(Received for publication, October 3, 1996)
From the Department of Biochemistry and Biophysics,
University of North Carolina School of Medicine, Chapel Hill, North
Carolina 27599 and the § Biology and Biotechnology Research
Program, Lawrence Livermore National Laboratory,
Livermore, California 94551
The human XPF-ERCC1 protein complex is one of
several factors known to be required for general nucleotide excision
repair. Genetic data indicate that both proteins of this complex are
necessary for the repair of interstrand cross-links, perhaps via
recombination. To determine whether XPF-ERCC1 completes a set of six
proteins that are sufficient to carry out excision repair, the human
XPF and ERCC1 cDNAs were coexpressed in
Sf21 insect cells from a baculovirus vector. The purified complex
contained the anticipated 5 junction-specific endonuclease activity
that is stimulated through a direct interaction between XPF and
replication protein A (RPA). The recombinant complex also complemented
extracts of XP-F cells and Chinese hamster ovary mutants assigned to
complementation groups 1, 4, and 11. Furthermore, reconstitution of the
human excision nuclease was observed with a mixture of five repair
factors (XPA, XPC, XPG, TFIIH, and RPA) and the recombinant XPF-ERCC1,
thus verifying that no additional protein factors are needed for the
specific dual incisions characteristic of human excision repair.
Nucleotide excision repair in humans consists of dual incisions on both sides of the lesion in the damaged strand, which results in excision of 24-32-nucleotide-long oligomers followed by repair synthesis and ligation (1-3). The enzyme system responsible for the dual incisions is referred to as excision nuclease (2). Individuals lacking excision nuclease suffer from xeroderma pigmentosum (XP),1 a disease that is characterized by photodermatoses and in some cases by neurological abnormalities (4, 5).
Recently, human excision nuclease has been reconstituted from highly
purified proteins including those encoded by XPA through XPG (6-8). These reconstitutions have helped define the
minimum essential set of proteins for the dual incision/excision
activity. It was found that all proteins encoded by the XP genes with
the exception of the XPE gene product were required for excision. Other
proteins necessary for this reaction pathway are replication protein A
(RPA, consisting of three subunits), transcription factor TFIIH
(composed of 5-8 subunits including XPB and XPD), and ERCC1 (which
together with XPF forms a distinct complex). These studies were
conducted with proteins purified mostly from human cells; therefore, a
requirement for additional proteins that were present in the
reconstitution fractions as "contaminants" was a realistic possibility. The need for additional unknown proteins was argued using
evidence that a factor (IF7) of one or more proteins that purified
through several columns with XPF-ERCC1 and finally separated from this
complex by a DEAE resin was essential for incision specificity by the
reconstituted human excision nuclease (8). However, a later report with
proteins purified to apparent homogeneity failed to confirm the need
for an additional factor (7). To clarify the discrepancy raised by
these studies, we attempted to use proteins expressed and purified from
heterologous systems because the presence of trace amounts of IF7
cannot be excluded with the most extensively purified human cellular
preparations of XPF-ERCC1. The recent cloning of XPF
(ERCC4) (9) and subsequent isolation of its cDNA
(10, 11) provide the opportunity to produce the recombinant XPF-ERCC1
complex in a surrogate host. Toward this goal, we expressed the
XPF-ERCC1 complex in abundance using a baculovirus vector, purified the
complex to homogeneity, and verified its activity as a
junction-specific endonuclease (12) that incises in the 5 direction of
a DNA lesion (7). Recombinant XPF-ERCC1, referred to hereafter as
r(XPF-ERCC1), also complemented extracts of XP-F cells and hamster
mutants in complementation groups 1, 4, and 11. The complex is fully
capable of reconstituting human excision nuclease when combined with
TFIIH, XPC, and recombinant XPA, RPA, and XPG proteins. Our data show that only these six factors are needed for damage-specific dual incision by human excision nuclease.
The NcoI-BamHI fragment of the XPF cDNA encoding the N-terminal 260 amino acids of XPF was expressed from pET26b (Novagen) in Escherichia coli BL21(DE3) and purified as described previously (10). This polypeptide antigen was used to produce mouse polyclonal antibodies. From one of the positive mice, a monoclonal cell line (6D12) was identified and subcloned by standard procedures at the University of California Berkeley Hybridoma Facility.
