From the Department of Genetics and Microbiology,
University Medical Centre, 9 ave de Champel,
1211 Geneva 4, Switzerland, the § Imperial Cancer
Research Fund, Clare Hall Laboratories, South Mimms,
Hertfordshire EN6 3LD, United Kingdom, and the ** Biomolecular
Modelling Laboratory, Imperial Cancer Research Fund, Lincoln's Inn
Fields, London WC2A 3PX, United Kingdom
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ABSTRACT |
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The human XPG endonuclease cuts on the 3' side of
a DNA lesion during nucleotide excision repair. Mutations in XPG can
lead to the disorders xeroderma pigmentosum (XP) and Cockayne syndrome. XPG shares sequence similarities in two regions with a family of
structure-specific nucleases and exonucleases. To begin defining its
catalytic mechanism, we changed highly conserved residues and
determined the effects on the endonuclease activity of isolated XPG,
its function in open complex formation and dual incision reconstituted
with purified proteins, and its ability to restore cellular resistance
to UV light. The substitution A792V present in two XP complementation
group G (XP-G) individuals reduced but did not abolish endonuclease
activity, explaining their mild clinical phenotype. Isolated XPG
proteins with Asp-77 or Glu-791 substitutions did not cleave DNA. In
the reconstituted repair system, alanine substitutions at these
positions permitted open complex formation but were inactive for 3'
cleavage, whereas D77E and E791D proteins retained considerable
activity. The function of each mutant protein in the reconstituted
system was mirrored by its ability to restore UV resistance to XP-G
cell lines. Hydrodynamic measurements indicated that XPG exists as a
monomer in high salt conditions, but immunoprecipitation of intact and
truncated XPG proteins showed that XPG polypeptides can interact with
each other, suggesting dimerization as an element of XPG function. The
mutation results define critical residues in the catalytic center of
XPG and strongly suggest that key features of the strand cleavage
mechanism and active site structure are shared by members of the
nuclease family.
The XPG protein is a DNA endonuclease with remarkable
structure-specific properties, cleaving near the junctions between
duplex and single-stranded DNA with a defined polarity. In its
N-terminal region (N region)1
and internal region (I region), XPG shares similarity in sequence with
a family of other nucleases. These include the bacteriophage T4 RNase H
and T5 D15 proteins (1), as well as the 5' to 3' exonuclease domains of
eubacterial DNA polymerases (2-4). Eukaryotic family members include a
family of small replication and repair nucleases (mammalian FEN-1/DNase
IV, Saccharomyces cerevisiae Rad27, and
Schizosaccharomyces pombe rad2) and larger proteins (vertebrate XPG, S. cerevisiae Rad2, and S. pombe
rad13) involved in nucleotide excision repair (NER). The FEN-1 group is
active on flap structures (5), whereas the NER enzymes cleave bubbles, splayed arms, and flaps (5-7).
In human cells, XPG functions to cleave on the 3' side of a damaged
site in DNA during NER, the process that removes injuries induced by UV
light and many chemical agents. Individuals with mutations in XPG are
almost equally divided between those with the inherited syndrome
xeroderma pigmentosum (XP) and those with a combination of both XP and
Cockayne syndrome (CS). The XP phenotype includes acute sun sensitivity
with a high incidence of cancers and, in some individuals, progressive
neurological degeneration. The even more severe CS is associated with a
characteristic constellation of skeletal, neurological, and
developmental disorders. Whether an individual is affected with XP only
or with both XP and CS is correlated with the severity of the mutation
in the XPG gene and its effect on protein function
(8-10).
During the NER process, lesions are recognized and removed as part of
an oligonucleotide fragment, and the resulting gap is subsequently
filled and ligated (11-13). XPA, XPC, and the single-stranded binding
protein RPA preferentially bind to damaged DNA. XPB and XPD proteins
are the two DNA helicase subunits of TFIIH, which has a dual function
both as a core NER factor and as a basal transcription factor for RNA
polymerase II. Its helicase activities and the XPC-hHR23B heterodimer
participate in the formation of a key open intermediate required for
dual incision (14, 15). The incisions made on each side of a lesion are
made by two different DNA endonucleases. The ERCC1-XPF heterodimer
makes the 5' incision (16, 17), whereas XPG protein cuts 3' to the
lesion (6, 18). The ERCC1-XPF complex is probably recruited through
interaction with XPA and RPA proteins (6, 18), whereas XPG co-purifies
with TFIIH under some conditions (19) and has been reported to interact with several TFIIH subunits (20). Both nucleases display structure specificity because they cut bubble, splayed arm, or flap structures near the border of single-stranded and duplex DNA. ERCC1-XPF (and its
S. cerevisiae counterpart Rad10-Rad1) cuts on the strand
that leads off from the junction in the 5' To gain insight into the important residues for the NER function of
XPG, we have introduced site-directed mutations into the conserved N
and I regions. A combination of biochemical and cellular tests was then
used to measure the function of the mutated proteins. Their intrinsic
endonuclease activity was measured on a model bubble substrate, and
their repair function was assayed by their ability to form an open
preincision complex and to complete dual incision of DNA containing a
single cisplatin adduct in the presence of purified NER components. We
further tested the mutated XPG proteins for their ability to restore UV
resistance to cell lines from two different XP-G individuals. Finally,
we used two approaches to examine the possibility that XPG is capable
of multimerization. The results emphasize that XPG normally works in
the context of a repair complex with other proteins and not as an
isolated peptide. We discuss the implications of the results for the
XPG incision mechanism and XP-G clinical phenotypes.
