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Molecular and Cellular Biology, April 2001, p. 2281-2291, Vol. 21, No. 7
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.7.2281-2291.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Strong Functional Interactions of TFIIH with XPC
and XPG in Human DNA Nucleotide Excision Repair, without a
Preassembled Repairosome
Sofia J.
Araújo,1,2
Erich A.
Nigg,3 and
Richard D.
Wood1,*
Imperial Cancer Research Fund, Clare Hall
Laboratories, South Mimms, Hertfordshire EN6
3LD,1 and MRC Centre for Developmental
Neurobiology, New Hunt's House, King's College London, Guy's
Hospital Campus, London SE1 1UL,2 United
Kingdom, and Department of Cell Biology, Max-Planck Institute
for Biochemistry, D-82152 Martinsried, Germany3
Received 6 December 2000/Returned for modification 9 January
2001/Accepted 16 January 2001
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ABSTRACT |
In mammalian cells, the core factors involved in the damage
recognition and incision steps of DNA nucleotide excision repair are
XPA, TFIIH complex, XPC-HR23B, replication protein A (RPA), XPG, and
ERCC1-XPF. Many interactions between these components have been
detected, using different physical methods, in human cells and for the
homologous factors in Saccharomyces cerevisiae. Several
human nucleotide excision repair (NER) complexes, including a
high-molecular-mass repairosome complex, have been proposed. However,
there have been no measurements of activity of any mammalian NER
protein complex isolated under native conditions. In order to assess
relative strengths of interactions between NER factors, we captured
TFIIH from cell extracts with an anti-cdk7 antibody, retaining TFIIH in
active form attached to magnetic beads. Coimmunoprecipitation of other
NER proteins was then monitored functionally in a reconstituted repair
system with purified proteins. We found that all detectable TFIIH in
gently prepared human cell extracts was present in the intact
nine-subunit form. There was no evidence for a repair complex that
contained all of the NER components. At low ionic strength TFIIH could
associate with functional amounts of each NER factor except RPA. At
physiological ionic strength, TFIIH associated with significant amounts
of XPC-HR23B and XPG but not other repair factors. The strongest
interaction was between TFIIH and XPC-HR23B, indicating a coupled role
of these proteins in early steps of repair. A panel of antibodies was
used to estimate that there are on the order of 105
molecules of each core NER factor per HeLa cell.
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INTRODUCTION |
Nucleotide excision repair
(NER) removes damage to mammalian DNA caused by UV light and some
chemical mutagens (10, 31). The complete NER reaction
involves damage recognition and formation of an open DNA structure
around the lesion, followed by dual incision, excision of an
oligonucleotide of 24 to 32 residues, and gap-filling DNA synthesis to
restore an undamaged DNA molecule. In human cells the minimal set of
components involved in performing this reaction comprises replication
protein A (RPA), XPA, XPC-HR23B, XPG, ERCC1-XPF, TFIIH, replication
factor C (RFC), PCNA, DNA polymerase 
or 
, and DNA ligase I
(2). These factors can be divided into two groups, the six
core factors necessary for damage recognition and dual incision (RPA,
XPA, XPC-HR23B, XPG, ERCC1-XPF, and TFIIH) and those for repair DNA
synthesis in the gap-filling step.
In a current NER model, the XPC-HR23B complex is likely to be the
initial damage recognition factor if lesions are situated in a
nontranscribed strand (5, 59). Subsequently, the DNA around the site of the lesion is opened asymmetrically in an
ATP-dependent manner, employing the TFIIH complex with its two
helicases, XPB and XPD (13, 14). This open DNA
complex creates the substrate for cleavage by the two
structure-specific endonucleases XPG (3' of the lesion) and ERCC1-XPF
(5' of the lesion), which cut near the junction between the single-and
the double-stranded DNA (13, 36, 43, 57). Once the
incisions have been placed, a damage-containing oligonucleotide of
approximately 24 to 32 nucleotides is released and the DNA structure is
restored by replicative DNA synthesis.
An array of different protein-protein interactions between the factors
involved in the first steps of NER has been detected by methods that
range from immunoprecipitation and affinity chromatography to
Saccharomyces cerevisiae two-hybrid systems
(3). Most investigations have been concerned only with
individual interactions between the proteins involved in NER and have
used detection methods that assess physical interactions but not
function. Complexes between NER proteins have been reported with
compositions that vary according to the isolation and the detection
methods used. For example, in S. cerevisiae, all of the core
NER factors have been detected after partial purification using
His-tagged TFIIH or Rad14 subunits, in an assembly designated a
repairosome (49, 61). An alternative view of the NER
mechanism in S. cerevisiae is that particular subcomplexes
of a few factors are sequentially assembled on DNA (16).
To date, a systematic comparison of relative strengths of interactions
has not been made. In this study we used immunoprecipitation from a
human cell extract active in NER in order to assess which core NER
proteins interact with one another most readily. Functionally relevant
interactions were analyzed by testing directly for NER activity.
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MATERIALS AND METHODS |
Immunoprecipitation.
Whole-cell extracts from lymphoblastoid
or fibroblast cells were made from approximately 109 cells
according to the method of Manley and coworkers (33) with
modifications as indicated in reference 66. Extracts had a
concentration of 20 to 40 µg of protein per µl in extract dialysis buffer (25 mM HEPES-KOH [pH 7.9], 0.1 M KCl, 17% glycerol
[vol/vol], 1 mM EDTA, 1 mM dithiothreitol, and 12 mM
MgCl2). M-450 paramagnetic Dynabeads (goat anti-mouse
immunoglobulin G [IgG]; DYNAL) were washed with extract dialysis
buffer and incubated with an anti-cdk7 monoclonal antibody (MO1-1;
Novocastra Laboratories) or an anti-XPG monoclonal antibody (8H7) at a
ratio of about 0.5 µg of antibody per 107 beads overnight
at 4°C. Cell extracts were diluted as necessary to 20 mg of protein
per ml with extract dialysis buffer and incubated with the MO1.1 beads
for 2 h at room temperature. For each 10 µg of extract protein,
1 µl (4 × 105) of beads was used. Beads were
collected on a magnetic particle concentrator (DYNAL MPC), and the
supernatant was removed for analysis. Beads were then washed three
times in 10 volumes of buffer W (25 mM HEPES-KOH [pH 7.6], 10%
glycerol, and 0.01% Triton X-100) containing the KCl concentration
indicated in the figures. Beads were resuspended in buffer W containing
50 mM KCl and used for sodium dodecyl sulfate-polyacrylamide gel
electrophoresis-immunoblot analysis or in vitro NER assays.
Immunoblotting.
Proteins were separated on sodium dodecyl
sulfate-10% polyacrylamide gels and transferred to Immobilon P
polyvinylidene difluoride (Millipore) membranes. Primary rabbit
polyclonal or mouse monoclonal antibodies were as follows: for XPA, a
1/1,000 dilution of polyclonal antibody AHP452 (Serotec), raised
against recombinant human XPA protein (30); for XPC, a
1/2,000 dilution of polyclonal antibody RW028 raised against residues
96 to 299 of human XPC protein (5); for HR23B, a 1/10,000
dilution of polyclonal antibody against Rad23 (49); for
XPG, a 1/250 dilution of monoclonal antibody 8H7 (13); for
XPB, a 1/1,000 dilution of monoclonal antibody 1B3; for cyclin H, a
1/2,000 dilution of monoclonal antibody 2D4; for p62, a 1/10,000
dilution of monoclonal antibody 3C9 (the last three were provided by
J.-M. Egly); for the RPA p34 subunit, a 1/250 dilution of monoclonal
antibody 34A (22); for XPF, a 1/3,000 dilution of
polyclonal antibody RA1 raised against residues 571 to 905 of human XPF
protein (24); and for ERCC1, a 1/1,500 dilution of
polyclonal antibody RW017 (24). The membranes were
incubated with the primary antibody for 1 to 2 h, followed by
incubation for 1 h with either a 1/25,000 dilution of
peroxidase-labeled anti-mouse IgG or a 1/50,000 dilution of
peroxidase-labeled anti-rabbit IgG (both from Sigma). Bands were
visualized by chemiluminescence (Amersham Pharmacia Biotech). The
intensity of the chemiluminescent signal was quantified using NIH Image
software after the X-ray film was scanned. Density units were plotted
against protein concentration, and the amount of each protein in the
immunoprecipitated fraction was estimated.
