From the Department of Biochemistry and Molecular Biology, Indiana University Cancer Center, and Walther Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana 46202
Received for publication, October 16, 2002, and in revised form, November 20, 2002
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ABSTRACT |
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XPA, XPC-hHR23B, RPA, and TFIIH all are the
damage recognition proteins essential for the early stage of nucleotide
excision repair. Nonetheless, it is not clear how these proteins work
together at the damaged DNA site. To get insight into the molecular
mechanism of damage recognition, we carried out a comprehensive
analysis on the interaction between damage recognition proteins and
their assembly on damaged DNA. XPC physically interacted with XPA, but failed to stabilize the XPA-damaged DNA complex. Instead, XPC-hHR23B was effectively displaced from the damaged DNA by the combined action
of RPA and XPA. A mutant RPA lacking the XPA interaction domain failed
to displace XPC-hHR23B from damaged DNA, suggesting that XPA and RPA
cooperate with each other to destabilize the XPC-hHR23B-damaged DNA
complex. Interestingly, the presence of hHR23B significantly increased
RPA/XPA-mediated displacement of XPC from damaged DNA, suggesting
that hHR23B may modulate the binding of XPC to damaged DNA. Together,
our results suggest that damage recognition occurs in a multistep
process such that XPC-hHR23B initiates damage recognition, which was
replaced by combined action of XPA and RPA. XPA and RPA, once forming a
complex at the damage site, would likely work with TFIIH, XPG, and
ERCC1-XPF for dual incision.
Nucleotide excision repair
(NER)1 is one of the major
repair pathways for removal of DNA damage caused by UV irradiation and a wide variety of bulky helix-distorting lesions such as cisplatin (1-4). In mammals, NER requires over 20 polypeptides, including damage
recognition and/or structure distortion factors (XPA, XPC-hHR23B, replication protein A (RPA), and a transcription factor, TFIIH), strand
separating helicases to create an open preincision complex (TFIIH
containing XPB and XPD DNA helicases), two structure-specific endonucleases (ERCC1-XPF and XPG), and the enzymes needed for gap
filling (DNA polymerase Both RPA and XPA are also known as damage recognition proteins because
they preferentially bind to cisplatin- or UV-damaged DNA (5-10). Both
proteins may also play a role in subsequent steps in NER through
interaction with other repair proteins (8, 11-15). The XPA-DNA
interaction is relatively weak and characterized by rapid dissociation,
whereas RPA formed a much more stable complex with UV-damaged DNA (16).
XPA physically interacts with RPA, which is necessary for efficient NER
action (14). Wild-type RPA, but not a mutant lacking the XPA
interaction domain, led to stabilization of XPA-damaged DNA complex,
implicating a unique role for RPA in stabilizing the XPA-damaged DNA
complex. The XPA-damaged DNA interaction is also likely necessary for
recruiting other DNA repair proteins such as XPG, ERCC1-XPF, and TFIIH
to the damaged site (8, 15, 17). RPA may also be involved in the later stage of NER, gap-filling, that requires proliferating cell nuclear antigen, replication factor C, and DNA polymerase XPC-hHR23B is a human homolog of yeast Rad4 and Rad23 proteins,
respectively, and forms a stable complex in solution. XPC-hHR23B exhibits the strongest affinity for damaged DNA (19-21), as does the
yeast counterpart, Rad4-Rad23 (22). Rad23 without Rad4 does not show
any DNA binding activity, suggesting that Rad4 is solely responsible
for recognition of damaged DNA. Rad23 is essential for XPC function in
NER and may also be necessary for the solubility of Rad4 (23).
XPC-hHR23B showed a remarkable preference to UV-damaged DNA
particularly in the presence of nondamaged competitor DNA and has been
suggested as the initiator of global genome NER (19, 24). A recent
immunohistochemistry study also strongly supports a role for XPC as a
global initiator in repair (25), while suggesting a role for XPA and
RPA as repair mediator proteins. XPC-hHR23B is also involved in the
recruitment of TFIIH to damaged DNA (26). TFIIH, once recruited, may
play a role in distinguishing the damaged strand from the nondamaged
one (27) as well as local unwinding of the damaged DNA region. TFIIH
with its DNA helicase activity likely generates a junction between
single-stranded DNA and duplex DNA that is recognized by two
structure-specific endonucleases, XPG and ERCC1-XPF, for dual incision
of damaged strand.
An ongoing challenge is to understand how DNA damage is recognized and
distinguished from nondamaged sites. In mammalian cells, XPC-hHR23B,
XPA, RPA, and TFIIH factors may all have roles in damage recognition
during the early stage of NER. In this study we carried out a
comprehensive analysis on the interaction between damage recognition
proteins and their assembly on damaged DNA. We found that XPC-hHR23B,
like RPA, physically interacted with XPA. However, the XPA-XPC
interaction, unlike the XPA-RPA interaction, failed to stabilize the
XPA-damaged DNA complex. Instead, XPA cooperates with RPA to promote
the destabilization of the XPC-hHR23B-damaged DNA interaction. This
finding supports a notion that the damage recognition process occurs in
a stepwise manner such that XPC-hHR23B initiates damage recognition,
which was replaced by the combined action of XPA and RPA.