Human Cells, Extracts, and Repair Proteins--
HeLa cells and
fibroblasts from XP-G/CS patient XPCS1RO transformed with an origin of
replication deficient SV40 (22) were cultured at 37 °C in the
presence of 5% CO2, in regular minimum Eagle's medium
(Seromed) supplemented with 5% fetal calf serum, 2 mM
L-glutamine, 100 units/ml penicillin, and 100 µg/ml
streptomycin. The LBL463 lymphoblastoid cell line was established by
Epstein-Barr virus immortalization of a frozen polymorphonuclear
lymphocyte blood sample from XP-G patient XP3BR (23). This cell line,
the XPG83 lymphoblastoid cell line from XP-G patient XP125LO (24), and
the Burkitt lymphoma Raji cell line were grown in suspension in RPMI
1640 medium (Seromed) with 10% fetal calf serum,
L-glutamine, and antibiotics. HeLa whole cell extract was
prepared as described (25). Purified recombinant XPA, RPA, and
ERCC1-XPF from Escherichia coli, XPG from baculovirus, and
XPC-hHR23B and TFIIH from HeLa cells were as previously noted (15).
Construction of XPG Mutants--
Mutations were created in
plasmid pBS-XPG, containing a full-length XPG cDNA with
67 base pairs of natural 5' leader (26), using the Sculptor in
vitro mutagenesis system (Amersham Pharmacia Biotech). Table
I lists the oligonucleotides used to
create the mutants. All were confirmed with a deaza-G/A T7 sequencing
kit (Amersham Pharmacia Biotech). Fragments containing a mutation were
excised by cleavage at nearby restriction sites and used to replace the
corresponding fragment in pBS-XPG.
For use with the vaccinia virus-based expression system, the coding and
3' untranslated regions of XPG were placed behind the U1A
leader (27) in the pGEM-3Zf(+) vector to yield clone pGEM-XPG. For
E791A, E791D, A792V, C794A, and C794S, the mutated sequences were
subcloned into pGEM-XPG using BspE1 and BsmI
sites. A BstBI-BstBI fragment was used for the
D77A and D77E substitutions. To generate the Expression of Mutated XPG Proteins--
84-mm-diameter Petri
dishes containing monolayers of HeLa cells or transformed XPCS1RO
fibroblasts were infected with 5 plaque-forming units/cell of vTF7-3,
a recombinant vaccinia virus expressing T7 RNA polymerase (28). At
1 h postinfection, the medium was replaced with a transfection mix
comprising 3 ml of minimum Eagle's medium, 5 µg of plasmid DNA (two
portions of 2.5 µg for co-expression), and 15 µl of TransfectACE
(29). After 3 h at 33 °C, an additional 7 ml of minimum
Eagle's medium supplemented with 5% fetal calf serum was added, and
incubation was continued at 33 °C. Cells were harvested 24 h
later in 1 ml of phosphate-buffered saline, centrifuged at 300 × g, and resuspended in one pellet volume of 50 mM
Tris-HCl (pH 8.0), 300 mM NaCl, 0.1% Nonidet P-40 (Buffer A) complemented with 10% glycerol, 1 mM EDTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl
fluoride, 2 µg/ml leupeptin, and 1 µg/ml pepstatin. The suspension
was incubated on ice for 30 min, and then nuclei were removed from the
cytosolic extracts by centrifugation at 14,000 × g for
20 min. For metabolic labeling, HeLa cells were infected-transfected as
described above. 20 h later, cells were washed and incubated for
1 h with minimum Eagle's medium lacking methionine and cysteine
before incubation for 4 h with 100 µCi/ml EasyTag Express
Protein labeling Mix (NEN Life Science Products) containing
L-[35S]methionine and
L-[35S]cysteine. Cells were lysed in 25 mM HEPES-KOH (pH 6.8), 10% glycerol, 2 mM
MgCl2, and 1 mM dithiothreitol (Buffer B)
containing 0.15 M KCl, 0.4% Nonidet P-40, 0.1 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, and
1 µg/ml pepstatin. Extracts were cleared as above at 14,000 × g for 20 min.
Immunoblotting, Immunopurification, and
Immunoprecipitation--
Immunoblotting was performed on cell extracts
(10-50 µg of protein), or 0-40 ng of purified XPG plus 20 µg of
bovine serum albumin (BSA) with the mouse anti-XPG monoclonal antibody
8H7 (14) essentially as described previously (10).
XPG proteins were immunopurified by mixing 4 µl of 8H7 antibody with
HeLa extracts completed to 100 µl with Buffer A. After incubation for
1 h at 4 °C, the XPG-8H7 antibody complex was coupled with 10 µl of protein A-Sepharose beads (CL-4B, Amersham Pharmacia Biotech)
for 1.5 h at 4 °C. Beads were subsequently washed twice in 500 µl of Buffer A and then twice in 500 µl of Buffer B and finally
resuspended in 20 µl of Buffer B for use in the endonuclease assay.
For wild-type XPG/ Endonuclease Assay--
The substrate was a "bubble" formed
by annealing two 90-mer oligonucleotides with a central 30-nucleotide
unpaired region (6). One strand was previously 5'-labeled with
polynucleotide kinase and [ In Vitro DNA Repair Assay--
Covalently closed circular DNA
containing a site-specific 1,3-intrastrand d(GpTpG)-cisplatin
cross-link has been described in detail (31, 32). Repair was
reconstituted in 7.5-µl reactions with purified proteins (per
reaction, 0.36 pmol of XPA, 0.04 pmol of XPC-hHR23B, 0.46 pmol of RPA,
1.5 µl of TFIIH heparin-5PW fraction, 0.06 pmol of ERCC1-XPF, and
0.35 pmol of XPG or vaccinia virus-overexpressed wild-type or mutant
XPG as indicated) in repair buffer containing 45 mM
HEPES-KOH (pH 7.8), 70 mM KCl, 7.5 mM
MgCl2, 0.9 mM dithiothreitol, 0.4 mM EDTA, 2 mM ATP, 22 mM
phosphocreatine (di-Tris salt), 0.375 µg of creatine kinase, 3.4%
glycerol, 0.02% Nonidet P-40, and 2.5 µg of BSA. Repair proteins
were preincubated for 10 min at 30 °C, 50 ng of DNA containing the
single cisplatin adduct were added, and incubation was continued for a
further 90 min. As a positive control, a reaction with HeLa whole cell
extract incubated for 30 min was usually included. Oligonucleotides
excised during repair were detected by end labeling with Sequenase as
described (32) and quantified using ImageQuant software (Molecular Dynamics).