Dual-incision NER assay.
Reconstituted repair reactions
(mixtures, 8.5 µl) were carried out in a buffer containing 45 mM
HEPES-KOH (pH 7.8), 70 mM KCl, 7 mM MgCl2, 1 mM
dithiothreitol, 0.3 mM EDTA, 12.5% (vol/vol) glycerol, 2.5 µg of
bovine serum albumin, 0.025% (vol/vol) NP-40, and 2 mM ATP. Unless
indicated otherwise, each reconstituted reaction mixture contained 50 ng of RPA, 22.5 ng of XPA, 10 ng of the XPC-HR23B complex, 50 ng of
XPG, 20 ng of the ERCC1-XPF complex, and 1.5 µl of Hep TFIIH
(heparin-Sepharose fraction IV from HeLa cells [34]).
Following preincubation for 10 min at 30°C, 50 ng of Pt-GTG DNA
(56) was added and reaction mixtures were incubated for 90 min at 30°C. Reactions were stopped by rapid freezing. Six nanograms
of an oligonucleotide complementary to the excised DNA fragment was
added to the reaction mixture. This oligonucleotide contains four extra
G residues at the 5' end and was annealed to the excised products by
heating at 95°C and gradually cooling the mixtures to 20°C.
Excision products were radiolabeled with 0.1 U of Sequenase version 2.0 polymerase (U.S. Biochemicals) and 1 µCi of
[
-32P]dCTP (3,000 Ci/mmol), separated on a denaturing
14% polyacrylamide gel, and visualized by autoradiography and with a
phosphorimager as described previously (56).
Phosphorimager data were used to quantify the results.
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RESULTS |
Immunoprecipitation of NER proteins from HeLa cell extracts with an
antibody against TFIIH.
Our object was to search for functional
interactions between NER proteins. We thought it important to use a
minimally disruptive but specific method under conditions where all
factors were present at relative concentrations similar to those in the
cell. The starting point was a human cell extract active in NER and
with no overproduced factors. TFIIH was the initial target factor for
several reasons. TFIIH is a core component of NER (14),
and there was some physical evidence that it interacts with other core
factors such as XPA (41, 46) and XPG (21,
39). TFIIH is composed of nine subunits, designated XPB, XPD,
p62, p52, p44, p34, cdk7, cyclin H, and Mat1. The last three subunits
comprise the cdk-activating kinase (CAK) subcomplex that phosphorylates
the C-terminal domain of RNA polymerase II during transcription
(60). The MO1.1 antibody raised against the cdk7 subunit
of TFIIH was a particularly attractive tool because this antibody can
be used to isolate transcriptionally active TFIIH from HeLa cells, even
while the TFIIH is still attached to the antibody-resin beads
(44). Further, the cdk7 subunit of TFIIH is not required
for the core NER reaction, and so an antibody attached to cdk7 was
unlikely to interfere with repair (2).
TFIIH from HeLa cell extracts was immunoprecipitated by cdk7 antibodies
bound to paramagnetic beads, and the input (HeLa cell extract), the
supernatant (HeLa cell extract after immunoprecipitation), and the
beads (the TFIIH-bound fraction) were analyzed by immunoblotting using
an antibody against the p62 subunit of TFIIH. Mouse whole IgG was used
as a precipitation control. TFIIH complex can be recovered in a
specific manner from HeLa cells in this way (Fig. 1, compare lanes 6 to 9). Essentially all
of the TFIIH present in HeLa cells could be immunoprecipitated by this
antibody, showing that the great majority of TFIIH includes CAK
subunits (Fig. 1, lanes 8 and 9). With control antibody, TFIIH was
detected in the supernatant but not in the immunoprecipitated fraction
(Fig. 1, lanes 5 and 6).
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FIG. 1.
Quantitative immunoprecipitation of TFIIH from HeLa cell
extracts with a cdk7 antibody. TFIIH was immunoprecipitated from a HeLa
whole-cell extract with a cdk7 antibody and detected using monoclonal
antibody 3C9 against the p62 subunit. Lane 1 contains 5 µl of Hep
TFIIH (fraction IV from HeLa cells), and lanes 2 and 3 contain 100 and
50 µg of HeLa whole-cell extract protein, respectively. HeLa cell
extract protein (400 µg in 20 µl) was added to 40 µl of antibody
(Ab) beads. Lanes 4 to 9 contain 20 µl of the supernatant (S) and
first-wash (W) fractions and 10 µl of the bead (B) fraction. The
control antibody was mouse whole IgG.
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We asked whether the immunoprecipitated TFIIH complex was associated
with other NER factors after washing was carried out
under relatively
mild conditions (50 mM KCl). TFIIH was immunoprecipitated
from HeLa
cell extract (Fig.
2, top panel, compare
lanes 3 to
5 with lanes 6 to 8). After being washed, the TFIIH attached
to
the beads remained in the nine-subunit form and contained, for
example, the core subunits XPB and p62 as well as the CAK subunit
cyclin H (lane 9).
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FIG. 2.
Coimmunoprecipitation of TFIIH and other NER factors.
TFIIH was immunoprecipitated from a HeLa whole-cell extract, and TFIIH
bound fractions were analyzed by immunoblotting. Detection was
performed by using the indicated antibodies against proteins involved
in the first steps of NER. Lanes 1 and 2 contain pure proteins (from
top to bottom) as follows: 2.5 and 5 µl of Hep TFIIH, 22.5 and 45 ng
of XPA, 75 and 150 ng of XPC-HR23B, 125 and 250 ng of RPA, 125 and 250 ng of XPG, and 50 and 100 ng of ERCC1-XPF. Lanes 3 to 5 contain 17.5, 35, and 70 µg of HeLa cell extract protein, respectively; lanes 6 to
8 contain 17.5, 35, and 70 µg of the supernatant protein from the
same extract after immunoprecipitation (Sup); and lane 9 contains 20 µl of antibody beads (B) with the immunoprecipitate.
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The presence of other NER factors in the immunoprecipitate was assayed
by immunoblotting. In the fraction bound to the beads,
some XPA, XPC,
HR23B, XPG, and ERCC1-XPF were found (Fig.
2, lane
9). A significant
amount of RPA was not detected; only weak cross-reacting
bands were
seen, migrating at positions different from those of
the p70 and p34
subunits. The immunoblots were quantified to estimate
the fractions of
NER proteins that were immunoprecipitated (with
the amount of TFIIH
recovered from HeLa cell extracts being considered
100%). The
fractions of NER proteins (in relation to the total
amount in the HeLa
cell extract) immunoprecipitated with the anti-cdk7
antibody are shown
in Table
1. About 36% of the total XPC
and
15% of the total XPG present in HeLa cell extracts were found
in
the TFIIH fraction. The amounts of XPA and ERCC1-XPF present
in this
fraction were below 5% of the total amount present in
HeLa cells. XPC
exists in a tightly bound complex with HR23B,
but the fraction of HR23B
precipitated was only about 1%. This
amount is consistent with the
approximately 10-fold excess of
HR23B over XPC in cells, where most of
the HR23B is not bound
to XPC (
62). The immunoblotting
measurements, quantified with
standard curves, also yielded estimates
of the number of molecules
of each core NER factor per HeLa cell. It is
interesting that
in nearly all cases there are on the order of
10
5 molecules per cell, with XPC possibly being the
limiting factor
(Table
1).