Preparation of Platinum-induced Damaged DNA--
To study the
interaction between damaged DNA and damage recognition factors, we
constructed a duplex DNA with the cisplatin lesion at a specific site
(Fig. 1A). Oligonucleotides containing an intrastrand
(ITR-60) cross-link were prepared according to the previously described
procedure with some modifications (28). For cross-linking, the top
strand was first incubated with cisplatin (in TE, pH 8.0, 2-fold molar
excess) at 37 °C in the dark for 48 h, and then
ethanol-precipitated. The damaged DNA was purified by 15% denaturing
polyacrylamide gel electrophoresis and annealed to the bottom strand
(5-fold molar excess). The duplex DNA was purified by 15% native
polyacrylamide gel electrophoresis. Purified duplex DNA,
5'-32P-labeled at top strand, was analyzed for intrastrand
cross-link by treatment with 10% dimethyl sulfate at room temperature
for 5 min and 1 M piperidine at 90 °C for 30 min,
followed by 15% denaturing polyacrylamide gel electrophoresis (Fig.
1B).
Purification of XPA, RPA, and XPC-hHR23B--
Both RPA and
XPC-hHR23B are multiprotein complexes that can be highly expressed in
insect cells by coinfecting recombinant baculoviruses (21, 29).
RPA complex was prepared by coinfecting recombinant baculoviruses
encoding the individual subunits (29) and the XPC-hHR23B complex was
from insect cell lysates infecting a recombinant baculovirus containing
both XPC and hHR23B genes (Ref. 21; recombinant baculovirus containing
XPC and hHR23B was kindly provided by Dr. A. Sancar, University of
North Carolina, Chapel Hill, NC). A high level of histidine-tagged XPA
was expressed in Escherichia coli under control of the T7
promoter (5). Both RPA and XPA have been isolated with more than 95%
purity using a series of conventional column chromatography as
described previously (5, 13, 29) (Fig. 1C). XPC-hHR23B
complex was purified using a series of column chromatography
(UV-damaged dsDNA cellulose, phosphocellulose (P11), and
hepharin-Sepharose) (Fig. 1C, the details of XPC-hHR23B
purification will be described elsewhere). All three proteins (XPA,
RPA, and XPC-hHR23B) were functionally active in supporting in
vitro NER activity with HeLa cell extracts lacking respective
protein (data not shown). All GST fusion proteins were purified using
glutathione-Sepharose affinity column as described previously (14).
TFIIH was isolated from HeLa cell nuclear extracts using an antibody
affinity procedure described previously (31). During purification,
TFIIH was monitored by Western blot using anti-XPB and -XPD antibodies
(Santa Cruz Biotechnology Inc., Santa Cruz, CA).
GST Fusion Pull-down Assay--
The indicated amount of GST
fusion protein was incubated with glutathione-Sepharose beads (25 µl)
in 0.5% nonfat milk at 4 °C for 30 min, and washed three times with
washing buffer (50 mM Tris-HCl, pH 8.0, 1 mM
EDTA, 1 mM dithiothreitol, 10% glycerol, 0.5% Nonidet
P-40, 150 mM NaCl, 200 µg/ml bovine serum albumin). After
washing the beads 3-5 times, beads were incubated with the indicated
protein either in the presence or absence of damaged DNA for an
additional 20 min at room temperature on a rocker and then washed 5 times with washing buffer. Beads were then mixed with 25 µl of
SDS-PAGE sample buffer, heated at 95 °C for 5 min, and proteins (or
DNA) were resolved on 10% SDS-PAGE. To quantitatively measure the
damaged DNA, the gel was directly scanned on a Hitachi FMBIO II
Fluorescent Image Scanner.
SDS-PAGE and Western Blot Analysis--
Protein samples were
separated by 10% SDS-PAGE as described previously (32). Proteins were
then transferred to polyvinylidene difluoride membranes, immunoblotted
with corresponding antibodies, and detected by ECL plus Western
blotting detection reagents (Amersham Biosciences).
Protein-damaged DNA Interaction: Surface Plasmon Resonance
Analysis--
Interactions of XPA, RPA, and XPC-hHR23B with DNA were
monitored using a surface plasmon resonance biosensor instrument,
Biacore 3000 (Biacore) as described previously (16, 34). For
preparation of the biosensor surface with DNA, 5'-biotinylated 60-mer
duplex DNA (prepared in a buffer containing 10 mM sodium
acetate, pH 4.8, and 1.0 M NaCl) was manually injected onto
a streptavidin-coated surface of a BIAcore sensor chip to the desired
density in different flow cells. One flow cell was left underivatized
to allow for refractive index change correction. Proteins were diluted
in the running buffer containing 10 mM Hepes, pH 7.4, 150 mM NaCl, 2 mM MgCl2, 0.005%
polysorbate-20, and 1 mM dithiothreitol. Each experiment
was repeated at least twice to assure reproducibility.
Interaction of Damage Recognition Factors with DNA--
To get
insight into the molecular mechanism of how damage recognition proteins
are assembled and working together at the damaged DNA site, we carried
out a comprehensive analysis on the interaction of XPA, RPA, and
XPC-hHR23B with damaged DNA. Binding affinity of damage recognition
factors to a duplex DNA containing an intrastrand platinum cross-link
(ITR-60, Fig. 1) was measured using an
electrophoretic mobility shift assay. As previously reported (Refs.
1-4 and references therein), both RPA and XPC-hHR23B showed
preferential binding to the cisplatin-damaged DNA over the
nondamaged one, whereas the XPA-DNA interaction was observed only in
the presence of excess amounts (Fig.