Open Intermediate Detection--
Intermediates with
single-stranded character were detected by their sensitivity to
KMnO4 as described previously (14). Covalently closed
circular double-stranded M13 DNA was cut at an AvaII site 140 base pairs 3' to the platinum lesion, radiolabeled at the 3' end
using Klenow fragment and [32P]dTTP, and purified over a
Sephadex G50 column. The opening reactions (30 µl) contained 200 ng
of DNA or 60 µg of Hela whole cell extract, 60 µg of BSA, or
purified DNA incision proteins (per reaction, 1.44 pmol of XPA, 0.16 pmol of XPC-hHR23B, 1.84 pmol of RPA, 6.0 µl of TFIIH
heparin-Sepharose fraction IV, 0.24 pmol of ERCC1-XPF, 1.4 pmol of XPG,
and 0.05 pmol of vaccinia virus-overexpressed wild-type or mutant XPG)
in repair buffer containing 20 µM each dATP, dCTP, dGTP,
and dTTP, 40 mM phosphocreatine (di-Tris salt), 1.5 µg of
creatine kinase, 3.4% glycerol, and 10 µg of BSA. Repair proteins
were preincubated for 10 min at 30 °C. DNA was added, and after a
further 15 min of incubation at 30 °C, KMnO4 (120 mM stock) was added to a final concentration of 6 mM. After 1 min, the oxidation was stopped by adding
UV Survival Assays--
Wild-type and mutant XPG
cDNAs were cloned into the NotI site of EBO-pLPP (33) or
of EBS-PL, a similar episomal vector containing the strong SR
For the fluorescence assay, duplicate 200-µl samples were incubated
at 37 °C in 24-well plates in 1.4 ml of RPMI medium containing 10%
fetal calf serum. 48 h later, duplicate 200-µl aliquots were incubated with 10 µl of Alamar blue (Accumed), a fluorometric growth
indicator, for 4 h at 37 °C in 96-well plates. Fluorescence was
measured in a Cytofluor (Millipore) with excitation and emission wavelengths of 530 and 590 nm, respectively (35). For the thymidine uptake assay, quadruplet 200-µl samples containing 105
cells were incubated for 48 h at 37 °C in 96-well plates. 1 µCi of [3H]thymidine was then added to 25 µl medium
in each well, and incubation was continued for a further 2 h. The
plates were frozen and thawed, and the lysed cells were recovered on
glass microfiber filters. After thorough washing, the filters were
assayed for 3H content by liquid scintillation counting
(36).
Hydrodynamic Measurements--
Velocity sedimentation was used
to determine an S value for the XPG protein. 3 µg of purified XPG
(15) or 425 µg of crude protein extract from infected-transfected
HeLa cells expressing wild-type XPG were layered onto 4.4 ml of
10-30% glycerol gradients in 25 mM HEPES-KOH (pH 7.4),
0.1 mM EDTA, and 1 mM dithiothreitol (Buffer C)
containing either 0.4 or 1 M KCl. After centrifugation in a
SW60 rotor at 47,000 rpm at 4 °C for 14 h, fractions (0.175 ml)
were collected and assayed for the presence of XPG by immunoblotting using the 8H7 monoclonal antibody. For determination of the Stokes radius, 15 µg of pure XPG were applied to a fast protein liquid chromatography Superose 6 column (Amersham Pharmacia Biotech). The
column was washed at 4 °C with Buffer C containing 1 M
KCl and 20% glycerol. The resulting 0.3-ml fractions were desalted and
concentrated using Microcon filtration units (Amicon), and XPG-containing fractions were identified by gel electrophoresis and
Coomassie Blue staining. Fractions were assayed for XPG
structure-specific nuclease cleavage of a splayed arm oligonucleotide
substrate as described (15).
Modeling the XPG Active Site--
Three nucleotide cleaving
enzymes with significant sequence similarity to XPG, the structures of
which have been solved by x-ray crystallography, were superimposed:
Thermus aquaticus DNA polymerase (37), T5 D15 exonuclease
(3), and T4 RNase H (1). The superposition was entirely automatic and
not biased to any particular region; it was performed using MULTISUP, a
program based on a pairwise superposition algorithm (38). A total of 103 equivalenced C Description and Expression of Mutated XPG Proteins--
XP125LO
and XP124LO are two sibling XP-G individuals with a very mild XP
clinical presentation (41). Their paternal XPG allele
contains a premature stop codon at position 960, whereas their maternal
allele codes for valine instead of alanine at position 792 (8, 10).
Immunoblots of cell extracts from these patients have revealed the
presence of full-length XPG carrying the A792V substitution but not the
truncated XPG protein, suggesting that the latter or its message is
unstable (10). Ala-792 is located in a conserved amino acid sequence
core within the I region and is surrounded by glutamate and cysteine
residues that have been found in the active site of various DNA
nucleases (Ref. 26 and Fig. 1). To test
the potential functional significance of these residues, site-directed
mutagenesis was used to convert Glu-791 to either Ala or Asp, and
Cys-794 to either Ala or Ser. Similarly, the highly conserved Asp-77 in
the N region was changed to either Ala or Glu. The mutations were
confirmed by sequencing, and appropriate sequenced restriction
fragments were subcloned into an otherwise wild-type XPG
cDNA background to ensure the absence of any additional undesired
mutations. The cDNAs were preceded by the 5' leader for the human
U1 snRNA-associated A protein (27) to improve translational efficiency
and were cloned in a plasmid vector behind a promoter for T7 RNA
polymerase.