Interactions between TFIIH and other NER components at low ionic
strength.
The NER activity of the immunoprecipitates was tested.
We found that the TFIIH bound to the cdk7 antibody beads was active in
an assay that detects the damaged oligonucleotides released by dual
incision during NER (Fig. 3, lanes 3 and
4). Repair was carried out in reaction mixtures including the purified
core factors XPA, XPC-HR23B, RPA, XPG, and ERCC1-XPF. Reaction mixtures
containing immunoprecipitated TFIIH had activity comparable to that of
reactions with purified TFIIH (Fig. 3, compare lane 2 with lane 3). To
test whether functionally significant amounts of other NER factors had
coprecipitated with the TFIIH complex, each purified factor was
individually omitted from a reconstituted reaction mixture containing
the immunoprecipitate. Significant dual incision, about 20 to 25% of
the full reaction, was found in the absence of either XPA, XPC-HR23B,
or XPG (Fig. 3, lanes 6 to 8). Limited repair, about 10% of the full
reaction, was found when ERCC1-XPF was omitted (Fig. 3, lane 9). This
result indicates that some XPA, XPC-HR23B, XPG, and ERCC1-XPF can bind
to TFIIH and are functional in NER after immunoprecipitates are washed
with 50 mM KCl. No dual-incision products were detected, however, when
RPA was omitted from reaction mixtures (Fig. 3, lane 5), showing that
no functionally significant amounts of this protein are present in the
immunoprecipitate.
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FIG. 3.
Functional interactions of TFIIH with other NER factors
in HeLa cell extracts. cdk7 antibody beads containing TFIIH and
associated proteins immunoprecipitated from HeLa cells were added to
dual-incision assays reconstituted with purified factors.
Protein-protein interactions were tested by omission of individual
repair factors as indicated. Lanes 1 to 4 contain XPA, RPA, XPC-HR23B,
XPG, and ERCC1-XPF, with no TFIIH (lane 1), 1.5 µl of Hep TFIIH (lane
2), or TFIIH beads (lanes 3 and 4). Excised fragments were detected by
a direct end-labeling procedure which extends the 24 to 32-nucleotide
products by 4 nucleotides. The quantification shown at the top was
normalized to lane 4, containing 3 µl of TFIIH beads. TFIIH
interactions in lanes 3 to 9 were measured on beads washed with buffer
containing 50 mM KCl. Lanes 5 to 9 contain 3 µl of beads.
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In each case in Fig.
3, lanes 6 to 9, where a single factor was
omitted, the NER activity was lower than in the reaction mixture
containing immunoprecipitate supplemented with all of the purified
proteins. This lower activity may largely be due to the fact that
in
each case, omitting the factor significantly reduced its concentration
in the reaction mixture so that it became rate limiting (Table
1).
Because the only core factor that was completely absent under these
conditions was RPA, we tested the NER activities of the
immunoprecipitates after addition of purified RPA only (Fig.
4).
The TFIIH immunoprecipitate was
indeed active for dual incision
when supplemented with RPA. A level of
NER activity was attained
(Fig.
4, lanes 4 and 5) that was similar to
the level found in
a reaction mixture reconstituted with purified
components (Fig.
4, lane 1). A severalfold-higher level of repair could
be attained
by adding the full set of purified NER proteins to the
immunoprecipitate
(Fig.
4, lane 3).
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FIG. 4.
RPA restores NER activity to HeLa immunoprecipitates
(IP). The results of dual-incision assays are presented as in Fig. 3.
Lanes 1 to 3 contain XPA, RPA, XPC-HR23B, XPG, and ERCC1-XPF, with 1.5 µl of Hep TFIIH (lane 1) or no TFIIH (lane 2). Magnetic beads (10 µl) containing TFIIH and associated factors were washed with buffer
containing 50 mM KCl and used in reconstituted dual-incision assays
either with all the other repair factors added (lane 3), with only RPA
added (lanes 4 to 5), or with no additions (lane 6). Lanes 4 and 5 contain 50 ng (0.45 pmol) and 125 ng (1.1 pmol) of RPA, respectively.
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As an independent test for interactions between these proteins, similar
experiments were performed using an antibody against
XPG protein. The
8H7 antibody can recover XPG in a form active
as a nuclease, without
inhibiting DNA repair (
8). In an immunoprecipitate
from
HeLa cell extracts with the 8H7 antibody, some XPA, XPC-HR23B,
ERCC1-XPF, and TFIIH activities were also detected (Fig.
5, lanes
5 to 8). As found with the
anti-cdk7 immunoprecipitates, there
was no activity in the absence of
RPA (Fig.
5, lane 4). Significantly,
although the relative amounts of
the recovered NER factors were
different with the 8H7 antibody (for
example, less XPC-HR23B and
more ERCC1-XPF), the same overall
functional group of factors
were present in immunoprecipitates after
the beads were washed
with buffer containing 50 mM KCl.
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FIG. 5.
Functional interactions between XPG and other NER
factors. XPG was immunoprecipitated from HeLa cell extracts, and the
8H7 antibody beads containing XPG and associated proteins were added to
reconstituted dual-incision assays. Functional interactions were tested
by omission of individual repair factors as indicated. Lanes marked
"Complete" contain all the repair factors. Lane 1 contains 45 ng of
purified recombinant XPG protein; lanes 3 to 8 contain 3 µl of XPG
magnetic beads; and lane M contains molecular size markers, with
lengths (in bases) noted at left.
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Strong interaction between TFIIH and XPC-HR23B.
The results
presented so far show that five of the core protein factors involved in
the first stages of NER could be coimmunoprecipitated after washing was
carried out with relatively mild ionic-strength buffer (50 mM KCl). The
immunoprecipitation approach offers the potential to discriminate
between stronger and weaker functional interactions by using
higher-stringency conditions. Figure 6A shows an experiment using a wash buffer containing 150 mM KCl, near
physiological ionic strength. The immunoprecipitated TFIIH remained as
active as the complex purified from HeLa cells (Fig. 6A, lanes 2 and
3). However, when each of the other NER factors was sequentially
omitted, NER activity was detected only in the absence of the XPC-HR23B
complex and in the absence of XPG (Fig. 6A, lanes 6 and 7). This
finding indicates that interactions between TFIIH and XPC-HR23B or XPG
are stronger than those between TFIIH and XPA or ERCC1-XPF. When the
salt concentration in the wash buffer was increased to 300 mM KCl, the
only remaining functionally significant interaction was with TFIIH-XPC
(Fig. 6B, lanes 5 to 9). The interaction between TFIIH and XPC was
finally disrupted by washing immunoprecipitate beads with 500 mM KCl
(Fig. 6B, lanes 12 to 16).
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FIG. 6.
Sensitivity of TFIIH interactions to ionic strength.
cdk7 antibody beads containing TFIIH and associated proteins
immunoprecipitated from HeLa cells were added to dual-incision assays
reconstituted with purified factors. Protein-protein interactions were
tested by omission of individual repair factors as indicated. (A) TFIIH
interactions on beads washed with buffer containing 150 mM KCl. Lanes 1 to 3 contain XPA, RPA, XPC-HR23B, XPG, and ERCC1-XPF, with no TFIIH
(lane 1), 1.5 µl of Hep TFIIH (lane 2), or TFIIH beads (lane 3). The
quantification shown at the top was normalized to lane 3, containing 3 µl of TFIIH beads. Lanes 4 to 8 contain 3 µl of beads. (B) TFIIH
interactions on beads washed with buffer containing 300 mM KCl (lanes 3 to 9) or 500 mM KCl (lanes 10 to 16). Lanes 1 to 4, 10, and 11 contain
XPA, RPA, XPC-HR23B, XPG, and ERCC1-XPF, with no TFIIH (lane 1), 1.5 µl of Hep TFIIH (lane 2), 1.5 µl of TFIIH beads (lanes 3 and 10),
or 3 µl of TFIIH beads (lanes 4 and 11). Lanes 5 to 16 contain 3 µl
of beads. Lane M contains molecular size markers, with lengths (in
bases) noted at left.
