates (2.6 mg)
were combined, dialyzed against buffer 4, and loaded onto a MonoQ HR5/5
column (1 ml) equilibrated with buffer 4. After washing the column with buffer 4 containing 0.1 M NaCl, the adsorbed proteins were
eluted with 10 ml of a linear salt gradient of 0.1-0.4 M
NaCl. CEN2 (2.3 mg) eluted around 0.25 M NaCl was pooled
and stored at
Recombinant hexa-histidine-tagged CEN2 (CEN2-His) was expressed in
E. coli cells transformed with pET24-CEN2H as described above. The cells were disrupted by sonication in buffer 6 (0.3 M NaCl, 50 mM Tris-HCl (pH 7.5), 0.5 mM PMSF, 0.1% Nonidet P-40, 0.4 µg/ml aprotinin, 0.4 µg/ml leupeptin, 0.2 µg/ml antipain, and 0.1 mM EGTA)
containing 10 µg/ml lysozyme. The lysate was cleared by
centrifugation, and the supernatant was loaded onto a nickel-chelating
Sepharose column (Amersham Pharmacia Biotech (1 ml)) equilibrated with
buffer 6 containing 20 mM imidazole. The column was washed
with the equilibration buffer, and the adsorbed proteins were eluted
with a linear gradient of 20-250 mM imidazole. The
protein-rich fractions were pooled and dialyzed against buffer 4. The
dialysate was loaded onto a Mono Q HR5/5 column equilibrated with
buffer 4. The column was washed with equilibration buffer, and the
adsorbed proteins were eluted with a linear salt gradient of 0-0.4
M NaCl. CEN2-His eluted around 0.25 M NaCl was
pooled and stored at Heat Inactivation Assay--
To examine the stability of XPC
protein, XPC was preincubated at various temperatures for 1 h in
preincubation buffer (0.1 M NaCl, 25 mM
Tris-HCl (pH 7.5), 10% glycerol, 0.01% Triton X-100, 1 mM
DTT, and 0.25 mM PMSF) in the presence or absence of CEN2 and/or HR23B. The residual activity was analyzed by the
XPC-complementing assay and the damaged DNA binding assay. The XPC
concentration during the preincubation was 12.5 nM and 50 nM for the XPC-complementing assay and the damaged DNA
binding assay, respectively. In both cases, the molar ratios of CEN2
and HR23B were 10 times higher than that of XPC. In the 1-h
preincubation, two tubes, the first one of which (tube A) contained XPC
with or without CEN2 and/or HR23B and the second one of which (tube B)
contained the proteins missing from the first tube, were incubated in a
water bath. For the XPC-complementing assay, 2 µl of solution from
tube A was combined with 2 µl of solution from tube B, added to the
reaction mixture containing the SV40 minichromosome, and incubated at
30 °C for 3 h under standard conditions. For the damaged
DNA binding assay, 4 µl of solution from tube A was combined with 4 µl of solution from tube B, added to the reaction mixture containing damaged or undamaged DNA, and incubated at 30 °C for 30 min.
These procedures are summarized in Figs. 5A and
6A. At these amounts of XPC, both XPC-complementing activity
and damaged DNA binding activity were dependent on XPC but not on CEN2
or HR23B, and the activity in both assays was not saturating but
proportional to the amount of added XPC (data not shown). Thus, in both
assays, the only limiting condition was the amount of XPC.
Cell-free NER Assay--
For the XPC complementation assay,
UV-irradiated (200 J/m2) simian virus 40 (SV40)
minichromosome (0.3 µg of DNA) was reacted with 80 µg of GM2248B
whole cell extract and/or other purified proteins (HeLa XPC-correcting
fraction, XPC, CEN2, and HR23B) in the standard reaction mixture (39).
After incubation at 30 °C for 3 h, DNA was purified from the
reaction mixture, linearized by EcoRI digestion, and then
electrophoresed in a 1% agarose gel. The gel was dried and
autoradiographed with Fuji RX x-ray films. DNA repair synthesis was
quantified using the Fuji BAS2500 Bio-Imaging analyzer.