2A). On the other hand,
surface plasmon resonance kinetic analysis indicated that XPC-hHR23B
compared with RPA exhibited much higher affinity to the damaged DNA
(Fig. 2B). This finding is in keeping with the previous
observation that XPC has a considerable preference for binding to the
damaged DNA over nondamaged competitor DNA (24, 25). XPC-hHR23B also
differs from RPA in its preferential binding to dsDNA over ssDNA (35),
whereas RPA has a much higher affinity to ssDNA than to dsDNA (Fig. 2,
A and B).
XPC Physically Interacts with XPA, but Not with RPA--
Because
all three proteins (RPA, XPA, and XPC-hHR23B) independently bind to the
damaged DNA, we examined a possibility whether all three damage
recognition proteins form a complex on the damaged DNA site (11-13).
Initially, we examined the interactions among damage recognition
factors without DNA. Because we know that XPA and RPA interact with
each other in solution as well as on damaged DNA (14, 17), our focus
was on the interaction of XPC-hHR23B with RPA or XPA (Fig.
3). GST·XPC or GST·XPC-hHR23B were
incubated with an increasing amount of XPA (or RPA), pulled down with
glutathione-Sepharose beads for co-precipitation of XPA (or RPA), and
analyzed by Western blot (Fig. 3). XPA was co-precipitated with
GST·XPC or GST·XPC-hHR23B complex, but not with GST (Fig.
3A), suggesting that XPA physically interacts with
XPC-hHR23B. In the XPA-XPC interaction, GST·XPC was almost comparable
with GST·XPC-hHR23B (Fig. 3A, lanes 3-4 versus
lanes 5 and 6), indicating that XPC not hHR23B
physically interacts with XPA. In keeping with this, hHR23B without XPC
failed to interact with XPA (data not shown). Unlike XPA, however, RPA had no physical interaction with GST·XPC-hHR23B or GST·XPC in solution (Fig. 3B).
Because both RPA and XPC interact with XPA (Refs. 11-13 and Fig. 3),
it is possible that the binding of XPC (or RPA) to XPA may
competitively exclude RPA (or XPC) from the complex. If so, we may not
be able to see a complex containing all three damage recognition
proteins. To examine this possibility, XPA was incubated with
GST·XPC-hHR23B in the presence of an increasing amount of RPA and
analyzed for the XPA-XPC interaction. Addition of a molar excess of RPA
had little or no effect on the XPA-XPC interaction as determined by the
GST·XPC-hHR23B pull-down assay (Fig. 3C), suggesting that
RPA and XPC-hHR23B may recognize two separate domains of XPA.
Nonetheless, we were not able to detect RPA in the GST·XPC pull-down
assay in the presence of XPA and RPA (Fig. 3C).
Presence of Damaged DNA Significantly Inhibits the XPA-XPC
Interaction--
The XPA-damaged DNA interaction is weak, but markedly
stimulated in the presence of a high affinity DNA-binding protein, RPA (14). The stimulatory effect of RPA on the XPA-damaged DNA interaction occurs through the RPA-XPA interaction (14, 16). Similar to RPA,
XPC-hHR23B exhibits a high affinity to damaged DNA (19-23) and
physically interacts with XPA (Fig. 3). We therefore examined whether
XPC-hHR23B affects the XPA-damaged DNA interaction. In the GST·XPA
pull-down assay, XPA had very little or no binding to the damaged DNA
(Fig. 4A, lane 2),
which was significantly stimulated by addition of RPA (Fig.
4A, lanes 3-5). In contrast, XPC-hHR23B showed
no effect on the XPA-damaged DNA interaction (Fig. 4A,
lanes 6-8). Interestingly, the stimulatory effect of RPA on
the XPA-damaged DNA interaction was markedly reduced in the presence of
XPC-hHR23B (Fig. 4A, lane 9), suggesting that XPC-hHR23B, unlike RPA, may not form a stable complex with XPA on
damaged DNA. To investigate this further, we examined the effect of
damaged DNA on the XPA-XPC interaction (Fig. 4B). XPA was
successfully co-precipitated with GST·XPC in the pull-down assay in
the absence of damaged DNA (Fig. 4B, lanes 4 and
5). In the presence of damaged DNA, however, the amount of
XPA co-precipitated with GST·XPC was significantly reduced to an
undetectable level (Fig. 4B, lanes 6 and
7), suggesting that the XPA-XPC interaction is destabilized in the presence of damaged DNA.
XPC-hHR23B Is Displaced from Damaged DNA by the Combined Action of
RPA and XPA--
Our result (Fig. 4) strongly suggests that XPA, RPA,
and XPC-hHR23B do not form a stable complex on the damaged DNA.
Instead, the damage recognition process may occur in a stepwise manner such that XPC-hHR23B interacts with damaged DNA first and then is
replaced by XPA and/or RPA. To test this, biotin-labeled
cisplatin-damaged DNA was first incubated with XPC-hHR23B, followed by
the addition of RPA (Fig. 5A,
lanes 3-5), XPA (lanes 6-8), or XPA + RPA
(lanes 9-11) to the reaction mixtures. The
streptavidin-Sepharose pull-down assay revealed that XPC-hHR23B formed
a stable complex with damaged DNA (Fig. 5A, top
panel, lane 2) and, the addition of RPA (lanes 3-5) or XPA (lanes 6-8) showed very little effect on
the XPC-hHR23B-damaged DNA interaction. In the presence of both XPA and
RPA, however, the XPC-hHR23B-damaged DNA interaction was significantly
inhibited (Fig. 5A, top panel, lanes
9-11). In contrast to XPC, the amount of XPA co-precipitated with
damaged DNA was markedly increased in the presence of RPA (Fig.