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Interactions between NER proteins in XPA-deficient cell
extracts.
In the experiments described above, HeLa cells were used
to examine interactions between NER proteins. In order to examine interactions in situations where one component was missing, a convenient and readily available resource is NER-defective
lymphoblastoid cell lines from XP patients. In control experiments,
TFIIH was immunoprecipitated from an extract of an NER-proficient
lymphoblastoid cell line, 705ori (45). As in the previous
experiments, it was possible to immunoprecipitate TFIIH from this cell
extract in active form (Fig. 7, lanes 2 to 4). Interactions were analyzed by separately omitting each of the
repair factors. The results were similar to those obtained with HeLa
cell extracts under the same conditions (50 mM KCl). TFIIH from
lymphoblastoid cells coimmunoprecipitated with some XPA, XPC-HR23B,
XPG, and ERCC1-XPF activities. When immunoprecipitated TFIIH was washed
with 250 mM KCl buffer, only XPC-HR23B activity was detected (Fig. 7B).
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FIG. 7.
Functional interactions of TFIIH with other NER factors
in extracts from normal lymphoblastoid cells. cdk7 magnetic beads
containing TFIIH and associated proteins immunoprecipitated from 705ori
cells were added to dual-incision assay mixtures reconstituted with
purified factors. Protein-protein interactions were tested by omission
of individual repair factors as indicated. Lanes marked "Complete"
contain all the factors. (A) TFIIH interactions on beads washed with
buffer containing 50 mM KCl. (B) TFIIH interactions on beads washed
with buffer containing 250 mM KCl. In each case, lane 2 contains 1.5 µl of Hep TFIIH; lanes 3 and 4 contain 1.5 and 3 µl of cdk7
magnetic beads, respectively; and lanes 5 to 9 contain 3 µl of
beads.
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XPA binds damaged DNA and plays a central role in NER, interacting with
many core repair factors (reviewed in reference
3).
To
study the influence of XPA in interactions between NER proteins,
we used the lymphoblastoid cell line GM2345, derived from patient
XP2OS. This cell line is completely defective in NER, showing
lower
levels of reduced-size XPA mRNA (
52) and no detectable
XPA
activity (reference
37 and D. Batty, unpublished results).
TFIIH immunoprecipitated from GM2345 lymphoblastoid cell extract
had an activity in NER similar to the activity of TFIIH purified
from
HeLa cells and normal lymphoblastoid cells (Fig.
8, lanes
2 to 4). As expected, the
immunoprecipitate was inactive when
XPA was omitted (Fig.
8, lane 6).
However, a strong interaction
between TFIIH and XPC complex was still
evident (Fig.
8, lane
7). Small amounts of XPG and ERCC1-XPF were also
coimmunoprecipitated
(Fig.
8, lanes 8 and 9).
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FIG. 8.
Functional interactions of TFIIH with other NER factors
in extracts from XP-A cells. cdk7 magnetic beads containing TFIIH and
associated proteins immunoprecipitated from GM2345 XP-A cells were
washed with buffer containing 50 mM KCl and added to dual-incision
assays reconstituted with purified factors. Protein-protein
interactions were tested by omission of individual repair factors as
indicated. Lanes marked "Complete" contain all the repair factors.
Lanes 3 to 9 contain reaction mixtures with TFIIH immunoprecipitated
from the XP-A cell extracts; lanes 10 to 16 contain reaction mixtures
with TFIIH immunoprecipitated from the XP-A extracts after addition of
1 ng of purified XPA per µg of cell extract protein and incubation
for 30 min at 30°C. Lane 2 contains 1.5 µl of Hep TFIIH, lanes 3 and 10 contain 1.5 µl of TFIIH antibody beads, and lanes 4 to 9 and
11 to 16 contain 3 µl of beads. Quantification was performed on a
phosphorimager relatively to the lane that contains 3 µl of TFIIH
beads (lane 4 for the first set of values and lane 11 for the second
set).
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To further investigate the influence of XPA on interactions between NER
components, pure recombinant XPA was incubated with
XP-A cell extract
to give an XPA concentration of 600 nM, the
same as used previously for
complementation for NER activity (
24).
Immunoprecipitation
was carried out with cdk7 antibody, and the
beads were washed with
buffer containing 50 mM KCl. In this case,
XPA immunoprecipitated with
TFIIH, indicating that an interaction
formed with recombinant XPA (Fig.
8, lane 13). Relatively more
XPA activity was found on the TFIIH
beads after incubation with
exogenous XPA in this way (compare Fig.
7A,
lane 6, with Fig.
8, lane 13). This would be the expected consequence
of a shift
in the equilibrium towards TFIIH-XPA association when excess
XPA
is present (normal cell extracts contain about 300 nM XPA).
Interactions
with the XPC complex and XPG were detected as previously
shown
(Fig.
8, lanes 14 and 15). The amounts of recovered ERCC1-XPF
complex were slightly increased. Comparison of the complexes isolated
before and after XPA complementation (lanes 9 and 16) suggests
that the
presence of XPA may provide a link between TFIIH and
ERCC1-XPF.
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DISCUSSION |
Interactions between components of human NER.
One objective of
our experiments was to look for evidence for a preassembled active NER
repairosome in human cells. The approach taken here had several
features. First, we used proteins at their native relative
concentrations in cell extracts active for repair, with no factors
being overproduced, tagged, or concentrated on a column. Second, a
gentle procedure to isolate a core factor was employed, using an
antibody against a part of TFIIH that is not necessary for repair.
Third, a functional assay for interactions was used for the first time,
and relative strengths of associations were assessed by adjusting the
ionic strength. No evidence was found for a preassembled human
repairosome complex. We did find that at low ionic strength, five of
the core factors could associate with one another (TFIIH, XPA, XPG,
XPC-HR23B, and ERCC1-XPF). With increasingly stringent washing in
higher-salt buffers, all of these interactions could be disrupted, with
the interaction between TFIIH and XPC-HR23B being the most stable and
that between TFIIH and XPG being the next most stable.
The existence of interactions of various strengths between all these
components is anticipated from the results of a study
that has looked
for pairwise interactions between them using affinity
tagging and
two-hybrid or antibody methods (
3). Many of the
pairwise
interactions have been characterized to the point of
mapping
interaction domains to defined regions of the proteins.
Thus, there are
specific contacts between components, many of
which may reflect those
contacts made between the core factors
as damage is recognized and an
incision complex assembles on a
damaged
site.
One somewhat unexpected finding of our experiments was that significant
amounts of RPA are not associated with the other factors,
even at low
ionic strength. It is known, however, that RPA can
interact with XPA
(
18,
25,
28,
35,
51,
58). This interaction
is most
significant in a ternary complex with damaged DNA (
18)
where the principal DNA contacts are made by RPA (
54). Our
results
simply indicate that in solution at normal concentrations, the
association of RPA with other repair factors is the weakest
interaction.
This was foreseeable, perhaps, from the earlier
observation that
when human whole-cell extracts are passed over a
phosphocellulose
column in buffer containing 0.1 M salt, all of the NER
recombination
and incision factors except for RPA are bound to the
matrix. RPA
quantitatively flows through the column (
55).