The corresponding XPG proteins were overexpressed in HeLa cells using a
vaccinia virus-based expression system (28). Cells were infected with a
recombinant vaccinia virus expressing T7 RNA polymerase and then were
transfected with the various mutated cDNAs. After overnight
incubation, cytosolic extracts were prepared and were examined by
immunoblotting to ensure that the wild-type and mutated XPG proteins
were equally expressed. All mutated proteins were recognized by the
anti-XPG mouse monoclonal antibody 8H7 (14) and migrated on gels at the
same position as wild-type XPG (Fig.
2A). All recombinant proteins
were strongly overexpressed compared with the endogenous HeLa XPG (Fig.
2A, compare the right lanes with lane
CSB).
A Pull-down Endonuclease Assay--
Previous studies showed that
purified XPG is capable of cleaving a DNA substrate containing a
centrally unpaired bubble of 30 nucleotides flanked by two duplex
regions of 30 base pairs, specifically at the 3' end of the unpaired
region (6, 14). Relatively pure XPG preparations are needed for this
type of assay because other nuclease activities in cruder fractions
degrade the bubble substrate. To circumvent this problem, we made use of the 8H7 monoclonal antibody to first immunopurify the overexpressed XPG proteins on protein A-Sepharose beads; the immobilized proteins were then mixed with the bubble substrate that had been 5'-labeled on
one strand. This antibody does not inhibit NER reactions in vitro (14) or the structure-specific cleavage of the bubble substrate by purified XPG in solution (data not shown). Similarly, binding of wild-type XPG to the antibody-protein A-Sepharose complex did not affect its endonuclease activity or change its substrate specificity: XPG still cut close to the 3' junction between single- and
double-stranded DNA, generating a labeled fragment of ~61 nucleotides
(Fig. 2B, lanes 4-6). The nuclease activity of the overexpressed wild-type XPG protein was very much higher than any
endogenous HeLa XPG background (Fig. 2B, compare lanes
4-6 with lanes 1-3).
The Alanine and Acidic Amino Acid Substitutions Abolish XPG
Intrinsic Endonuclease Activity--
Because all mutated XPG proteins
were immunoprecipitated by the monoclonal antibody with equal
efficiency, we were able to test their intrinsic endonuclease activity
using this pull-down assay. Conversion of the highly conserved Asp-77
and Glu-791 residues to Ala abolished endonuclease activity (Fig.
2B, lanes 7-9 and 13-15). Changing each of
these residues to the other acidic amino acid (namely D77E and E791D)
also abolished activity in this assay (Fig. 2B, lanes 10-12
and 16-18). In contrast, amino acid changes at Cys-794 only
partially reduced endonuclease activity, with C794S being slightly less
active than C794A (Fig. 2B, lanes 22-24 and
25-27). The natural A792V substitution present in XP125LO and XP124LO cells also appeared to abolish intrinsic endonuclease activity (Fig. 2B, lanes 19-21). Because wild-type XPG can
utilize either Mg2+ or Mn2+as cofactor, but not
Co2+ (30), we assayed the mutated proteins with
Mn2+ as divalent cation. This stimulated endonuclease
activity of the overexpressed wild-type XPG protein (Fig. 2C,
lanes 1-4). Proteins carrying the Asp-77 or Glu-791 substitutions
again showed no detectable activity in this assay (data not shown),
but interestingly, weak but measurable activity was found for the
A792V protein in the presence of Mn2+ (Fig. 2C, lanes
5 and 6).
The Acidic Amino Acid Substitutions, but Not the Alanine
Substitutions, Permit Dual Incision Activity in Reconstituted NER in
Vitro--
To test the activity of the various mutant XPG proteins in
NER, we used a dual incision assay with DNA containing a single cisplatin adduct, purified repair proteins (XPA, XPC-hHR23B, RPA, ERCC1-XPF, and TFIIH), and recombinant XPG produced in the vaccinia virus system. Pilot studies showed that although production of XPG
mutant proteins in HeLa cells gave the highest yields, production in
XPG-defective XPCS1RO fibroblasts was preferable as it eliminated any
residual interference from endogenous XPG activity.
The amount of vaccinia virus-expressed wild-type XPG in cytosolic
extracts was determined by comparison to known amounts of purified XPG
by immunoblotting. To determine the appropriate amounts of protein to
use in repair reactions, the wild-type vaccinia-produced XPG was
titrated in the reconstituted repair system (data not shown). Based on
this titration, we compared the activity of 1.5 ng of vaccinia-produced
wild-type and mutant XPG proteins.
Oligonucleotides excised during repair were detected by end labeling as
described (32). Vaccinia-expressed wild-type XPG (Fig.
3, lane 2) showed the same
pattern of excision products as found with baculovirus-expressed XPG
(lane 11) or HeLa whole cell extract (lane 12).
No incision products were observed when XPG was omitted (lane
1) or when an extract containing overexpressed CSB protein was
used (lane 3). The mutant XPG proteins differed in their
repair activity. The nonconservative D77A and E791A changes completely
inactivated repair function (Fig. 3, lanes 4 and
6), but the acidic substitution mutants D77E and E791D
retained considerable activity in the reconstituted system. In this
particular experiment, they exhibited 37 and 97% of normal
(lanes 5 and 7), but in other experiments, both
mutants were close to 100% of normal. The A792V mutant XPG gave a very
weak repair signal (~8% of normal), which could be increased by
using more mutant protein (data not shown). The C794A and C794S XPG
proteins were intermediate, displaying 63-73% of normal repair
activity in this experiment (Fig. 3, lanes 9 and
10).