Plasmid ConstructsFor the overexpression of the recombinant XPF-ERCC1 complex in insect cells, both cDNAs were inserted into the p2Bac vector (Invitrogen). First, the SacI-HindIII fragment of the pMal-ERCC1 construct (13) containing the ERCC1 open reading frame (ORF) was placed behind the PH promoter of p2Bac, resulting in the plasmid p2Bac-E1. The ApaI-XbaI fragment of pcER4-34 containing the human XPF ORF (10) was then inserted into p2Bac-E1, placing XPF under the control of the P10 promoter and creating the final expression construct p2Bac-FE1. Recombinant baculovirus was obtained by standard methods (14) using the BaculoGold transfection kit (PharMingen).
For in vitro transcription-translation, the ERCC1
and XPF ORFs were inserted into the pIBI24 vector (IBI). In
order to create the appropriate restriction sites for subcloning of
XPF, 974 bp encoding the N-terminal one-third of the ORF was
amplified from the 5-end of pcER4-34 using the following polymerase
chain reaction primers:
5
-CGCGAATTCAAGGATATAGCTCTAGAGATGGAGTCAGGCAGCCGGCTCGACGGATT and 5
-AAACAGCCAACCTGAATTCTGACCAAACGCTTTTTCCGTT. The
XbaI-BamHI fragment of this polymerase chain
reaction product and the BamHI-ApaI fragment
excised from pcER4-34 carrying the 3
section of the XPF
ORF were cloned into pIBI24, placing XPF under the control of the T7 promoter and creating pTB-F. The ERCC1 cDNA
was similarly placed behind the T7 promoter by inserting the
SalI-XbaI fragment of pMal-ERCC1 into pIBI24,
creating pTB-E1.
Sf21 cells (5 × 108) were infected with the baculovirus expression
construct p2Bac-FE1 at a multiplicity of infection of 20, and cells
were harvested 48 h after infection. Cell-free extract was
prepared as described previously (15) and loaded onto a 20-ml
SP-Sepharose column, and the column was developed with a 200-ml linear
gradient from 0.1 to 1.0 M KCl in Buffer A (25 mM Hepes-KOH, pH 7.9, 12 mM MgCl2,
0.5 mM EDTA, 2 mM dithiothreitol, and 15%
glycerol). The r(XPF-ERCC1) complex that eluted at about 0.4 M KCl was located with polyclonal anti-XPF antibodies (10) and polyclonal anti-ERCC1 antibodies (16). Fractions containing r(XPF-ERCC1) were combined, dialyzed against Buffer A + 100 mM KCl, and then loaded onto a 5-ml glutathione
S-transferase-XPA affinity column (17). The affinity column
was washed with 200 ml of Buffer B (25 mM Hepes-KOH, pH
7.4, 100 mM KCl, 1 mM EDTA, 10 mM
-mercaptoethanol, and 10% glycerol). r(XPF-ERCC1) complex was
eluted with 1.0 M KCl in Buffer B as indicated by both
silver staining and immunoblotting of SDS-polyacrylamide gels.
Fractions containing r(XPF-ERCC1) were collected and dialyzed against
Buffer A plus 100 mM KCl. The yield was typically 150 µg
of protein of high purity from 20 mg of total CFE.
A 3-end-labeled 90-bp duplex and a 90-bp
DNA fragment with a 30-nucleotide unpaired sequence ("bubble") in
the middle as described previously (12) were used as substrates to test
for nonspecific nuclease and junction-specific nuclease activities of
r(XPF-ERCC1), respectively. The reaction mixture (7.5 µl) contained 5 fmol of DNA and the indicated amount of r(XPF-ERCC1) in nuclease buffer
(25 mM Hepes-KOH, pH 7.9, 10 mM KCl, 3 mM MgCl2, 1 mM dithiothreitol, 100 µg/ml bovine serum albumin, and 6.5% (v/v) glycerol). The reaction
mixture was incubated at 37 °C for 30 min, and the products were
analyzed on 8% denaturing polyacrylamide gels followed by autoradiography.
Reaction mixtures contained 50 µg of mutant CFE, 20 ng of r(XPF-ERCC1), and 25 fmol of the 140-bp cholesterol A substrate (7) in 25 µl of excision buffer (6, 7). The reaction was carried out at 30 °C for 60 min. For cross-complementation, 25 µg of each mutant CFE was mixed in 25 µl of excision buffer with substrate, and the reaction was carried out in an identical manner. Excised fragments were analyzed on 8% denaturing polyacrylamide gels. The mutant CFEs used in this study were XP-A (GM02345B), XP-F (GM08437), ERCC-1 (UV20), ERCC-3 (UV24), ERCC-4 (UV41), ERCC-5 (UV135), and ERCC-11 (UVS1).