Some previous investigations have failed to distinguish clearly between
the tight, salt-resistant interactions that hold together
the subunits
of core factors and the looser interactions between
different core
factors. The strong interactions between XPC and
HR23B, between the
three subunits of RPA, or between ERCC1 and
XPF appear to be dominated
by hydrophobic forces, and in most
cases the associations are formed as
the subunits fold together
soon after they are synthesized. As shown
here, interactions between
the core factors are dominated by ionic
forces which nevertheless
involve specific regions of the interacting
pairs.
A consequence of not making a distinction between the strengths
of different interactions has been the occasional report that
many
human DNA repair proteins can be found together in very
high-molecular-weight
complexes, accompanied by numerous DNA
replication, transcription,
and recombination factors from other
pathways (
32,
63). Neither
the functional activity nor the
relative stability of such associations
has been
investigated.
He and Ingles (
19) loaded HeLa cell extracts onto an
affinity column made with XPA and found that unquantified amounts of
many NER proteins could be found bound to the matrix after the
column
was washed with 0.1 M salt. The XPA column may have worked
partially by
ion exchange, as this is essentially the same result
as that obtained
with a phosphocellulose column (
55). The difference
is
that in this case some RPA was bound. This would be the consequence
of
a very high local XPA concentration, which would shift the
equilibrium
to favor XPA-RPA
complexes.
At the other extreme, XPB was tagged with a hemagglutinin epitope and
TFIIH was isolated from whole-cell extracts with a hemagglutinin
antibody attached to antibody beads. Following extensive washing
with
0.1 M KCl buffer, no XPC, XPG, or any other NER factors were
detected
by immunoblotting (
64). This is consistent with the
fact
that at low concentration, XPC and XPG will eventually dissociate
from
TFIIH. It also seems possible that the use of a tagged XPB
may not
always be ideal for capturing a TFIIH-XPC interaction
under native
conditions.
We believe that most evidence points towards a mechanism for human NER
in which individual core factors come together to be
assembled at a
site of DNA damage, rather than being part of a
larger preassembled
compex. In living cells, for example, the
ERCC1-XPF factor diffuses
freely and rapidly until a site of damage
is encountered, where it
remains bound for several minutes before
dissociating upon completion
of repair. The molecular mass of
freely diffusing ERCC1-XPF matches
that of its two subunits, indicating
that ERCC1-XPF is not normally
resident in a high-molecular-weight
complex (
20).
TFIIH in cell extracts is in the nine-subunit form and active in
NER.
It is noteworthy that almost all of the TFIIH in a HeLa
whole-cell extract was immunoprecipitated with cdk7 antibody, showing that nearly 100% of the TFIIH present in the cell includes the CAK
components. We found that this nine-subunit TFIIH complex is active in
NER even when anchored to beads. TFIIH immunoprecipitated in a similar
way has also been shown to be active in transcription (44). However, several previous studies have detected
other versions of TFIIH, including a six-subunit form, a nonfunctional five-subunit form lacking XPD, and a separate complex of XPD with CAK
(1, 11, 48, 50, 67). Indeed, a six-subunit form of TFIIH
lacking the CAK subunits cdk7, cyclin H, and MAT1 can function in NER
in a reconstituted system when isolated from HeLa cells
(38) or as a recombinant protein complex (2).
By analogy with some studies of yeast, it has been suggested that this
six-subunit form of TFIIH is the one that normally functions in NER.
This is because a form of yeast TFIIH lacking the kinase components is
found preferentially associated with other NER factors in a high-molecular-mass fraction from yeast cell extracts (15,
61).
However, the current situation with regard to yeast TFIIH is as
follows. Transcriptionally active
S. cerevisiae TFIIH is
also
composed of nine subunits, each of which has an ortholog in human
TFIIH (shown in parentheses), as follows: Tfb1 (p62), Tfb2 (p55),
Tfb3
(Mat1), Tfb4 (p34), Rad3 (XPD), Ssl1 (p44), Ssl2 (XPB), Kin28
(cdk7),
and Ccl1 (cyclin H). The Kin28 and Ccl1 subunits contain
CAK kinase
activity and dissociate more readily from the other
seven subunits
(
15,
61). Upon further purification of yeast
TFIIH, the
Ssl2 and Tfb4 subunits are lost, leaving a five-subunit
core of Tfb1,
Tfb2, Tfb3, Rad3, and Ssl1 (
7). This core is
quite
different from the composition of smaller forms of human
TFIIH. The
six-subunit form of human TFIIH lacks MAT1, while its
homolog Tfb3 is,
in contrast, a tightly bound component of five-subunit
yeast TFIIH
(
7). Further, a five-subunit subcomplex of the
most firmly
associated human TFIIH subunits can be formed (
53)
comprising XPB, p62, p52, p44, and p34. Only three of these correspond
to components of the five-subunit yeast TFIIH (Tfb1, Tfb2, and
Ssl1).
These major differences seem unlikely to reflect basic alterations in
the mechanism of TFIIH action in yeast versus mammalian
cells. Instead
it seems likely that in general the different TFIIH
subcomplexes in
mammalian cells and yeast arise during purification,
as subunits
gradually dissociate. It is possible that none of
the smaller forms is
physiologically relevant. We find that the
nine-subunit form is the
predominant form in cell extracts, that
a nine-subunit form is
functional in NER (
2), and that this
form of TFIIH can
readily associate with other NER
factors.
Functional interactions of TFIIH with XPC and XPG.
Interactions detected between core components in cell extracts do not
necessarily represent preformed complexes in the cell. Instead they may
reflect the interactions between protein components which normally take
place on DNA. Nevertheless, the firmer interactions, between TFIIH and
XPC and between TFIIH and XPG, have special functional significance.
About 36% of all the XPC and 15% of all the XPG present in a HeLa
whole-cell extract is complexed with TFIIH at 50 mM KCl. For the other
NER factors, only fractions smaller than 10% were detected in the
TFIIH bound fraction. When the ionic strength was increased to the more
physiological level of 150 mM, the only functionally detectable
interactions were between TFIIH and XPC-HR23B and TFIIH and XPG. The
TFIIH-XPC complex interaction is the strongest. This is consistent with
a model of the preincision opening reaction where the XPC complex, as the primary damage recognition factor, binds to a distorted site and
then recruits TFIIH (14). In that study, both of these
factors were required to see the earliest DNA opening or "bubble"
formation around an adduct. XPC-HR23B as a primary recognition factor
may bring the TFIIH onto the DNA, where the other factors may bind and
perform opening of the DNA around the lesion. Recently, support for
this model was presented by Yokoi et al. (68), who found that although TFIIH by itself has little DNA-binding activity, XPC
could recruit TFIIH to a DNA-bound form. Further, Li et al. (30) showed that in a cell extract, the binding of XPA and
TFIIH to damaged DNA is dependent on the presence of XPC. As shown in Fig. 8, the functional TFIIH-XPC interaction is independent of the
presence of XPA and so XPA is most likely brought into the complex
after XPC and TFIIH.
The fact that TFIIH interacts more strongly with XPC and XPG than with
other NER factors probably accounts for the observation
that XPC and
XPG are sometimes found in partially purified TFIIH
preparations
(
12,
39), depending on the particular chromatography
steps. The mechanistic significance of the TFIIH-XPG interaction
is not
entirely clear. Like TFIIH, XPA, and RPA, XPG is a component
of the NER
preincision complex and must be in place before ERCC1-XPF
can act
(
8,
40). On the other hand, mutation data indicate
that
XPG has at least one additional function (
42) so the
TFIIH-XPG
interaction may be relevant to an entirely different process.
Both TFIIH and XPG also act in a separate pathway of
transcription-coupled
repair of oxidative damage (
9,
26).
It is noteworthy that
in
S. cerevisiae, the Rad4 and Rad2
proteins (yeast homologs of
XPC and XPG, respectively) can associate
with yeast TFIIH under
some conditions (
4,
17).