For the NER reaction with purified proteins, the standard
reaction mixture (20 µl) was as indicated above except that
radiolabeled dCTP and crude cell extracts were omitted. The proteins
used were 540 ng of RPA-His, 10 ng of XPA-His (23), 6 ng of
XPF-ERCC1-His (23), 10 ng of XPG (23), and 880 ng of partially purified TFIIH, and/or the indicated amounts of recombinant proteins composing the XPC complex (28, 29). After incubation at 30 °C for 3 h,
the DNA was purified from the reaction mixture, and the gaps on the
DNA, generated by the excision of the damaged DNA, were filled in by
gap-filling DNA synthesis with radioactive dNTPs (23). DNA was purified
again, linearized by EcoRI digestion, subjected to 1%
agarose gel electrophoresis, and analyzed as described above.
The recombinant RPA trimeric complex carrying a hexa-histidine
tag at the C-terminal end of the p32 subunit (RPA-His) was expressed in
bacterial cells and purified by three column chromatographic steps
(nickel-chelating Sepharose, single-stranded DNA-agarose, and MonoQ).
The activity of RPA-His was indistinguishable from that of the
nontagged form both in the in vitro NER assay and in the
in vitro replication
assay.2 HeLa TFIIH was
partially purified from HeLa nuclear extracts as far as the TSK-DEAE
5PW column step, corresponding to two column chromatographic steps
before the final purification step (40). Using the previously purified
fraction, we obtained results similar to those of the partially
purified fraction (data not shown).
Damaged DNA Binding Assay--
The damaged DNA binding assay was
carried out in the standard reaction mixture containing 200 ng of
pBS.XPC Other Methods--
SDS-PAGE was performed as described by
Laemmli (41). For immunoblot analyses, proteins separated by SDS-PAGE
were electrically transferred onto polyvinylidene difluoride membranes
(Millipore Immobilon-P) and analyzed as described previously (23).
The anti-CEN2(rabbit) and anti-CSA antibodies were obtained by
immunization of rabbits with recombinant CEN2 and histidine-tagged CSA,
respectively, and purified by antigen-coupled affinity column chromatography from the antiserum. The CEN2(rat) antiserum was obtained
by immunization of rats with recombinant CEN2. The anti-HR23B and
anti-XPC affinity-purified antibodies were obtained as described previously (28).
XPC Forms a Trimeric Complex with CEN2 and HR23B--
The XPC
complex was purified from HeLa nuclear extracts after four column
chromatographic steps (Fig.
1A). In this study, we
obtained a fraction that was approximately 10 times more concentrated than previous preparations (25). The addition of the MonoQ fractions to
XPC cell extracts showed significant recovery of NER synthesis, whereas
the extracts alone did not show any NER synthesis (Fig. 1B).
The elution profile of XPC-complementing activity (NER activity) correlated with the presence of XPC and HR23B proteins as detected by
silver staining SDS-polyacrylamide gels containing samples from the
elution peak (compare Fig. 1, B and C). However,
several extra bands, which were not detected in the previous study,
were observed in these fractions (Fig. 1C). Most of these
could be accounted for XPC-derived degradation products and HR23A,
another human homologue of S. cerevisiae Rad23, by
immunoblot analyses (data not shown). However, protein sequencing
analysis revealed that the 18-kDa protein was the centrosome protein
centrin 2/caltractin 1 (CEN2).
CEN2 co-sedimented with both XPC and HR23B proteins in a complex with a
sedimentation co-efficient of 6.2 S during glycerol density gradient
centrifugation (Fig. 2A).
Using hydrodynamic methods (42), Shivji et al. (43)
determined that the XPC-complementing complex is 160 kDa, in good
agreement with the calculated mass of these proteins (169 kDa).
To confirm that the XPC-HR23B-CEN2 complex is naturally present in the
cells, we performed co-immunoprecipitation analyses. CEN2 was
co-precipitated from HeLa cell extracts by resin containing anti-XPC
antibody or by anti-HR23B antibody, but not by anti-CSA antibody (Fig.