In Vitro Transcription-Translation[35S]Methionine-labeled XPF and ERCC1 proteins were prepared by the TNT Coupled Reticulocyte Lysate System (Promega) using pTB-F and pTB-E1 as templates, either added separately or together in the reaction mixture following the manufacturer's instructions. 10 fmol of ERCC1, 4 fmol of XPF, and 4 fmol of XPF-ERCC1 were used for protein-protein interaction study.
DNA Binding AssayA 90-bp duplex was terminally labeled
with [-32P]ATP and T4 polynucleotide kinase as
described (12). To obtain UV-damaged DNA, the 90-bp duplex was
irradiated with 2500 J/m2 of 254 nm light from a germicidal
lamp at a fluence rate of 250 milliwatts/cm2. For binding
assays, 3 fmol of DNA was incubated with r(XPF-ERCC1) in Buffer A
containing 100 mM KCl for 10 min on ice. The reaction mixtures were directly analyzed on 5% native polyacrylamide gels run
in 1 × TBE buffer for 90 min at room temperature. The E. coli UvrA protein that binds specifically to UV-irradiated DNA
(18) was used as a positive control. Binding was quantified using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
To obtain a highly
purified sample of XPF-ERCC1, we constructed a baculovirus expression
vector (p2Bac-FE1) containing the cDNAs from both ERCC1
and the recently cloned XPF (from pcER4-34; Ref. 10).
Infection of Sf21 insect cells resulted in overexpression of both
proteins, which purified as a complex through SP-Sepharose followed by
XPA-affinity columns (16, 17). The use of the XPA-affinity step enabled
us to isolate 150 µg of r(XPF-ERCC1) from 20 mg of cellular protein
with greater than 95% homogeneity. This compares favorably with the
yield of <1.0 µg of protein purified from 22 g of HeLa CFE
(from a 250-liter cell culture; Ref. 17). After separating the proteins
by SDS-gel electrophoresis, analysis by silver staining verified that
the preparation was free of protein contaminants (Fig.
1). Antibodies to each of the two subunits detected
proteins from the purified preparation that migrated exactly as the
38-kDa (ERCC1) and 112-kDa (XPF) proteins extracted from HeLa cells.
This agreement indicates that neither one of the overexpressed proteins
undergoes post-translational modification in Sf21 cells, and that the
insect cells are capable of carrying out the same post-translational
modification as human cells.
Functional Properties of r(XPF-ERCC1)
Two related enzymatic
activities have been attributed to XPF-ERCC1 that help to explain its
function in excision repair. The complex incises the 5 junction of DNA
bubble and loop structures four nucleotides 5
to the junction (12) and
makes the 5
incision of the dual incision human excision nuclease (7,
19). Purified r(XPF-ERCC1) was tested for these properties. When a DNA
bubble substrate was used, r(XPF-ERCC1) made the specific cleavage
expected, and this activity was stimulated 40-50-fold by the addition
of RPA (Fig. 2A). The complex did not nick
double-stranded DNA (Fig. 2A, lanes 7 and 8). Incision
activity was severely inhibited by the anti-XPF monoclonal antibody,
demonstrating the dependence of the reaction on the XPF protein (Fig.
2B).
We also tested r(XPF-ERCC1) for complementation of excision nuclease
(20), an activity that we consider to be the most specific for the
native complex (17). r(XPF-ERCC1) was found to complement extracts from
XP-F cells and the Chinese hamster ovary mutants of complementation
groups 4 (ERCC4/XPF) and 1 (ERCC1), but not extracts from XP-A cells and groups 3 (ERCC3/XPB) and 5 (ERCC5/XPG) (Fig. 3). The recombinant protein
also complemented the Chinese hamster ovary complementation group 11 mutant (21), which is now known to be a member of group 4 (10, 11).
Protein and DNA Interactions of XPF and ERCC1
As we have
observed previously, XPF-ERCC1 complex binds tightly to XPA,
e.g. during its purification on the XPA-affinity column. Moreover, XPF-ERCC1 appears to interact with the RPA protein, since the
junction endonuclease activity is greatly stimulated by RPA (12). To
study the contribution of the two members of the complex in these
binding interactions, the proteins were expressed either singly or in
combination in an in vitro transcription-translation system
and tested for binding to XPA or RPA (Fig. 4). In
agreement with earlier reports (16, 22, 23), we found that ERCC1
(lane 1) and XPF-ERCC1 (lane 3) bound to XPA. XPF
by itself weakly adsorbed to the XPA-affinity resin (lane
2), suggesting that the association of the complex with XPA is
mediated mostly by ERCC1. In contrast, ERCC1 did not bind to RPA
(lane 5), as XPF did (lane 6), and therefore XPF
must mediate the binding of the complex to RPA (lane 7).