Activity of Mutant XPG Proteins in Open Intermediate
Formation--
The mutant XPG proteins were tested for their ability
to sustain formation of a key preincision open intermediate in NER. Open intermediates with single-stranded character were detected by
their sensitivity to a 1-min pulse of KMnO4 following a
15-min incubation of damaged DNA with repair proteins, as described
(14).
Because of the distortion caused by the cisplatin adduct, there is an
intrinsic permanganate sensitivity of the T residues at positions
When XPG protein with nuclease activity was included in reaction
mixtures, 3' incisions were formed and accumulated throughout the
15-min incubation period (Fig. 4A, lane 4). To ensure the assignment of these bands, reactions were done without permanganate treatment so that incision products could be visualized separately. The
3' incision bands were absent in a reaction mixture containing HeLa
cell extract instead of purified incision proteins (Fig. 4B, lane
1), because the extract can carry out DNA synthesis and ligation
after incision. The results show that vaccinia-expressed wild-type XPG
(Fig. 4B, lanes 5 and 6) and the mutants D77E
(lane 9), E791D (lane 11), A792V (lanes
12 and 13), C794A (lane 14), and C794S
(lane 15) are all capable of carrying out 3' incision during
this 15-min period, in the context of the rest of the repair complex.
Two mutants, D77A and E791A, gave no detectable 3' incision (Fig.
4B, lanes 8 and 10). However, bands indicating
opening were even more intense, indicating accumulation of open
intermediates (Fig. 4A, lanes 7 and 9). The band
at T(+5) was particularly intense, and further bands at T(+7) and T(+8)
were detected. These are the most 3' T residues before the sites of the
major 3' incisions (Fig. 4C). These bands were not
detectable in the absence of KMnO4 treatment (Fig.
4B), showing that they represent oxidation-sensitive T
residues with single-stranded character.
Because DNA is labeled at the 3' end in these experiments, it is also
possible to detect "uncoupled" 5' incisions that occur in the
absence of 3' incision. These are normally rare events (31). With most
XPG mutant proteins, uncoupled 5' incisions occurred at similarly low
levels. However, 5' incisions readily accumulated in reaction mixtures
with the D77A and E791A XPG proteins (Fig. 4B, lanes 8 and
10). Indeed, the intensity of these uncoupled 5' incisions
was similar to the intensity of the 3' incisions seen with wild-type
XPG protein. This indicates that the NER reaction proceeds normally
with the alanine substituted XPG proteins, except that the 3' incision
does not occur. The D77A and E791A proteins therefore are well folded
and assemble normally into the repair complex. Uncoupled 5' incisions
were barely if at all detectable with the A792V protein (Fig. 4B,
lane 12), even with a 4-fold excess (lane 13),
suggesting that this mutant XPG protein does not remain stably bound in
the repair complex.
The Acidic Amino Acid Substitutions, but Not the Alanine
Substitutions, Permit Complementation of XP-G Cell Lines in
Vivo--
To test the in vivo relevance of these results,
we cloned the various mutated XPG cDNAs into plasmid
EBO-pLPP, an Epstein-Barr virus-based episomal vector (33), and
transfected them into two lymphoblastoid cell lines derived from XP-G
patients XP3BR and XP125LO (23, 24). Following hygromycin selection,
stable transfectants were irradiated with 254-nm light, and their
survival was assayed 48 h later with Alamar blue, a redox
indicator that both fluoresces and changes color in response to
chemical reduction of the medium resulting from cell growth (35). As
revealed by immunoblotting, all mutated forms of XPG were expressed in
the transfectants, and in levels comparable to that of XPG in a
wild-type lymphoblastoid line (data not shown).
In both XP-G cell lines, transfectants containing the EBO-pLPP vector
alone were highly sensitive to UV, whereas the presence of wild-type
XPG restored UV resistance to normal levels (Ref. 26 and Fig.
5A). UV resistance was not
regained by transfectants expressing either the D77A or E791A forms of
XPG but was partially restored by the C794A and C794S substitutions
(Fig. 5, A and B). These results were
corroborated by an alternative assay for cell viability, the uptake of
[3H]thymidine during a short pulse 2 days after UV
irradiation (36). This assay measures the proportion of cells that
enter S-phase and is a more direct and sensitive assay of the effect of
DNA damage on viable cell number than the redox indicator. XP125LO cells expressing the EBO-pLPP vector alone or XPG with the D77A or
E791A substitutions were comparably UV-sensitive by this assay. UV
resistance was again partially restored in the C794A and C794S transfectants, with the C794A protein being slightly more active than
XPG carrying the C794S substitution (Fig. 5, C and
D). In contrast to their inactivity on the bubble substrate
(Fig. 2), the D77E and E791D XPG proteins were able to fully restore UV resistance to both XP-G cell lines in both assays (Fig. 5,
A-D). Identical results were obtained (data not shown) when
the mutant XPG cDNAs were transfected into lymphoblastoid line
AG08802 from XP-G/CS patient XP20BE (43). These in vivo
results thus corroborate the conclusion that the acidic substitutions
permit XPG endonuclease activity when in the presence of the other NER
proteins.
The Alanine Substitutions Render Transfected Raji Cells
UV-sensitive--
The accumulation of uncoupled 5' incisions found
with the D77A and E791A proteins (Fig. 4) raised the possibility that
these alanine substitutions may be able to sequester the NER machinery into inactive complexes to generate a dominant negative phenotype in
wild-type cells. To test this possibility, cDNAs for the various XPG proteins were cloned behind the strong SR The A792V Substitution Permits Weak Complementation in
Vivo--
The A792V protein is the only stably expressed form of XPG
in XP124LO and XP125LO lymphoblasts (10). This mutant protein is of
particular interest because it is unable to complement these cells by
one fluorescence assay (8), yet the Alamar blue assay suggests that it
is able to restore UV resistance to lymphoblasts from patient XP3BR
(47). To investigate this further, we examined transfectants of both
cell lines with the [3H]thymidine uptake assay. This more
sensitive assay revealed that the XP3BR cells are significantly more
UV-sensitive than the XP125LO cells (Fig. 5G). As expected,
and consistent with the earlier fluorescence assay (8), expression of
the A792V protein in XP125LO cells did not alter their UV sensitivity.