2B). Quantitative analyses revealed that almost 100% of
CEN2 in the cell extract was co-precipitated with the anti-XPC
antibody. Thus, most of the CEN2 in this extract was complexed to XPC.
The anti-HR23B resin precipitated lower amounts of CEN2 than did the
anti-XPC resin (Fig. 2B). This was also true for the
anti-HR23A antibody, which could also precipitate low but nonetheless
detectable amounts of CEN2 (data not shown). These results are
consistent with the observation that only a small proportion of the XPC
complex in the final purification step contained HR23A (Fig.
1C).
CEN2 Directly Interacts with XPC--
To understand XPC complex
formation, we studied the properties of complexes reconstituted from
each component that were purified as a recombinant protein (see
"Experimental Procedures"). CEN2-His was bound to nickel-chelating
Sepharose resin, as detected by immunoblotting (Fig.
3, A and B),
whereas neither XPC nor HR23B bound to the resin. In the presence of
CEN2-His, however, XPC was retained on nickel-chelating resin (Fig.
3A) but HR23B was not (Fig. 3B), indicating that
CEN2 interacts with XPC but not HR23B. CEN2 has four EF-hand domains
that are known to be important for calcium binding. However, the XPC
binding activity of the recombinant CEN2, which was purified in the
absence of calcium, was not affected by the presence of 5 mM calcium (data not shown).
NER Activities of the XPC Complex Components--
From the above
studies, we surmised that CEN2 might have some function in NER because
it is present in virtually all of the purified XPC complexes. To
directly test the role of CEN2 in NER, we purified recombinant
nontagged CEN2 as described under "Experimental Procedures" (Fig.
4A). NER activity was measured
on UV-irradiated SV40 minichromosomes in the cell-free NER assay
reconstituted with purified NER factors (23). As shown in Fig. 4,
B and C, in reactions containing all factors
except the XPC complex, the addition of recombinant XPC alone resulted
in weak NER activity, as reported previously (30). In the absence of
XPC, CEN2 alone, HR23B alone, or a mixture of CEN2 and HR23B indicated
no significant NER activity. However, HR23B stimulated the NER activity
of XPC, as previously reported (30). On the other hand, the addition of
CEN2 either alone or in combination with HR23B failed to produce any
significant effect on XPC-dependent NER activity in
vitro.
HR23B Stabilizes XPC--
As shown above, HR23B has an
XPC-dependent NER stimulatory activity in defined
conditions. Though the mechanism of NER stimulation by HR23B has not
been well understood, several observations suggested that HR23B might
function by stabilizing XPC. Previous studies showed that the XPC
binding region of HR23B was necessary and sufficient for the
stimulation of XPC-dependent NER activity in a
semi-reconstituted reaction (29) and that the interaction of HR23B with
XPC was important for the stimulation of NER by HR23B (30). Recently,
we observed that the capacity of HR23B to stimulate
XPC-dependent NER activity was greater during long incubations than short ones (data not shown). Taken together, these
observations suggested that HR23B might function by protecting XPC
protein from heat denaturation during the reaction.
To examine this possibility more directly, we designed a heat
inactivation assay. As shown in Fig.
5A, XPC, CEN2, and HR23B were
preincubated at various temperatures either alone or in various combinations. After preincubation, the proteins were combined and added
to the XP-C whole cell extracts, and NER activity was measured (Fig. 5,
B and C). When XPC was preincubated in the
absence of either CEN2 and HR23B, ~75% activity was lost during a
1-h incubation at temperatures
To determine the reason for the loss of XPC function during the
preincubation, we examined XPC binding to damaged DNA after preincubation (Fig. 6A). After
preincubation, the components of the XPC complex were incubated with
both UV-irradiated DNA and unirradiated DNA in reaction mixtures, and
the DNA-XPC complexes were immunoprecipitated with resin conjugated to
XPC antibodies (see "Experimental Procedures"). The DNA present in
the precipitated complexes was purified and analyzed by Southern
blotting (Fig. 6, B and C). The results showed
that specific binding activity of XPC to damaged DNA was more resistant
to preincubation at high temperatures in the presence of HR23B than in
its absence. Moreover, CEN2 had a small but reproducible effect on XPC
stabilization activity at 42 °C when combined with HR23B, whereas
CEN2 alone had no significant effect on XPC stabilization. Finally,
there was good correlation between the heat inactivation profiles of XPC as assayed by the NER assay and the DNA binding assay (compare Fig.