These results demonstrate that the binding observed previously (in
which XPF-ERCC1 bound to RPA with particularly high affinity in the presence of the DNA bubble substrate (12)), occurs through a direct
interaction between XPF and RPA.
Although XPA (24), RPA (25, 26), and XPC (27) have been found to bind
somewhat specifically to damaged DNA, the issue of damage recognition
remains one of the unsolved questions of the human excision nuclease
reaction mechanism. We tested for binding of r(XPF-ERCC1) to UV-damaged
DNA to find out if it contributed to damage recognition. However,
unlike the UvrA subunit of E. coli excision nuclease that
discriminates between damaged and undamaged DNA, r(XPF-ERCC1) bound
nearly as well to both substrates (Fig. 5). Thus, we
conclude that even though XPF-ERCC1 has an intrinsic affinity to DNA,
it does not contribute to locating lesions and in this respect is
comparable to the homologous Rad1-Rad10 protein complex of
Saccharomyces cerevisiae (28).
Reconstitution of Human Excision Nuclease with Purified Proteins and r(XPF-ERCC1)
Although significant progress has been made
recently in identifying and assigning functions to all of the proteins
involved in human excision repair (6-8) and the highly homologous
S. cerevisiae system (29, 30), the two reports on human
excision nuclease differed on an important point. Mu et al.
(6, 7) found that XPA, RPA, TFIIH, XPC, XPG, and XPF-ERCC1 were
necessary and sufficient for reconstituting human excision nuclease,
whereas Aboussekhra et al. (8) reported that, in addition to
these six proteins, an incision factor (IF7) was required. In the
latter report, this factor appeared to be tightly bound to XPF-ERCC1
and was separated from it at the last step of a 7-column purification
scheme. Therefore, the possibility remained that the nominally pure
XPF-ERCC1 complex used in other studies might contain IF7, which would
explain the discrepancy between the two reports. Hence, we reasoned
that r(XPF-ERCC1) purified from a heterologous source would be useful
for solving this problem. We combined r(XPF-ERCC1) with the other known
factors: XPA, RPA, and XPG, overexpressed and purified from E. coli or insect cells, and XPC and TFIIH, purified to
near-homogeneity from HeLa cells (7). This set of highly purified
proteins was able to carry out precise excision of an adducted
oligonucleotide, thus giving firm evidence that XPF-ERCC1 protein is
both necessary and sufficient to reconstitute human excision nuclease
when added to the other five excision repair factors (Fig.
6). Indeed, studies with the highly homologous S. cerevisiae system have arrived at the same conclusion; that is,
the yeast homologs of the six factors are necessary and sufficient for
specific incision/excision of UV-damaged DNA (29, 30).
Human excision nuclease has been reconstituted from purified repair factors and characterized in considerable detail (2, 3). However, the repair factors are of low cellular abundance, precluding detailed mechanistic studies with proteins purified from natural sources. Of the six repair factors, XPA, RPA, XPG, and XPC are currently available in recombinant forms that can be purified in abundant quantities. As reported here, the availability of the XPF cDNA (10) has enabled us to demonstrate direct interaction of XPF with RPA, a finding that may help explain how RPA stimulates the nuclease function of XPF-ERCC1 and how it may target it to its proper site of action in the excision nuclease complex. The use of recombinant protein in the reconstituted system has also confirmed that no additional factors are needed for reconstituting human excision nuclease. When the recombinant form of TFIIH becomes available, it will be possible to perform mechanistic experiments with human excision nuclease such as those that have been conducted with prokaryotic excision nuclease for many years.
We thank Dr. T. Matsunaga for recombinant XPG, Dr. D. Mu for performing the reconstitution experiment shown in Fig. 6, and David Hsu for UvrA protein and UvrB-maltose-binding protein fusion protein. We also thank Kerry Brookman for the XPF cDNA, Dr. Alex Karu (University of California Berkeley Hybridoma Facility) for expert guidance, and Mona Hwang for assistance in XPF-monoclonal antibody production.