However, expression of this mutant protein in XP3BR cells increased
their UV resistance up to the XP125LO level (Fig. 5G). We
conclude that the A792V XPG protein has very low but detectable
nuclease activity in cells that permits removal of some UV damage.
The Monomeric versus Multimeric State of Human XPG
Protein--
The 5' cut in human NER is made by a structure-specific
nuclease formed by a heterodimer of the XPF and ERCC1 proteins (16, 17). Although XPG is able to make a structure-specific 3' cut without
additional proteins (6, 14), and Fig. 2), it is conceivable that it
does so as a multimer. It is also noteworthy that some related
nucleases, such as FEN-1/DNase IV, load onto a single-stranded DNA end
(48), whereas XPG requires an open structure within a contiguous
stretch of DNA (14). For these reasons, we used two experimental
approaches to investigate whether XPG could multimerize.
In the first approach, we measured the Stokes radius and the
sedimentation coefficient of purified XPG recombinant protein produced
in the baculovirus system. Gel filtration chromatography on a fast
protein liquid chromatography Superose 6 column revealed that in 1 M KCl, XPG protein and structure-specific endonuclease activity eluted as a single peak (Fig.
6A) with an estimated Stokes radius of 70.3Å. For S value determination, pure XPG protein was loaded onto a 10-30% glycerol gradient in either 0.4 or 1 M KCl, and after centrifugation, XPG-containing fractions
were revealed by immunoblotting. The protein sedimented at 5.1 S in
both salt concentrations (Fig. 6, B and C).
Overexpressed XPG in a crude vaccinia-infected HeLa extract migrated in
a gradient with 0.4 M KCl to exactly the same fractions as
the pure protein (Fig. 6C). Combining the two measurements
made in identical buffer conditions (1 M KCl), the native
molecular mass of the protein was calculated to be 146 kDa by the
equation of Siegel and Monty (49), assuming a partial specific volume
of 0.725 cm3/g. This figure is close to the predicted
monomeric molecular mass of 133 kDa. Thus, under these conditions, the
bulk of XPG appears to exist most stably as a monomer.
In the second approach, we asked whether interactions between XPG
polypeptides could be detected by immunoprecipitation. For this
approach, we engineered a truncated XPG protein lacking the N-terminal
136 amino acids. This deletion eliminates the conserved N region,
including the first 109 amino acids that were used previously as the
immunogen to raise an anti-XPG rabbit polyclonal antibody (30).
Recombinant proteins were again produced in the vaccinia expression
system, but in this case, the infected-transfected HeLa cells were
incubated overnight and then labeled with [35S]methionine
and [35S]cysteine for 4 h before cell lysis. As
revealed by autoradiography after gel electrophoresis, the wild-type
and
When separately expressed, wild-type XPG protein, but not the
To estimate the stoichiometry of the interaction, infected HeLa cells
were co-transfected with various ratios of the two cDNAs to try to
obtain equimolar expression of the full-length and truncated proteins.
When this was achieved (Fig. 7A, lane 4), the subsequent immunoprecipitate was found to contain the wild-type and Conserved Catalytic Residues in the XPG Family--
The results
presented here provide insight into human XPG function. Structurally,
the active site geometry of this protein is expected to be very similar
to that of other nucleotide cleaving enzymes in the XPG family. Fig.
8A shows a superposition of
three relevant proteins for which structures have been solved by x-ray crystallography: T. aquaticus DNA polymerase (37), T5 D15
exonuclease (3), and T4 RNase H (1). The central
Fig. 8B shows a model of XPG around the conserved active
site region, which consists of both the N and I regions. These come together with remarkable exactness despite the very long sequence (>600 amino acids) between these two regions. All residues mutated in
this study lie within this highly conserved active site region. It is
possible that XPG co-ordinates two metal ions as found for T4 RNase H
(Fig. 8B). Although not necessarily expected to have high
precision at the atomic level, the model provides a useful visual aid
for interpreting the consequences of each mutation examined in this
study, and it helps to predict additional mutations that potentially
could be useful in the future to probe the XPG incision mechanism in
finer detail.
Consequences of Mutating the Conserved Acidic Residues--
In
most nucleolytic enzymes for which detailed information is available, a
carboxylate is required either to directly capture a proton from a
water molecule (in this case the divalent ion stabilizes the reaction
intermediate) or to bind a divalent ion that promotes the formation of
a hydroxyl ion. In either case, the resulting OH
The other mutations generated in the present study, C794A and C794S,
had only mild effects on XPG function (Figs. 2-5). The Cys-794 residue
is conserved within most eukaryotic members of the XPG family (Fig. 1)
but is predicted to be some distance down the
Changes have been made in positions corresponding to Asp-77 and Glu-791
in other XPG family members. D73N and D125N substitutions in DNA
polymerase I of Mycobacterium tuberculosis and D63A and D115A in the E. coli enzyme abolish 5' nuclease activity
(57, 58). Similarly, mutations of the equivalent residues in T4 RNase H
(D71N and D132N) and human FEN-1 (D86A and E160A) cause the complete
loss of nuclease activity but do not affect binding to DNA (59, 60).