5A, second panel, lanes 9-11),
whereas the RPA-damaged DNA interaction was hardly affected by the
presence of XPA (Fig. 5A, third and fourth
panels, lanes 9-11). Together, this result suggests
that XPC-hHR23B can be effectively displaced from damaged DNA by the
combined action of RPA and XPA.
We further analyzed the effect of XPA and/or RPA on the interaction
between XPC-hHR23B and damaged DNA by measuring damaged DNA
co-precipitated with GST·XPC-hHR23B in the glutathione-Sepharose pull-down assay. Fluorescence (TET)-labeled damaged DNA was first incubated with GST·XPC-hHR23B before the addition of either XPA or
XPA + RPA and analyzed quantitatively following GST·XPC pull-down assay (Fig. 5B). Addition of both XPA and RPA drastically
reduced the amount of damaged DNA interacting with GST·XPC-hHR23B,
whereas XPA alone marginally affected the XPC-damaged DNA interaction (Fig. 5B), suggesting that both RPA and XPA are necessary to
destabilize the interaction between XPC-hHR23B and damaged DNA. Effect
of XPA and/or RPA on the GST·XPC-hHR23B-damaged DNA interaction was also analyzed by an antibody supershift assay using an anti-GST antibody (Fig. 5C). On a native gel electrophoresis,
GST·XPC-hHR23B-damaged DNA complex was identified as a distinct band
(Fig. 5C, lane 2) that was supershifted in the
presence of an anti-GST antibody (Fig. 5C, lane
3). The GST·XPC-hHR23B-damaged DNA complex (lanes 12 and 13) or its supershifted complex (lanes 14 and
15) was significantly reduced by addition of an increasing
amount of both RPA and XPA, even though the supershift may be in part
because of the reaction of GST antibody to XPA and/or RPA (Fig.
5C). This result is in keeping with Fig. 5, A and
B, indicating that XPC-hHR23B is displaced from damaged DNA
by the combined action of RPA and XPA.
hHR23B Is Necessary for XPA/RPA-mediated Displacement of
XPC from Damaged DNA--
XPC (Rad4) forms a stable complex with
hHR23B (Rad23), which appears to be essential for NER (23).
Nonetheless, damaged DNA binding activity of the XPC-hHR23B complex
belongs to the XPC subunit (23) and the exact role for hHR23B in repair
is unclear. In an effort to explore the role for hHR23B in DNA repair, we examined whether the presence of hHR23B affects RPA/XPA-mediated displacement of XPC from the damaged DNA. GST·XPC-hHR23B (or
GST·XPC) was incubated with fluorescence (TET)-labeled damaged DNA in
the presence of RPA and XPA, and the amount of damaged DNA precipitated with GST·XPC was measured (Fig.
6A). Both XPC and
XPC-hHR23B showed strong interaction with damaged DNA (Fig.
6A, lanes 2 and 5). In the presence of
RPA + XPA, however, the damaged DNA·XPC-hHR23B complex, not the
damaged DNA·XPC complex, was significantly reduced (Fig.
6A, lanes 3 and 4 versus
6 and 7), suggesting that hHR23B is somehow
involved in the displacement of XPC from damaged DNA in the presence of
RPA + XPA. To further investigate this, purified hHR23B was added to
the reaction mixtures containing GST·XPC for its effect on the
XPC-damaged DNA interaction in the presence of RPA + XPA (Fig.
6B). GST·XPC without hHR23B formed a stable complex with
damaged DNA (Fig. 6B, lane 2), which was hardly
affected by RPA + XPA (Fig. 6B, lanes 3 and
4). In contrast, addition of purified hHR23B to the reaction
mixtures significantly decreased the binding of XPC to the damaged DNA
in the presence of RPA + XPA (Fig. 6B, lanes 6 and 7). This result strongly suggests that hHR23B is
necessary for the RPA/XPA-mediated displacement of XPC from damaged
DNA.
The RPA-XPA Interaction Is Essential for the Displacement of
XPC-hHR23B from Damaged DNA--
Interaction between RPA and XPA is
not only required to stabilize the XPA-damaged DNA interaction, but
also necessary for NER activity (14, 16). Because both RPA and XPA are
essential for the displacement of XPC from damaged DNA, we examined
whether the RPA-XPA interaction is necessary for it. For this,
wild-type RPA and two RPA mutants (RPAp34C33 lacking the C terminus of
p34 subunit (XPA interaction domain); ZFM4, a mutant with cysteine to
alanine substitution at the zinc finger domain of the p70 subunit) were
compared in the displacement of XPC-hHR23B from damaged DNA. Although
both mutants poorly supported NER activity in vitro (14, 36), ZFM4 supported the displacement of XPC-hHR23B from damaged DNA,
whereas RPAp34C33 did not (Fig. 7). This
result not only suggests that RPA and XPA cooperate with each other in
the displacement of XPC-hHR23B from damaged DNA, but also supports that
the RPA-XPA interaction may be necessary for such cooperation.
Displacement of XPC-hHR23B from Damaged DNA Is Not
Affected by the Order of Protein Assembly on Damaged DNA--
Our
finding that XPA and RPA cooperate with each other to displace
XPC-hHR23B from the damaged DNA supports a notion that XPC-hHR23B is
the initiator of global genome NER (19, 23, 24). To investigate this
further, we examined whether the order of protein assembly at the
damaged DNA site affects the XPC-hHR23B-damaged DNA interaction (Fig.