5C and 6C). We could not exclude the possibility
that highly denatured XPC protein was not co-precipitated as
efficiently as native XPC by the anti-XPC antibody conjugated resin.
Therefore, the precipitated DNA may not reflect the real DNA binding
activity of XPC for damaged DNA. However, we examined the DNA binding
affinity of XPC for either damaged or nondamaged DNA in a filter
binding assay and found that DNA binding activity decreased with
increasing preincubation temperature. We also observed that CEN2 and
HR23B prevented heat inactivation of XPC as measured by a filter
binding assay (data not shown). Thus, CEN2 and HR23B are important for the stability of XPC protein and, especially, for the preservation of
the binding activity of XPC for damaged DNA, which is essential for
NER.
CEN2 Stabilizes XPC in Co-operation with HR23B--
As shown in
Figs. 5 and 6, CEN2 enhanced, albeit weakly, the ability of HR23B to
stabilize XPC. To confirm this point, we performed the heat
inactivation assay at different temperatures. As shown in Fig.
7, XPC preincubated only with HR23B and
lost more than 55% of its activity at 42 °C, 89% of its activity
at 44 °C, and all of its activity at 46 °C compared with the
activity of the control incubated at 0 °C. On the other hand, XPC
preincubated with both CEN2 and HR23B retained nearly 69% of its
activity at 42 °C, 47% of its activity at 44 °C, and 7%
of its activity at 46 °C compared with the activity of the
control incubated at 0 °C. These results further support our
conclusion that CEN2 contributes to NER by binding to and stabilizing
XPC.
Roles of CEN2 in the XPC Complex--
In this study, we have
demonstrated that CEN2 is a component of the XPC complex (Figs. 1 and
2) that directly interacts with XPC (Fig. 3). In contrast, we could
detect neither XPC nor HR23B in a partially purified centrosome
fraction from HeLa cells (data not shown). These observations strongly
suggest that CEN2 has a role in NER in human nuclei that is distinct
from its function in the centrosome. Actually, CEN2 contributed to XPC
stability, thereby increasing the efficiency of NER (Fig. 5), whereas
CEN2 did not show significant activity under standard conditions for in vitro NER. CEN2 stabilized XPC in vitro in
co-operation with HR23B. Although the direct interaction was not
observed between CEN2 and HR23B, it is possible that both proteins
interact on XPC protein. It is also likely that the binding of CEN2 to
XPC confers a proper interaction between XPC and HR23B. We suspect that
CEN2 might be important for XPC complex formation in vivo, although CEN2 contributes relatively little to XPC stabilization in vitro (Fig. 5-7). Unprotected XPC is quite labile (Figs.
5 and 6) and susceptible to degradation during purification (Fig.
1C). Consequently, we believe that XPC is stabilized in
human cells by virtue of association with CEN2 and HR23B.
Moreover, the complex reconstituted from the purified recombinant
proteins showed lower activity in in vitro NER than the XPC
complex purified from HeLa cells (data not shown). From our previous
studies, we know this is mainly due to incomplete complex assembly
in vitro (30). To examine the role of CEN2 in complex assembly, we purified XPC complexes from insect cells infected with
recombinant baculoviruses expressing XPC and HR23B with or without CEN2
and measured XP-C complementation activity. The XPC complexes
containing both CEN2 and HR23B showed slightly higher NER activity and
solubility than XPC complexes containing HR23B alone.3 These observations
support the results shown in this paper and strongly suggest that CEN2
plays a role in XPC complex assembly within cells.