Sequence of events in NER in human cells.
Figure
9 shows a schematic drawing of the
interactions between TFIIH and other core factors, where interactions
are shaded according to strength. The strongest interaction is between
XPC-HR23B and TFIIH, followed by the interaction of XPG with TFIIH
complex. Weaker interactions are between XPA and ERCC1-XPF. XPA can
interact with ERCC1 (6, 19, 27, 29, 47, 51) and with TFIIH complex (41, 46). In XPA-defective cell extracts, we
detected hardly any ERCC1-XPF in the absence of XPA protein (Fig. 8).
So far no direct interaction between ERCC1-XPF and TFIIH has been found
in mammalian cells, and this is reflected in the figure. It seems
likely that encounters between the NER factors and damaged DNA
encourage the formation of new and sequential interactions and that
weaker interactions cooperate to form strong temporary associations on
DNA. During the present study, all of the interactions were studied in
extracts from nonirradiated cells. It is possible of course that other
transient NER complexes form after a cell is damaged by UV irradiation
or a chemical agent, and this is an area for future study.
View larger version (30K):
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|
FIG. 9.
Protein-protein interactions between human NER factors.
Presented is a summary of the most stable interactions between core NER
factors in human cell extracts; darker tones represent stronger
interactions with TFIIH, and lighter tones represent weaker
interactions with TFIIH, as detected by functional assays in the
present study.
|
|
| |
FOOTNOTES |
*
Corresponding author. Present address: University of
Pittsburgh Cancer Institute, S867 Scaife Hall, 3550 Terrace St.,
Pittsburgh, PA 15261. Phone: (412) 648-9248.
| |
REFERENCES |
| 1.
|
Adamczewski, J. P.,
M. Rossignol,
J. P. Tassan,
E. A. Nigg,
V. Moncollin, and J. M. Egly.
1996.
MAT1, cdk7 and cyclin H form a kinase complex which is UV light-sensitive upon association with TFIIH.
EMBO J.
15:1877-1884[Medline].
|
| 2.
|
Araújo, S. J.,
F. Tirode,
F. Coin,
H. Pospiech,
J. E. Syväoja,
M. Stucki,
U. Hübscher,
J.-M. Egly, and R. D. Wood.
2000.
Nucleotide excision repair of DNA with recombinant human proteins: definition of the minimal set of factors, active forms of TFIIH and modulation by CAK.
Genes Dev.
14:349-359[Abstract/Free Full Text].
|
| 3.
|
Araújo, S. J., and R. D. Wood.
1999.
Protein complexes in nucleotide excision repair.
Mutat. Res.
435:23-33[Medline].
|
| 4.
|
Bardwell, A. J.,
L. Bardwell,
N. Iyer,
J. Q. Svejstrup,
W. J. Feaver,
R. D. Kornberg, and E. C. Friedberg.
1994.
Yeast nucleotide excision repair proteins Rad2 and Rad4 interact with RNA polymerase II basal transcription factor b (TFIIH).
Mol. Cell. Biol.
14:3569-3576[Abstract/Free Full Text].
|
| 5.
|
Batty, D. P.,
V. R. Otrin,
A. S. Levine, and R. D. Wood.
2000.
Stable binding of human XPC-hHR23B complex to irradiated DNA confers strong discrimination for damaged sites.
J. Mol. Biol.
300:275-290[CrossRef][Medline].
|
| 6.
|
Bessho, T.,
A. Sancar,
L. H. Thompson, and M. P. Thelen.
1997.
Reconstitution of human excision nuclease with recombinant XPF-ERCC1 complex.
J. Biol. Chem.
272:3833-3837[Abstract/Free Full Text].
|
| 7.
|
Chang, W. H., and R. D. Kornberg.
2000.
Electron crystal structure of the transcription factor and DNA repair complex, core TFIIH.
Cell
102:609-613[CrossRef][Medline].
|
| 8.
|
Constantinou, A.,
D. Gunz,
E. Evans,
P. Lalle,
P. A. Bates,
R. D. Wood, and S. G. Clarkson.
1999.
Conserved residues of human XPG protein important for nuclease activity and function in nucleotide excision repair.
J. Biol. Chem.
274:5637-5648[Abstract/Free Full Text].
|
| 9.
|
Cooper, P. K.,
T. Nouspikel,
S. G. Clarkson, and S. A. Leadon.
1997.
Defective transcription-coupled repair of oxidative base damage in Cockayne Syndrome patients from XP group G.
Science
275:990-993[Abstract/Free Full Text].
|
| 10.
|
de Laat, W. L.,
N. G. J. Jaspers, and J. H. J. Hoeijmakers.
1999.
Molecular mechanism of nucleotide excision repair.
Genes Dev.
13:768-785[Free Full Text].
|
| 11.
|
Drapkin, R.,
G. Le Roy,
H. Cho,
S. Akoulitchev, and D. Reinberg.
1996.
Human cyclin-dependent kinase-activating kinase exists in three distinct complexes.
Proc. Natl. Acad. Sci. USA
93:6488-6493[Abstract/Free Full Text].
|
| 12.
|
Drapkin, R.,
J. T. Reardon,
A. Ansari,
J. C. Huang,
L. Zawel,
K. J. Ahn,
A. Sancar, and D. Reinberg.
1994.
Dual role of TFIIH in DNA excision-repair and in transcription by RNA-polymerase-II.
Nature
368:769-772[CrossRef][Medline].
|
| 13.
|
Evans, E.,
J. Fellows,
A. Coffer, and R. D. Wood.
1997.
Open complex formation around a lesion during nucleotide excision repair provides a structure for cleavage by human XPG protein.
EMBO J.
16:625-638[CrossRef][Medline].
|
| 14.
|
Evans, E.,
J. G. Moggs,
J. R. Hwang,
J.-M. Egly, and R. D. Wood.
1997.
Mechanism of open complex and dual incision formation by human nucleotide excision repair factors.
EMBO J.
16:6559-6573[CrossRef][Medline].
|
| 15.
|
Feaver, W. J.,
W. Y. Huang,
O. Gileadi,
L. Myers,
C. M. Gustafsson,
R. D. Kornberg, and E. C. Friedberg.
2000.
Subunit interactions in yeast transcription/repair factor TFIIH requirement for Tfb3 subunit in nucleotide excision repair.
J. Biol. Chem.
275:5941-5946[Abstract/Free Full Text].
|
| 16.
|
Guzder, S. N.,
P. Sung,
L. Prakash, and S. Prakash.
1996.
Nucleotide excision-repair in yeast is mediated by sequential assembly of repair factors and not by a pre-assembled repairosome.
J. Biol. Chem.
271:8903-8910[Abstract/Free Full Text].
|
| 17.
|
Habraken, Y.,
P. Sung,
S. Prakash, and L. Prakash.
1996.
Transcription factor TFIIH and DNA endonuclease Rad2 constitute yeast nucleotide excision-repair factor-3implications for nucleotide excision-repair and Cockayne-syndrome.
Proc. Natl. Acad. Sci. USA
93:10718-10722[Abstract/Free Full Text].
|
| 18.
|
He, Z.,
L. A. Henricksen,
M. S. Wold, and C. J. Ingles.
1995.
RPA involvement in the damage-recognition and incision steps of nucleotide excision repair.
Nature
374:566-569[CrossRef][Medline].
|
| 19.
|
He, Z. G., and C. J. Ingles.
1997.
Isolation of human complexes proficient in nucleotide excision repair.
Nucleic Acids Res.
25:1136-1141[Abstract/Free Full Text].
|
| 20.
|
Houtsmuller, A. B.,
S. Rademakers,
A. L. Nigg,
D. Hoogstraten,
J. H. J. Hoeijmakers, and W. Vermeulen.
1999.