8). The 5'-fluorescence (TET)-labeled
damaged DNA (ITR-60) was incubated with GST·XPC-hHR23B (Fig. 8,
lanes 3-5), XPA + RPA (Fig. 8, lanes 6-8), or
all three proteins (Fig. 8, lanes 9-11), and 20 min later,
the remaining factor(s) were added to the reaction mixtures. The
mixtures were then analyzed by the GST·XPC pull-down assay for the
interaction of GST·XPC-hHR23B with damaged DNA. Regardless of the
order of assembly, all assembly groups showed remarkably similar
patterns in that the addition of increasing amounts of XPA + RPA
proportionally displaced XPC-hHR23B from damaged DNA (Fig. 8). This
result not only supports the multistep damage recognition in NER, but
is also in keeping with a notion that XPC-hHR23B is the global
initiator in the damage recognition process.
Recognition of damaged DNA is a complex process involving a number
of proteins (XPA, RPA, XPC-hHR23B, and TFIIH), all of which can
independently bind to the damaged DNA (Refs. 1-4 and references therein). Although some biochemical properties of damage recognition proteins are known, the molecular mechanism of how these proteins function at the damaged DNA site is not clear. In this study we carried
out a comprehensive biochemical analysis on the interaction of damage
recognition factors themselves and with damaged DNA.
XPC as a Global Initiator in NER--
Recent in vitro
studies strongly point to a role for XPC-hHR23B as the initiator of
global genomic repair (25). This is primarily based on the findings
that: 1) preincubation of UV-damaged plasmid DNA with XPC was
preferentially repaired in an in vitro kinetic experiment,
and 2) XPC shows a considerable preference for binding to UV-damaged
DNA in the presence of nondamaged competitor DNA (24, 25). On the other
hand, a separate in vitro study demonstrated that
preincubation of damaged DNA with RPA and XPA, compared with that of
XPC-hHR23B, led to a faster repair (10).
From the study described here and the previous studies by others (9,
16, 19-23), it is evident that XPC-hHR23B and RPA share basic
properties in damage recognition: 1) preferential binding to the
damaged DNA, and 2) the physical interaction with XPA (11-13) (Fig.
3), which makes both XPC-hHR23B and RPA eligible for a global initiator
in NER (10, 19). On the other hand, these two proteins exhibit quite
different biochemical characteristics: first, XPC has a considerable
preference for binding to UV- or cisplatin-damaged DNA in the presence
of nondamaged competitor DNA (24, 25) (Fig. 2B), whereas RPA
retains only a moderate preference to damaged DNA over nondamaged DNA
(16). Second, RPA has a significant preference to ssDNA over dsDNA,
whereas XPC (or XPC-hHR23B) shows a higher affinity to dsDNA over ssDNA (35) (Fig. 2). The latter finding suggests that XPC-hHR23B functions at
an early stage of duplex DNA damage recognition, whereas RPA is
involved in a later stage of damage recognition including a structural
distortion of damaged DNA that leads to an unwinding of duplex DNA (or
a generation of ssDNA). Third, XPC-hHR23B and RPA differ in their
interaction with XPA such that the RPA-XPA interaction stabilizes weak
binding of XPA to the damaged DNA (14, 16) (Fig. 4) necessary for NER
action (14), whereas the interaction between XPC-hHR23B and XPA did not
contribute to a stability of the XPA-damaged DNA complex (Fig. 4).
Instead, the presence of XPC-hHR23B interferes with the formation of
the RPA-XPA-damaged DNA complex (Fig. 4). Moreover, the XPA-XPC
interaction may eventually lead to the displacement of XPC-hHR23B from
damaged DNA (Fig. 5). The difference between XPC-hHR23B and RPA in
their biochemical characteristics described above not only reflects their unique role(s) in the early stage of NER, but also supports the
role for XPC-hHR23B as a global initiator in NER.
Multistep Damage Recognition Process in Early Stage of
NER--
XPC physically interacted with XPA (Fig. 3), but the
interaction was significantly inhibited by the presence of damaged DNA (Fig. 4). This observation suggests that the interaction between XPA
and XPC may be necessary to recruit XPA to the damaged DNA site. The
XPC-hHR23B, once binding to the damaged DNA, likely recruits XPA (Fig.