Roles of HR23B in the XPC Complex--
During the analyses of CEN2
function in in vitro NER, we found that HR23B stimulates NER
mainly by stabilizing XPC and that CEN2 supports HR23B in this
function. This is the first paper that clearly shows how HR23B
stimulates in vitro NER by XPC-dependent manner,
but we cannot rule out the possibility that HR23B has additional
functions for NER in vivo (discussed below).
Studies in S. cerevisiae led to the idea that Rad23, the
yeast counterpart of HR23B(A), has a function carried out by its N
terminus ubiquitin-like (UbL) domain via interaction with proteasome. Previous studies showed that the UbL domains of Rad23 are important for
cell survival after UV irradiation (44) and that Rad23 interacts with
the 26 S proteasome through its UbL domain (45). Recently, two
completely different models have been proposed to explain the function
of the UbL region of HR23B. Russell et al. (46) show that
the proteasome 19 S subunit plays a direct role in NER both in
vitro and in vivo and that the ATPase activity, but not the proteolytic activity of the 19 S subunit, is essential for NER.
They suggest that the molecular chaperone function, but not the
proteolytic function, of the 19 S subunit is important for NER.
However, Ortolan et al. (47) find that Rad23 can inhibit the
degradation of proteasome substrates and show that a proteasome subunit
mutation can partially rescue the UV sensitivity of a Rad23 deletion
mutant. Taken together, they proposed that the UbL domain of Rad23
negatively regulates the degradation of repair factors after DNA
damage, which may promote NER events. In neither report is there direct
evidence of how an interaction between Rad23 and the proteasome acts to
promote survival after UV irradiation. In human cells, a
protein-protein interaction between HR23B and the 26S proteasome was
observed (48), indicating that the above-mentioned mechanisms might be
conserved in human cells.
Although most of the XPC-complementing complex contains CEN2 and HR23B,
we also purified an XPC-HR23A-CEN2 complex (Fig. 1C) that
was detected by co-immunoprecipitation (data not shown). HR23A was
first identified as another human homologue of Rad23 by PCR methods
(25) and was subsequently shown to be functionally redundant to HR23B
in NER both in the cell-free system (30) and in mouse embryonic
fibroblast cells derived from knock out mice.4 Thus, we expect that
HR23A can stabilize in a manner XPC similar to HR23B. However, the
functional differences between these two isoforms and the mechanism
that generates the alternative complex still remain unclear.
Centrosome Duplication and the XPC-HR23B-CEN2 Complex--
Genetic
studies in yeast suggest that XPC complex might have a role in
centrosome duplication in human cells. In S. cerevisiae, mutations in the gene encoding a counterpart of human centrin, CDC31, were also isolated as DSK1, dominant
suppressors of the Kar1-
In human cells, the intracellular localization of XPC changes
dramatically during mitosis (51). During anaphase and telophase, the
XPC protein becomes preferentially associated with chromatin, whereas
it is distributed throughout the cell during metaphase. Thus, we can
imagine that localization of XPC (Rad4 in yeast) complex is strictly
regulated via CEN2 (Cdc31 in yeast) during mitosis and that the failure
to regulate the Rad4 complex might cause some defect in SPB duplication
in yeast. Further studies may show that XPC cancer-prone phenotypes are
partly due to defects in the mechanism that couples cell division to
NER and/or in the repair reaction involving the XPC-HR23B-CEN2 complex.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C. Protein concentrations were determined by the method of Bradford (34) using the Bio-Rad reagent.
80 °C.
80 °C. Throughout the purification, column
chromatographies were performed with fast protein liquid chromatography
system, and the proteins were analyzed by SDS-PAGE.