The D77A and E791A XPG proteins are less active on the bubble substrate
than endogenous XPG in the CSB control (Fig. 2), they reduce the UV
resistance of transfected Raji cells (Fig. 5), and although they are
inactive as a 3' endonuclease (Figs. 2-4), they allow the 5' NER cut
to be made very efficiently (Fig. 4). These results strongly suggest
that the D77A and E791A substitutions, like the homologous mutations in
FEN-1 and T4 RNase H, still permit substrate binding, and they provide
further evidence that XPG needs to be in the preincision complex to
permit ERCC1-XPF to make the 5' cut (56).
XPG proteins carrying the D77E and E791D substitutions were unable to
cut the bubble substrate but were highly active in the dual incision
assay and in vivo (Figs. 2, 4, and 5). This is in accord
with a strict requirement for carboxylates at positions 77 and 791. These results further suggest that other NER components are needed to
reveal the endonuclease activity of these mutated proteins. Minor
modifications in the active surface of the protein are likely to affect
the strict relative orientations of the single-stranded DNA, the metal
ion, and the attacking water molecule needed for efficient catalysis.
Apparently, the repair partners of the D77E and E791D XPG proteins can
modulate the impact of these mutations on nucleolysis. One way this
might occur is to subtly adjust the conformation of the mutated XPG
proteins to better fit the substrate. Another possibility is that even
if the kinetic parameters (Km and
Vmax) for the acidic substitution mutants are
low for the isolated enzymes, their effects are negligible compared
with the thermodynamic gain of having specific contacts with other
repair proteins and with an optimally positioned DNA substrate within the open complex.
Implications for the XPG Incision Mechanism--
In the crystal
structure of T4 RNase H, the short peptide between the N and I regions,
located above the cleft with the two Mg2+ ions, shows nine
disordered residues that have been proposed to be involved in substrate
binding (1). The corresponding sequence in the Taq DNA
polymerase also shows disordered residues (37). In the T5 D15
exonuclease, the corresponding region is part of a helical arch that
could perfectly accommodate DNA (3). It has been proposed that this
arch may allow T5 D15 nuclease to thread-through single-stranded DNA
until it reaches the branch point, where it cleaves. Evidence for a
thread-through mechanism has been found for other members of the
nuclease family. The flap endonuclease activity of FEN-1 is inhibited
by protein binding or by annealing of a complementary strand to the
single-stranded arm (61). Proliferating cell nuclear antigen physically
associates with FEN-1 and stimulates its nuclease activity at branch
substrates (48). It has been proposed that FEN-1 diffuses down the
single-stranded flap from the 5' terminus to the single-stranded
double-stranded junction, where it is stabilized by proliferating cell
nuclear antigen (48). T4 gene 32 single-stranded DNA-binding protein inhibits the flap endonuclease of T4 RNase H (62). Consistent with a
thread-through model, the 5' nucleases of Taq and E. coli polymerase I (58, 63), FEN-1 (5), and T5 D15 exonuclease (3)
absolutely require a free single-stranded terminus to display nucleolytic activity.
In contrast, XPG must load onto DNA sites where there is no free 5' end
and therefore may use a different mechanism to accomplish the 3' cut.
During NER, a heterodimer composed of XPF and ERCC1 proteins is
responsible for the 5' incision (16, 17). Given that XPG is able to
make a structure-specific 3' cut without additional proteins (Refs. 6
and 15 and Fig. 2), the ability of XPG to multimerize (Fig. 7) may be
relevant to its incision mechanism. Also relevant may be a
helix-loop-helix motif found within the I region of XPG (64). Although
no evidence was found for XPG multimers in the latter study, a
recombinant peptide containing this helix-loop-helix motif was able to
dimerize (64). In the present work, co-expressed full-length XPG
protein and XPG lacking the conserved N region were found together by
immunoprecipitation. Such multimers, most likely dimers, were resistant
to high concentrations of detergent and salt and to ethidium bromide,
thereby indicating that the interactions are specific and are not
mediated by DNA. However, these results are not in accord with
hydrodynamic measurements indicating that XPG exists as a monomer in
high ionic strength (Fig. 6). This may simply mean that the observed
self-interaction is reversible. Indeed, condition-dependent
variability of native structure has been observed for many proteins,
including the E. coli RuvC endonuclease (65) and members of
the Bcl-2 family (4, 66). Further studies with XPG protein and its
substrates will be required to test the potential relevance of
multimerization to the XPG incision mechanism.
Implications for XP-G Clinical Phenotypes--
A striking feature
of individuals belonging to XP group G is that they rarely develop skin
cancers, even though their cells are among the most UV-sensitive of the
eight XP complementation groups. Several XP-G individuals suffer from a
very severe early onset form of Cockayne syndrome, which is correlated
with an inability to produce full-length XP-G protein (10, 67) and an
inability to carry out transcription-coupled repair of oxidative base
damage (9). Due to their hospitalization and early demise, these
XP-G/CS patients had quite limited exposure to UV. In contrast, the
XP124LO and XP125LO siblings have been exposed to UV for over two
decades, yet despite only occasional use of sun protection, they have
no skin cancers.
The results presented here suggest an explanation for this paradox. At
a cellular level, transcription-coupled repair of UV damage cannot be
detected in XP125LO fibroblasts (10), and unscheduled DNA synthesis is
at background levels after UV treatment of XP125LO lymphoblasts (26).
The thymidine uptake assay showed, however, that the XP125LO
lymphoblasts, although very UV-sensitive, are significantly more
UV-resistant than lymphoblasts from XP-G patient XP3BR (Fig.
5G). Moreover, when the A792V protein, the only stably expressed form of XPG detectable in the XP124LO and XP125LO siblings (10), was expressed in XP3BR lymphoblasts, it increased their UV
resistance up to the XP125LO level (Fig. 5G). These results strongly suggest that the XP124LO and XP125LO siblings exhibit a very
mild XP phenotype because their A792V XPG protein retains some residual
repair capacity.