3) and other repair factors such as TFIIH and XPG (38) to the damaged
DNA site (Fig. 9). XPC-hHR23B is also involved in the recruitment of TFIIH to damaged DNA (26). The XPA-RPA,
once introduced to the damaged DNA site, cooperates with each other to
destabilize the XPC-hHR23B-damaged DNA interaction. A recent in
vitro study also supported the absence of XPC-hHR23B in the final
incision complex (39). Displacement of XPC-hHR23B from the damaged DNA
likely requires a physical interaction between XPA and RPA on damaged
DNA because a mutant RPA lacking XPA interaction domain poorly
functioned in the displacement of XPC from damaged DNA (Fig. 7). The
XPA-RPA interaction on the damaged DNA would likely force XPC to
dissociate itself from XPA and the damaged DNA (Figs. 5 and 9). These
lines of evidence suggest that XPA, RPA, and XPC-hHR23B do not form a
stable three-protein complex at the damaged DNA site. Instead, it
supports a notion that the damage recognition process occurs in a
stepwise manner. However, we cannot rule out a possibility that XPA,
XPC-hHR23B, and RPA form a transient complex on the damaged DNA prior
to the displacement of XPC-hHR23B (Fig. 9) because RPA and XPC likely
recognize two separate domains of XPA (Fig. 3). We should also point
out that the in vitro study described here did not include
TFIIH, a key damage recognition protein involved in distinguishing the
damaged strand from the nondamaged one (27) as well as local unwinding of the damaged DNA region. The interaction between XPC and TFIIH appears to be essential for nucleotide excision repair (40). Nonetheless, addition of TFIIH appeared to have no effect on the RPA/XPA-mediated displacement of XPC-hHR23B from damaged
DNA.2
Role for hHR23B in Damage Recognition--
The XPC forms a stable
complex with hHR23B (41, 42). Although the XPC subunit is solely
responsible for the binding of the XPC-hHR23B complex to the damaged
DNA (23) and the interaction with XPA (Fig. 3), hHR23B (Rad23) is
essential for XPC function in NER (23). Very large amounts of XPC
without hHR23B showed some repair activity, but equimolar hHR23B led
about 10-fold higher activity (20). Rad23 contains a
ubiquitin-associated domain that may play a role in controlling NER
through proteosome-mediated degradation of repair factors (33, 43). In
addition, hHR23B interacts with the base excision repair protein,
N-methylpurine-DNA glycosylase, suggesting that it may have
a role in mediating various repair pathways (37). In this study we
found that hHR23B is necessary for XPA/RPA-mediated displacement of the
XPC-hHR23B complex from damaged DNA (Fig. 6). hHR23B does not directly
interact with XPA or RPA; we do not know what specific role hHR23B
plays in the displacement of XPC from damaged DNA. A recent
study showed that the DNA binding domain of XPC overlaps with the
hHR23B interaction domain (40), suggesting that hHR23B may facilitate
the displacement of XPC through the modulation of its DNA binding
activity in the presence of XPA + RPA (Fig. 3).
Physiologic significance of the displacement of XPC from damaged DNA in
NER is yet to be determined, however, it at least provides a crucial
information that damage recognition occurs in an ordered, multistep
process. Our finding that the order of protein assembly had no effect
on the displacement of XPC from damaged DNA (Fig. 8) supported a notion
that XPC-hHR23B is the initiator of global genomic repair (19, 24).
Because XPC, compared with RPA and XPA, exhibited exceptionally strong
affinity to damaged DNA (24), it is quite possible that the role for XPC-hHR23B is to effectively identify DNA damage in vivo.
RPA-XPA-mediated displacement of XPC may be necessary for the formation
of the stable XPA·RPA complex at the damaged site, which would allow a proper positioning of the endonucleases (XPG and ERCC1-XPF) for
accurate and efficient incisions. It should be pointed out, however,
that the RPA-XPA-mediated displacement of XPC from damaged DNA could be
a part of the alternative global genomic NER. For example, although
XPC-HR23B does not preferentially bind to CPD DNA (24), repair of
cis-syn cyclobutane dimer-containing DNA was dependent on
XPC-hHR23B, suggesting that there may be alternative pathways for the
global genomic NER that requires XPC-HR23B but can be replaced by XPA
and RPA (30). Further functional analysis would be necessary to
validate the role for damage recognition complex in NER action.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
, proliferating cell nuclear antigen, replication factor C, and RPA).
(or
)
(18).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Panel A, cisplatin-induced intrastrand
cross-linked DNA (60-mer; ITR-60). Underlined guanine
residues (G) are the reactive sites for platination.
Panel B, the duplex DNA with intrastrand cross-link (ITR-60)
showed slower mobility on a 15% denatured polyacrylamide gel
electrophoresis (lanes 1 versus 3). The Maxam-Gilbert
G-residue reaction (DMS + piperidine treatment) showed protection of
32P-labeled DNA from cleavage in intrastrand cross-linked
DNA (lanes 2 versus 4). Cisplatin-damaged duplex DNA was
labeled at the 5'-end of the nondamage-containing strand with
fluorescence dye (TET, synthesized from the Integrated DNA
Technologies, Coralville, IA). Panel C, SDS-PAGE and Western
blot analysis of purified XPA, RPA, and XPC and/or hHR23B. Individual
proteins were electrophoretically separated by 12% SDS-PAGE and
visualized by Coomassie Blue staining (left lane; see
"Materials and Methods" for the details) as well as by immunoblot
using the corresponding polyclonal antibody (right
lane).
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Fig. 2.
Binding characteristics of XPA, RPA, and
XPC-hHR23B to the cisplatin-damaged DNA (ITR-60). Panel A,
increasing amounts of XPC-hHR23B (top panel; lanes
1, 6, and 11, 0 ng; lanes 2,
7, and 12, 20 ng; lanes 3,
8, and 13, 40 ng; lanes 4,
9, and 14, 60 ng; lanes 5,
10, and 15, 100 ng), RPA (middle
panel; lanes 1, 6, and 11, 0 ng;
lanes 2, 7, and 12, 10 ng; lanes
3, 8, and 13, 20 ng; lanes 4,
9, and 14, 40 ng; lanes 5,
10, and 15, 80 ng), or XPA (bottom
panel; lanes 1, 6, and 11, 0 ng;
lanes 2, 7, and 12, 35 ng; lanes
3, 8, and 13, 70 ng; lanes 4,
9, and 14, 175 ng; lanes 5,
10, and 15, 350 ng) was incubated with 100 fmol
of 5'-32P-damaged duplex DNA (ITR-60; lanes
1-5), 5'-32P-nondamaged duplex DNA (lanes
6-10), or 5'-32P-ssDNA (lanes 11-15) for
15 min at room temperature. The protein-DNA complex was analyzed by 4%
polyacrylamide gel in 0.5× TBE (acrylamide:bisacrylamide = 43.2:0.8). For quantification, regions corresponding to protein-DNA
complex were excised and measured for radioactivity.