(500 J/m2 UV-irradiated) and 200 ng of pHM14
(unirradiated) (24). After incubation at 30 °C for 30 min, plasmid
DNA bound to XPC was successively immunoprecipitated by protein
A-Sepharose 4FF conjugated to anti-XPC antibody, washed with ice-cold
immunoprecipitation wash buffer, eluted by 1% SDS treatment at
50 °C for 1 h, purified, linearized by BamHI
digestion, separated by 0.8% agarose gel electrophoresis, and blotted
onto a Hybond-N+ membrane (Amersham Pharmacia Biotech) by alkali
transfer (24). The blots were hybridized to 32P-labeled
pBluescript KS(+) (Stratagene) sequences.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (32K):
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Fig. 1.
Purification of the XPC complex.
XPC-complementing activity was determined by the cell-free NER assay
system. UV-irradiated (200 J/m2) SV40 minichromosomes were
reacted with 80 µg of XPC cell-free extract in reaction mixture (see
"Experimental Procedures") in the presence or absence of 0.1 µl
of a MonoQ fraction. A, schematic representation of the
purification of the XPC complex from HeLa nuclear extracts. NER
activity was determined by measuring 32P-labeled dCMP
incorporation by endogenous DNA polymerases, which fill in the gaps
generated by the damaged DNA excision reaction. Successively, SV40 DNA
was purified from the reaction mixture, linearized by EcoRI
digestion, and subjected to 1% agarose gel electrophoresis.
ssDNA, single-stranded DNA. B, an autoradiogram
of the gel. C, silver staining of the MonoQ fraction
(fr). A portion (1 µl) of the MonoQ fraction was subjected
to 8-18% SDS-PAGE and stained with silver. The proteins were
identified by immunoblot analysis (XPC, HR23B, and HR23A) and amino
acid sequencing (CEN2). Asterisks represent the XPC-derived
degraded proteins. The indicated molecular weights were obtained using
standard high molecular mass markers (Amersham Pharmacia
Biotech).
View larger version (26K):
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Fig. 2.
CEN2 is part of the XPC-correcting
complex. A, MonoQ fraction 22 (Fig. 1) was subjected to
glycerol density gradient centrifugation, and portions (16 µl) of the
collected fractions were analyzed by 8-18% SDS-PAGE followed by
silver staining. The sedimentation positions of marker proteins in a
parallel gradient are indicated. A portion of the MonoQ fraction
(Q) was subjected to SDS-PAGE as a control. The molecular
weights were obtained using a 10-kDa ladder marker (Life Technologies,
Inc.). BSA, bovine serum albumin. B,
co-immunoprecipitation analyses of the XPC complex from HeLa whole cell
extracts. HeLa whole cell extracts (200 µg) were incubated with
protein A beads conjugated with affinity-purified anti-XPC, anti-HR23B,
or anti-CSA antibody. The precipitates were subjected to 14% SDS-PAGE
followed by immunoblotting with anti-CEN2(rat) antibody. Anti-CSA
antibody was used for a negative control of the affinity-purified
antibody. Ten ng of recombinant CEN2 protein was loaded onto the gel as
a positive control. The recombinant CEN2 was prepared under a
calcium-free condition, and it was reported that the migration profile
of endogenous CEN2 is not affected by calcium during electrophoresis
(12). Therefore, the presence of a doublet band could not be due to a
calcium binding state of CEN2. It is rather suggested that the upper
band of the doublet is full-length CEN2 and that the lower band is a
truncated version of CEN2 or another centrin isoform closely related to
CEN2. Asterisks indicate the protein that cross-reacted with
anti-CEN2(rat) serum but not with affinity-purified anti-CEN2(rabbit)
(data not shown). Molecular weights were calculated from a set of
prestained molecular weight standards (Life Technologies, Inc.).
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Fig. 3.
CEN2 binds directly to XPC but not to
HR23B. Hexa-histidine-tagged recombinant CEN2 (1 pmol) was mixed
with 0.2 pmol of recombinant XPC (A) or 0.2 pmol of
recombinant HR23B (B) in binding buffer (80 µl) containing
the nickel-chelating Sepharose resin. Unbound (U) and bound
(B) materials were recovered, and a portion (16 µl) of
each sample was subjected to SDS-PAGE (14% for CEN2 and 8% for XPC
and HR23B). Proteins were detected by immunoblotting with
anti-CEN2(rabbit), anti-XPC, or anti-HR23B.