Action of DNA repair endonuclease ERCC1/XPF in living cells.
Science
284:958-961[Abstract/Free Full Text].
|
| 21.
|
Iyer, N.,
M. S. Reagan,
K. J. Wu,
B. Canagarajah, and E. C. Friedberg.
1996.
Interactions involving the human RNA-polymerase-II transcription/nucleotide excision-repair complex TFIIH, the nucleotide excision repair protein XPG, and Cockayne syndrome group-B (CSB) protein.
Biochemistry
35:2157-2167[CrossRef][Medline].
|
| 22.
|
Kenny, M. K.,
U. Schlegel,
H. Furneaux, and J. Hurwitz.
1990.
The role of human single-stranded DNA binding protein and its individual subunits in simian virus 40 DNA replication.
J. Biol. Chem.
265:7693-7700[Abstract/Free Full Text].
|
| 23.
|
Kimura, H.,
Y. Tao,
R. G. Roeder, and P. R. Cook.
1999.
Quantitation of RNA polymerase II and its transcription factors in a HeLa cell: little soluble holoenzyme but significant amounts of polymerases attached to the nuclear substructure.
Mol. Cell. Biol.
19:5383-5392[Abstract/Free Full Text].
|
| 24.
|
Köberle, B.,
J. R. W. Masters,
J. A. Hartley, and R. D. Wood.
1999.
Defective repair of cisplatin-induced DNA damage caused by reduced XPA protein in testicular germ cell tumours.
Curr. Biol.
9:273-276[CrossRef][Medline].
|
| 25.
|
Lee, S. H.,
D. K. Kim, and R. Drissi.
1995.
Human xeroderma-pigmentosum group-A protein interacts with human replication protein-A and inhibits DNA-replication.
J. Biol. Chem.
270:21800-21805[Abstract/Free Full Text].
|
| 26.
|
LePage, F.,
E. E. Kwoh,
A. Avrutskaya,
A. Gentil,
S. A. Leadon,
A. Sarasin, and P. K. Cooper.
2000.
Transcription-coupled repair of 8-oxoguanine: requirement for XPG, TFIIH, and CSB and implications for Cockayne syndrome.
Cell
101:159-171[CrossRef][Medline].
|
| 27.
|
Li, L.,
S. J. Elledge,
C. A. Peterson,
E. S. Bales, and R. J. Legerski.
1994.
Specific association between the human DNA repair proteins XPA and ERCC1.
Proc. Natl. Acad. Sci. USA
91:5012-5016[Abstract/Free Full Text].
|
| 28.
|
Li, L.,
X. Y. Lu,
C. A. Peterson, and R. J. Legerski.
1995.
An interaction between the DNA repair factor XPA and replication protein A appears essential for nucleotide excision repair.
Mol. Cell. Biol.
15:5396-5402[Abstract].
|
| 29.
|
Li, L.,
C. A. Peterson,
X. Y. Lu, and R. J. Legerski.
1995.
Mutations in XPA that prevent association with ERCC1 are defective in nucleotide excision repair.
Mol. Cell. Biol.
15:1993-1998[Abstract].
|
| 30.
|
Li, R. Y.,
P. Calsou,
C. J. Jones, and B. Salles.
1998.
Interactions of the transcription/DNA repair factor TFIIH and XP repair proteins with DNA lesions in a cell-free repair assay.
J. Mol. Biol.
281:211-218[CrossRef][Medline].
|
| 31.
|
Lindahl, T., and R. D. Wood.
1999.
Quality control by DNA repair.
Science
286:1897-1905[Abstract/Free Full Text].
|
| 32.
|
Maldonado, E.,
R. Shiekhattar,
M. Sheldon,
H. Cho,
R. Drapkin,
P. Rickert,
E. Lees,
C. W. Anderson,
S. Linn, and D. Reinberg.
1996.
A human RNA-polymerase-II complex-associated with srb and DNA-repair proteins.
Nature
381:86-89[CrossRef][Medline].
|
| 33.
|
Manley, J. L.,
A. Fire,
M. Samuels, and P. A. Sharp.
1983.
In vitro transcription: whole cell extract.
Methods Enzymol.
101:568-582[Medline].
|
| 34.
|
Marinoni, J. C.,
M. E. Rossignol, and J. M. Egly.
1997.
Purification of the transcription/repair factor TFIIH and evaluation of its associated activities in vitro.
Methods
12:235-253[CrossRef][Medline].
|
| 35.
|
Matsuda, T.,
M. Saijo,
I. Kuraoka,
T. Kobayashi,
Y. Nakatsu,
A. Nagai,
T. Enjoji,
C. Masutani,
K. Sugasawa,
F. Hanaoka,
A. Yasui, and K. Tanaka.
1995.
DNA-repair protein XPA binds replication protein-A (RPA).
J. Biol. Chem.
270:4152-4157[Abstract/Free Full Text].
|
| 36.
|
Matsunaga, T.,
D. Mu,
C. H. Park,
J. T. Reardon, and A. Sancar.
1995.
Human DNA-repair excision nucleaseanalysis of the roles of the subunits involved in dual incisions by using anti-XPG and anti-ERCC1 antibodies.
J. Biol. Chem.
270:20862-20869[Abstract/Free Full Text].
|
| 37.
|
Miura, N.,
I. Miyamoto,
H. Asahina,
I. Satokata,
K. Tanaka, and Y. Okada.
1991.
Identification and characterization of XPAC protein, the gene product of the human XPAC (xeroderma pigmentosum group A complementing) gene.
J. Biol. Chem.
266:19786-19789[Abstract/Free Full Text].
|
| 38.
|
Mu, D.,
D. S. Hsu, and A. Sancar.
1996.
Reaction-mechanism of human DNA-repair excision nuclease.
J. Biol. Chem.
271:8285-8294[Abstract/Free Full Text].
|
| 39.
|
Mu, D.,
C. H. Park,
T. Matsunaga,
D. S. Hsu,
J. T. Reardon, and A. Sancar.
1995.
Reconstitution of human DNA-repair excision nuclease in a highly defined system.
J. Biol. Chem.
270:2415-2418[Abstract/Free Full Text].
|
| 40.
|
Mu, D.,
M. Wakasugi,
D. S. Hsu, and A. Sancar.
1997.
Characterization of reaction intermediates of human excision-repair nuclease.
J. Biol. Chem.
272:28971-28979[Abstract/Free Full Text].
|
| 41.
|
Nocentini, S.,
F. Coin,
M. Saijo,
K. Tanaka, and J. M. Egly.
1997.
DNA-damage recognition by XPA protein promotes efficient recruitment of transcription factor IIH.
J. Biol. Chem.
272:22991-22994[Abstract/Free Full Text].
|
| 42.
|
Nouspikel, T.,
P. Lalle,
S. A. Leadon,
P. K. Cooper, and S. G. Clarkson.
1997.
A common mutational pattern in xeroderma pigmentosum group G/Cockayne syndrome patients: implications for a second XPG function.
Proc. Natl. Acad. Sci. USA
94:3116-3121[Abstract/Free Full Text].
|
| 43.
|
O'Donovan, A.,
A. A. Davies,
J. G. Moggs,
S. C. West, and R. D. Wood.
1994.
XPG endonuclease makes the 3' incision in human DNA nucleotide excision repair.
Nature
371:432-435[CrossRef][Medline].
|
| 44.
|
Ossipow, V.,
J. P. Tassan,
E. A. Nigg, and U. Schibler.
1995.
A mammalian RNA polymerase II holoenzyme containing all components required for promoter-specific transcription initiation.
Cell
83:137-146[CrossRef][Medline].
|
| 45.
|
Otrin, V.,
I. Kuraoka,
T. Nardo,
M. McLenigan,
A. Eker,
M. Stefanini,
A. Levine, and R. 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].
|
| 46.
|
Park, C. H.,
D. Mu,
J. T. Reardon, and A. Sancar.
1995.