Asterisk indicates the ssDNA fragment (60-mer) generated
from ITR-60 during gel purification. Panel B, biomolecular
interaction (BIAcore) analysis of the interaction of XPC-hHR23B and RPA
with damaged DNA (ITR-60). XPC-hHR23B or RPA (25 nM) was
injected into ssDNA (SS), nondamaged duplex DNA (ND), or
cisplatin-damaged DNA (platinum; Pt) surface (50 response
units) using the KINJECT function of BIAcore 3000. The
association phase was allowed for 180 s followed by a 120-s buffer
injection period for dissociation (see "Materials and Methods" for
details).
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Fig. 3.
XPC interacts with XPA, but not with RPA.
Panel A, 2 pmol of GST·XPC or GST·XPC-hHR23B was mixed
with glutathione-Sepharose beads (25 µl) and gently rocked in the
presence of XPA (2 pmol (lanes 3 and 5) and 5 pmol (lanes 4 and 6)) at room temperature for 20 min. After incubation, beads were washed with buffer (50 mM
Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM
dithiothreitol, 10% glycerol, 0.5% Nonidet P-40, 150 mM
NaCl, 200 µg/ml bovine serum albumin), and the bound XPA was analyzed
by Western blot using an anti-XPA polyclonal antibody. Panel
B, GST·XPC or GST·XPC-hHR23B (2 pmol) was mixed with
glutathione beads (25 µl) in the presence of RPA (2 pmol (lanes
3 and 5) or 5 pmol (lanes 4 and
6)) at room temperature for 20 min. After incubation, beads
were washed with buffer and bound RPA was analyzed by Western blot
using an anti-RPA p34 polyclonal antibody. Lane 1 contained
purified RPA as a control.
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Fig. 4.
Interaction between XPC-hHR23B and XPA on
damaged DNA. Panel A, RPA not XPC stabilizes the
XPA-damaged DNA interaction. GST·XPA (2 pmol) was incubated with 2 pmol of 5'-TET-labeled cisplatin-damaged DNA (60-mer) in the presence
of increasing amounts of RPA (2, 5, and 10 pmol in lanes
3-5, respectively) or XPC-hHR23B (2, 5, and 10 pmol in
lanes 6-8, respectively). Following the GST pull-down assay
(as described in the legend to Fig. 3A), precipitates were
separated by 10% SDS-PAGE and analyzed for TET-labeled damaged DNA
using a fluorescent image scanner (Hitachi FMBIO II) (top
panel) or GST·XPA using an anti-GST antibody (bottom
panel). Panel B, the presence of damaged DNA
significantly inhibits the XPA-XPC interaction. Two pmol of GST
(lane 2) or GST·XPC-hHR23B (lanes 3-7) was
incubated with increasing amount of XPA (2 pmol in lanes 2,
4, and 7; 5 pmol in lanes 5 and
7) for 20 min at room temperature. Where indicated, 2 pmol
of cisplatin-damaged DNA (ITR-60) was included. Following the GST
pull-down, precipitates were separated (10% SDS-PAGE) and analyzed by
Western blot using an anti-XPA (top panel) or -GST
(bottom panel) antibody.
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Fig. 5.
XPC-hHR23B is displaced from the damaged DNA
by a combined action of XPA and RPA. Panel A, Western blot
analysis of individual damage recognition factors associated with
biotin-labeled damaged DNA. Biotin-labeled damaged DNA (ITR-60; 2 pmol)
was preincubated with streptavidin-Sepharose prior to the addition of
GST·XPC-hHR23B (800 ng in lanes 1-11), RPA (2, 5, and 10 pmol in lanes 3-5, respectively), XPA (2, 5, and 10 pmol in
lanes 6-8, respectively), and XPA + RPA (2, 5, and 10 pmol
each in lanes 9-11, respectively). Following the pull-down
of beads, proteins were analyzed by Western blot using an anti-GST (for
GST·XPC), -XPA, -RPAp70, or RPAp34 antibody. In lane M,
purified protein (GST·XPC, XPA, or RPA for p70 and p34) was included.
Panel B, glutathione-Sepharose beads (25 µl) were mixed
with GST·XPC-hHR23B (400 ng) and fluorescence (TET)-labeled damaged
DNA (2 pmol), and gently rocked for 20 min at room temperature. After
challenging the complex with either XPA (2, 5, and 10 pmol in
lanes 3-5, respectively) or XPA + RPA (2, 5, and 10 pmol
each in lanes 6-8, respectively), beads were pulled down
and analyzed by 10% SDS-PAGE for TET-labeled damaged DNA using
fluorescence image scanner (top panel) or Western blot using
an anti-GST antibody (bottom panel). Relative amounts of
TET-labeled damaged DNA are indicated at the bottom of the
figure. Panel C, effect of RPA and XPA on the interaction
between XPC and damaged DNA in a gel mobility shift assay.