View larger version (23K):
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Fig. 4.
NER activities of XPC complex components in
the reconstituted reaction. UV-irradiated (200 J/m2)
SV40 minichromosomes were incubated in a reaction mixture with purified
NER factors (see "Experimental Procedures") and increasing amounts
(0, 20, 60, and 180 fmol) of recombinant XPC along with various
combinations of recombinant CEN2 (200 fmol) and HR23B (200 fmol).
Recombinant XPC and HB23B were purified as described previously (24,
40), and CEN2 was purified as described under "Experimental
Procedures." A, Coomassie Brilliant Blue staining of CEN2
(0.5 µg). After incubation at 30 °C for 3 h, DNA was
purified, and the gaps generated by the excision of damaged DNA were
labeled by T4 DNA polymerase with radioactive dNTPs. DNA was purified
again, linearized by EcoRI digestion, and subjected to 1%
agarose gel electrophoresis. B, an autoradiogram of the gel.
C, incorporation of dCMP into SV40 minichromosomes was
calculated, and the results are shown on the graph. Symbols:
diamond, titration of XPC in the absence of both CEN2
and HR23B; triangle, in the presence of CEN2;
square, in the presence of HR23B; circle, in the
presence of both CEN2 and HR23B. Averages and experimental errors were
calculated from two independent experiments.
37 °C. With a preincubation in the presence of HR23B, XPC retained most of its activity at 37 °C and
showed a certain activity at higher temperatures. After a 1-h
preincubation at 30 °C, a small but reproducible increase in XPC
activity was observed in the presence of HR23B. These observations strongly suggest that the NER stimulatory activity of HR23B is due
mainly to its ability to stabilize XPC. CEN2 alone did not show a
significant effect on XPC stabilization. During the 42 °C
preincubation, however, CEN2 increased the stability of XPC protein
when HR23B was concomitantly present in the preincubation mixture,
suggesting that CEN2 may help HR23B to stabilize XPC (see
below).
View larger version (31K):
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Fig. 5.
Heat inactivation of XPC. Various
combinations of recombinant proteins of the XPC complex were
preincubated at various temperatures (0, 30, 37, 42, and 50 °C) for
1 h, and residual NER activity was measured by the
XPC-complementing assay. XPC (12.5 nM) with or without 125 nM CEN2 and/or 125 nM HR23B were added to one
tube, the omitted proteins were added to another tube, and both tubes
were preincubated as a set. After preincubation, a portion (2 µl) of
solution from each tube was combined, and the residual NER activity of
XPC was measured in the complementing assay. A, schematic
representation for the heat inactivation and XPC-complementing assays.
The XPC-complementing assay was carried out under standard conditions
using UV-irradiated SV40 minichromosomes as described under
"Experimental Procedures." B, an autoradiogram of the
gel. C, incorporation of dCMP into SV40 minichromosomes was
calculated, and the results are represented in graph form. Symbols:
diamond, XPC was preincubated in the absence of both
CEN2 and HR23B; triangle, in the presence of CEN2;
square, in the presence of HR23B; circle, in the
presence of both CEN2 and HR23B. Averages and experimental errors were
calculated from two independent experiments.
View larger version (34K):
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Fig. 6.
Loss of damaged DNA binding activity by
heat-inactivation. Various combinations of recombinant proteins of
the XPC complex were preincubated at various temperatures (0, 30, 37, 42, and 50 °C) for 1 h, and residual binding activity to
damaged DNA was measured. XPC (50 nM) with or without 500 nM CEN2 and/or 500 nM HR23B were placed in one
tube, and the omitted proteins were placed in a second tube. After
preincubation, a portion (4 µl) of solution from each tube was
combined, and the residual amount of XPC binding activity to damaged
DNA was measured. A, schematic representation of the heat
inactivation and DNA binding assays. The damaged DNA binding assay was
carried out under standard conditions (see "Experimental
Procedures") using UV-irradiated (500 J/m2)
(UV+) or unirradiated (UV ) plasmid DNA. After
incubation at 30 °C for 30 min, plasmid DNA bound to XPC was
co-immunoprecipitated with anti-XPC antibody. Plasmid DNA purified from
the precipitates was linearized by BamHI digestion,
subjected to 0.8% agarose gel electrophoresis, and analyzed by
Southern blot. B, autoradiogram of the blot. C,
the percentage amounts of DNA recovered by the anti-XPC antibody.