The general transcription-repair factor TFIIH is recruited to the excision-repair complex by the XPA protein independent of the TFIIE transcription factor.
J. Biol. Chem.
270:4896-4902[Abstract/Free Full Text].
|
| 47.
|
Park, C. H., and A. Sancar.
1994.
Formation of a ternary complex by human XPA, ERCC1, and ERCC4(XPF) excision-repair proteins.
Proc. Natl. Acad. Sci. USA
91:5017-5021[Abstract/Free Full Text].
|
| 48.
|
Reardon, J. T.,
H. Ge,
E. Gibbs,
A. Sancar,
J. Hurwitz, and Z. Q. Pan.
1996.
Isolation and characterization of 2 human transcription factor IIH (TFIIH)-related complexesERCC2/CAK and TFIIH.
Proc. Natl. Acad. Sci. USA
93:6482-6487[Abstract/Free Full Text].
|
| 49.
|
Rodriguez, K.,
J. Talamantez,
W. Huang,
S. H. Reed,
Z. Wang,
L. Chen,
W. J. Feaver,
E. C. Friedberg, and A. E. Tomkinson.
1998.
Affinity purification and partial characterization of a yeast multiprotein complex for nucleotide excision repair using histidine-tagged Rad14 protein.
J. Biol. Chem.
273:34180-34189[Abstract/Free Full Text].
|
| 50.
|
Rossignol, M.,
I. Kolb-Cheynel, and J. M. Egly.
1997.
Substrate specificity of the cdk-activating kinase (CAK) is altered upon association with TFIIH.
EMBO J.
16:1628-1637[CrossRef][Medline].
|
| 51.
|
Saijo, M.,
I. Kuraoka,
C. Masutani,
F. Hanaoka, and K. Tanaka.
1996.
Sequential binding of DNA-repair proteins RPA and ERCC1 to XPA in vitro.
Nucleic Acids Res.
24:4719-4724[Abstract/Free Full Text].
|
| 52.
|
Satokata, I.,
K. Tanaka,
N. Miura,
I. Miyamoto,
Y. Satoh,
S. Kondo, and Y. Okada.
1990.
Characterization of a splicing mutation in group-A xeroderma pigmentosum.
Proc. Natl. Acad. Sci. USA
87:9908-9912[Abstract/Free Full Text].
|
| 53.
|
Schultz, P.,
S. Fribourg,
A. Poterszman,
V. Mallouh,
D. Moras, and J. M. Egly.
2000.
Molecular structure of human TFIIH.
Cell
102:599-607[CrossRef][Medline].
|
| 54.
|
Schweizer, U.,
T. Hey,
G. Lipps, and G. Krauss.
1999.
Photocrosslinking locates a binding site for the large subunit of human replication protein A to the damaged strand of cisplatin-modified DNA.
Nucleic Acids Res.
27:3183-3189[Abstract/Free Full Text].
|
| 55.
|
Shivji, M. K. K.,
M. K. Kenny, and R. D. Wood.
1992.
Proliferating cell nuclear antigen is required for DNA excision repair.
Cell
69:367-374[CrossRef][Medline].
|
| 56.
|
Shivji, M. K. K.,
J. G. Moggs,
I. Kuraoka, and R. D. Wood.
1999.
Dual incision assays for nucleotide excision repair using DNA with a lesion at a specific site, p. 373-392.
In
D. S. Henderson (ed.), DNA repair protocols: eukaryotic systems, vol. 113. Humana Press, Totowa, N.J.
|
| 57.
|
Sijbers, A. M.,
W. L. de Laat,
R. R. Ariza,
M. Biggerstaff,
Y.-F. Wei,
J. G. Moggs,
K. C. Carter,
B. K. Shell,
E. Evans,
M. C. de Jong,
S. Rademakers,
J. de Rooij,
N. G. J. Jaspers,
J. H. J. Hoeijmakers, and R. D. Wood.
1996.
Xeroderma pigmentosum group F caused by a defect in a structure-specific DNA repair endonuclease.
Cell
86:811-822[CrossRef][Medline].
|
| 58.
|
Stigger, E.,
R. Drissi, and S. Lee.
1998.
Functional-analysis of human replication protein-A in nucleotide excision-repair.
J. Biol. Chem.
273:9337-9343[Abstract/Free Full Text].
|
| 59.
|
Sugasawa, K.,
J. M. Y. Ng,
C. Masutani,
I. S.,
P. J. van der Spek,
A. P. M. Eker,
F. Hanaoka,
D. Bootsma, and J. H. J. Hoeijmakers.
1998.
Xeroderma pigmentosum group C protein complex is the initiator of global genome nucleotide excision repair.
Mol. Cell
2:223-232[CrossRef][Medline].
|
| 60.
|
Svejstrup, J.,
P. Vichi, and J.-M. Egly.
1996.
The multiple roles of transcription/repair factor TFIIH.
Trends Biochem. Sci.
21:346-350[CrossRef][Medline].
|
| 61.
|
Svejstrup, J. Q.,
Z. Wang,
W. J. Feaver,
X. Wu,
D. A. Bushnell,
T. F. Donahue,
E. C. Friedberg, and R. D. Kornberg.
1995.
Different forms of TFIIH for transcription and DNA repair: holo-TFIIH and a nucleotide excision repairosome.
Cell
80:21-28[CrossRef][Medline].
|
| 62.
|
van der Spek, P. J.,
A. Eker,
S. Rademakers,
C. Visser,
K. Sugasawa,
C. Masutani,
F. Hanaoka,
D. Bootsma, and J. H. J. Hoeijmakers.
1996.
XPC and human homologs of Rad23intracellular localization and relationship to other nucleotide excision repair complexes.
Nucleic Acids Res.
24:2551-2559[Abstract/Free Full Text].
|
| 63.
|
Wang, Y.,
D. Cortez,
P. Yazdi,
N. Neff,
S. J. Elledge, and J. Qin.
2000.
BASC, a super complex of BRCA1-associated proteins involved in the recognition and repair of aberrant DNA structures.
Genes Dev.
14:927-939[Abstract/Free Full Text].
|
| 64.
|
Winkler, G. S.,
W. Vermeulen,
F. Coin,
J. M. Egly,
J. H. J. Hoeijmakers, and G. Weeda.
1998.
Affinity purification of human DNA-repair transcription factor TFIIH using epitope-tagged xeroderma-pigmentosum B-protein.
J. Biol. Chem.
273:1092-1098[Abstract/Free Full Text].
|
| 65.
|
Wold, M. S.
1997.
Replication protein A: a heterotrimeric single-stranded DNA binding protein required for eukaryotic DNA metabolism.
Annu. Rev. Biochem.
66:61-92[CrossRef][Medline].
|
| 66.
|
Wood, R. D.,
M. Biggerstaff, and M. K. K. Shivji.
1995.
Detection and measurement of nucleotide excision repair synthesis by mammalian cell extracts in vitro.
Methods
7:163-175.
|
| 67.
|
Yankulov, K. Y., and D. L. Bentley.
1997.
Regulation of CDK7 substrate specificity by MAT1 and TFIIH.
EMBO J.
16:1638-1646[CrossRef][Medline].
|
| 68.
|
Yokoi, M.,
C. Masutani,
T. Maekawa,
K. Sugasawa,
Y. Ohkuma, and F. Hanaoka.
2000.
The xeroderma pigmentosum group C protein complex XPC-HR23B plays an important role in the recruitment of transcription factor IIH to damaged DNA.
J. Biol. Chem.
275:9870-9875[Abstract/Free Full Text].
|
Molecular and Cellular Biology, April 2001, p. 2281-2291, Vol. 21, No. 7
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.7.2281-2291.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