GST·XPC-hHR23B (20 ng) was incubated with 100 fmol of
32P-platinum-damaged DNA (ITR-60) and incubated for 20 min
at room temperature. Where indicated, XPA (20 ng in lanes 4,
10, 12, and 14; 50 ng in lanes
5, 11, 13, and 15), RPA (20 ng in
lanes 7, 10, 12, and 14; 50 ng in lanes 8, 11, 13, and
15), or an anti-GST antibody (5 µl; 100 µg/ml) was added
to the mixtures and further incubated for 20 min at room temperature
prior to gel electrophoresis. The protein-DNA complex was analyzed by
4% polyacrylamide gel in 0.5× TBE (acrylamide:bisacrylamide = 43.2:0.8). GST/XPC-damaged DNA complex and its supershifted complexes
are indicated by arrows. No protein was included in
lane 1.
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Fig. 6.
hHR23B is necessary for the displacement of
XPC from damaged DNA. Panel A, XPC-hHR23B not XPC is
effectively displaced from damaged DNA. Two pmol of either GST·XPC
(lanes 2-4) or GST·XPC-hHR23B (lanes 5-7) was
mixed with glutathione-Sepharose beads (25 µl) and TET-labeled
cisplatin-damaged DNA (2 pmol) and rocked for 20 min at room
temperature. After adding increasing amounts of RPA + XPA (2 pmol
(lanes 3 and 6) or 5 pmol (lanes 1,
4, and 7)), beads were washed and analyzed for
TET-labeled cisplatin-damaged DNA by 10% SDS-PAGE. Relative amounts of
TET-labeled DNA are indicated at the bottom of the figure.
Panel B, hHR23B stimulates XPA/RPA-mediated displacement of
XPC from the damaged DNA. Two pmol of GST·XPC (lanes 2-7)
or GST·XPC-hHR23B (lanes 8-10) was mixed with glutathione
beads (25 µl) and TET-labeled damaged DNA (2 pmol), and where
indicated, 2 pmol of purified hHR23B was included. After rocking for 20 min at room temperature, equimolar amounts
of RPA + XPA (2 pmol (lanes 3, 6, and
9) or 5 pmol (lanes 4, 7, and
10)) were added to the mixtures, and beads were pulled down
and analyzed for TET-labeled damaged DNA by 10% SDS-PAGE. Relative
amounts of TET-labeled DNA are indicated at the bottom of
the figure.
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Fig. 7.
A mutant RPA lacking the XPA interaction
domain did not support the displacement of XPC from damaged DNA.
GST·XPC-hHR23B (400 ng) was mixed with glutathione-Sepharose beads
and TET-damaged DNA (2 pmol), and rocked for 20 min at room
temperature. The mixtures were then challenged with increasing amounts
of XPA (2 pmol (lanes 3, 5, and 7) or
5 pmol (lanes 4, 6, and 8)) in the
presence of wild-type RPA (lanes 3 and 4), a
mutant lacking C terminus of p34 (lanes 5 and 6),
or a RPA zinc finger mutant (ZFM4) (lanes 7 and
8). Following the GST pull-down, beads were washed and
analyzed for TET-labeled damaged DNA by 10% SDS-PAGE. Relative amounts
of TET-labeled DNA are indicated at the bottom of the
figure.
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Fig. 8.
Displacement of XPC-hHR23B from damaged DNA
is not affected by the order of assembly of damage recognition
proteins. The components indicated inside the
boxes were incubated together for 20 min at room temperature
prior to the addition of the remaining components. Where indicated, 400 ng of GST·XPC-hHR23B, 2 pmol of TET-labeled damaged DNA, and
equimolar amounts of XPA + RPA (2 pmol (lanes 3,
6, and 9), 5 pmol (lanes 4,
7, and 10), or 10 pmol (lanes 5,
8, and 11)) were added. Following the GST
pull-down, beads were washed and analyzed for TET-labeled damaged DNA
by 10% SDS-PAGE. Relative amounts of TET-labeled DNA are indicated at
the bottom of the figure.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 9.
A proposed molecular mechanism of damage
recognition process in the early stage of nucleotide excision
repair. Transient steps are indicated with
brackets.
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ACKNOWLEDGEMENTS |
---|
We thank B. Hwang for providing purified XPC-hHR23B protein, A. Riemen for technical assistance, and A. Sancar and J. T. Reardon for XPC-hHR23B expressing recombinant baculoviruses and anti-XPC and hHR23B antibodies. We also thank C-H. Kim for assistance in earlier experiments.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant CA92111 and United States Army Grant DAMD17-00-1-0295.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.
Supported by the National Institutes of Health Postdoctoral
Fellowship F32 GM20167-01.
§ To whom correspondence should be addressed: 1044 W. Walnut St., Rm. 153, Indiana University Cancer Research Institute Building, Indianapolis, IN 46202. Tel.: 317-278-3464; Fax: 317-274-8046; E-mail: slee@iupui.edu.
Published, JBC Papers in Press, December 13, 2002, DOI 10.1074/jbc.M210603200
2 J-S. You and S-H. Lee, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: NER, nucleotide excision repair; ERCC, excision repair cross-complementing; GST, glutathione S-transferase; hHR23, human homolog of the yeast RAD23; ITR-60, intrastrand cross-linked duplex DNA of 60-mer; RPA, replication protein A; ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; TFIIH, transcription factor IIH; XP, xeroderma pigmentosum; TET, tetrachlorofluorescein.
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