Symbols: diamond, XPC was preincubated in the absence of
both CEN2 and HR23B; triangle, in the presence of CEN2;
square, in the presence of HR23B; circle, in the
presence of both CEN2 and HR23B. Open symbols represent the
percentage amounts of UV-irradiated plasmid bound to XPC, and
solid symbols represent those of unirradiated DNA. Averages
and experimental errors were calculated from two independent
experiments.
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Fig. 7.
CEN2 stabilizes XPC. XPC (50 nM) and HR23B (500 nM) were preincubated with
or without CEN2 (500 nM), and residual XPC binding activity
to damaged DNA was measured. Preincubation was carried out at various
temperatures as indicated. A, autoradiogram of the blot.
B, the percentages of DNA recovered by the anti-XPC
antibody. Symbols: square, XPC was preincubated in the
presence of HR23B; circle, in the presence of both CEN2 and
HR23B. Open symbols represent the percentage amounts of
UV-irradiated plasmid bound to XPC, and solid symbols
represent those of unirradiated DNA. Averages and experimental errors
were calculated from two independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
17 allele, which
produces defects in SPB duplication (49). Another dominant suppressor
of Kar1-
17 is DSK2, which encodes a
protein that is very similar to Rad23 within the N-terminal UbL domain.
The double deletion mutant,
rad23
dsk2
shows temperature-sensitive lethality caused by defects in SPB
duplication, although neither UbL protein is essential for cell growth
(50). These observations suggest that RAD23 has a role that
is redundant to DSK2 in SPB duplication and that
RAD23 engages in an indirect genetic interaction with
CDC31. Our findings appear to support the yeast genetic
studies, and we suspect that Rad4 (the XPC counterpart in
S. cerevisiae) interacts with Cdc31. Therefore, the Rad4
complex might have a role in SPB duplication in yeast.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Robin P. Wharton for critical reading of the manuscript, Takafumi Maekawa, Yoshinori Watanabe, Daisuke Sakai, Rika Kusumoto, and Ayumi Yamada for technical suggestions, and Masayuki Yokoi and Hideki Hiyama for critical discussions. We also thank other members of Dr. Hanaoka's laboratory at the Institute for Molecular and Cellular Biology and RIKEN for helpful discussions.
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FOOTNOTES |
---|
* This work was supported by grants from the Ministry of Education, Science, Sports, and Culture of Japan, CREST, and the Bioarchitect Research Project of RIKEN.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.
§ A Research Fellow of the Japan Society for the Promotion of Science. Present address: Dept. of Genetics, Duke University Medical Center, P. O. Box 3657, Durham, NC 27710.
§§ To whom correspondence should be addressed. Tel.: 81-6-6879-7975; Fax: 81-6-6877-9382; E-mail: fhanaoka@imcb.osaka-u.ac.jp.
Published, JBC Papers in Press, February 27, 2001, DOI 10.1074/jbc.M100855200
2 K. Tatsuta, T. Maekawa, and F. Hanaoka, unpublished observation.
3 M. Araki, C. Masutani, and F. Hanaoka, unpublished observations.
4 J. H. J. Hoeijmakers, personal communication.
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ABBREVIATIONS |
---|
The abbreviations used are: SPB, spindle pole body; NER, nucleotide excision repair; XP, xeroderma pigmentosum; UbL, ubiquitin-like; RPA, replication protein A; CSA, Cockayne's syndrome A protein; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction.
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