The latter suggestion implies that the A792V protein has some 3'
endonuclease activity, and this is borne out by the in vitro studies. Although A792V XPG was unable to cleave the bubble substrate in the presence of Mg2+, it was able to do so to some
extent with Mn2+ as metal ion cofactor (Fig. 2). Similar
Mn2+-driven partial activity has been reported for active
site mutants of BamHI (68), EcoRV (69), T4 RNase
H (59), and MunI (70) nucleases. Residue Ala-792 is at the
beginning of an
INTRODUCTION
Top
Abstract
Introduction
References
3' direction (21),
whereas XPG (and its S. cerevisiae homolog Rad2) makes
endonucleolytic incision close to the 3' junction, where
single-stranded DNA meets duplex DNA (6, 7).
EXPERIMENTAL PROCEDURES
Oligonucleotides used for site-directed mutagenesis
N136 truncation,
XPG cDNA was cut with BstBI, filled in with
Klenow enzyme and then cut with HindIII, and the resulting
3298-base pair fragment was subcloned between the NcoI (filled-in) and HindIII sites of pGEM-XPG.
N136 XPG co-immunoprecipitation, cells were
labeled for 4 h with L-[35S]methionine
and L-[35S]cysteine and then lysed as
described above. Approximately 200 µg of HeLa extracts were
immunoprecipitated with 3 µl of crude rabbit polyclonal anti-N
terminus XPG antibody (30) in Buffer B containing 0.15 M
KCl and 0.4% Nonidet P-40. After extensive washing with the same
buffer, the beads were resuspended in SDS gel loading buffer, boiled
for 5 min, and electrophoresed on 5% polyacrylamide gels.
-32P]ATP. The bubble
substrate (0.2-0.5 ng) was incubated with the immunopurified and
immobilized XPG proteins for 3 h at 37 °C. Reactions were
stopped by addition of 30 mM EDTA, 0.6% SDS, and 0.4 mg/ml
proteinase K. After 30 min at 37 °C followed by phenol extraction
and ethanol precipitation, samples were resuspended in formamide dye
mix, heated at 95 °C, and loaded onto denaturing 12% polyacrylamide
gels. Maxam-Gilbert reactions (A+G and C+T) were performed on the
5'-labeled oligonucleotide to localize the site of XPG cleavage.
-mercaptoethanol to 1 M. Purification of the DNA and
analysis on a 6% sequencing gel was performed as described previously
(14).
promoter (34). 107 XP-G or Raji cells were transfected by
electroporation with a Gene Pulser (Bio-Rad) at 250 V, 960 microfarads
in RPMI 1640 medium containing 20 µg/ml recombinant DNA and 400 µg/ml E. coli tRNA. Transfectants were selected with
increasing doses of hygromycin B (Calbiochem) up to 200 µg/ml.
Selected cells were UV-irradiated at 254 nm in Hanks' buffer (without
phenol red) at a dose rate of 0.12 J/m2/s for various times.
positions were superimposed to under 1.0 Å root
mean squared deviation. Visual inspection of the superposition (see
Fig. 8A) indicated that all 103 equivalenced residues lie in
one domain of each enzyme. This domain is the catalytic domain and
consists of the conserved N and I regions (Fig. 1), which come together
to form the active site. The XPG sequence was manually aligned in this
region by maintaining the conserved hydrophobic and expected charged
residues of the catalytic center. A secondary structure prediction (39)
of the XPG sequence agreed with the superposition and was also used as
a guide to the alignment. The XPG sequence has ~16% sequence
identity within this region; this drops to ~11% for the complete
alignment. Hence, it was decided to model only the active site region,
as this covered all of the mutations made and also the most conserved
region. Only small differences were found between models based on each
of the three crystal structures or the average of their superimposed
coordinates. Because the positions of two Mg2+ metals have
been assigned in the active site region of T4 RNase H (1), it was
decided to model directly from the RNase H structure. The ligands to
both Mg2+ sites are conserved in all sequences. Side chains
were replaced between RNase H and XPG using 3D-JIGSAW (40), a program
that optimizes the interaction between all side chains by considering all possible side chain conformer interactions.
RESULTS
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Fig. 1.
Locations of the amino acid substitutions in
XPG. A, schematic representation of the conserved
N-terminal (N) and internal (I) regions of some
members of the structure-specific nuclease family. The total number of
amino acids in each protein is indicated. B, amino acids
substitutions in the N and I regions of XPG. The three proteins for
which the structures have been determined are compared with the
eukaryotic members of this nuclease family. The conserved sequences
encompassing the XPG substitutions are aligned. Numbers at
left and right correspond to the first and last
amino acids, respectively, in that segment. A792V results from a
natural mutation in the maternal XPG allele of patients
XP125LO and XP124LO. All other substitutions were created by
site-directed mutagenesis.
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Fig. 2.
Intrinsic endonuclease activity of mutant XPG
proteins in the pull-down assay. A, immunoblot analysis
of overexpressed wild-type and mutated XPG proteins. Extracts were
prepared from vTF7-3-infected HeLa cells transfected with cDNAs
for CSB as a negative control, wild-type XPG, or various mutated XPG
proteins as indicated. Lysates were electrophoresed through a 5%
polyacrylamide gel, blotted, and probed with the mouse anti-XPG
monoclonal antibody 8H7. Note that the amount of endogenous HeLa XPG
(lane CSB) is negligible compared with the overexpressed
proteins and that XPG, which has a predicted molecular mass of 133 kDa,
migrates very close to the 205-kDa myosin marker (lane M).
B, endonuclease activity of mutated XPG proteins. The bubble
substrate was 5'-labeled on one strand, gel purified, and incubated for
3 h with increasing amounts of immunopurified XPG. The autograph
in A was first scanned to evaluate the amount of XPG
relative to the total protein concentration in each HeLa extract.
Equivalent 1:2:3 ratios of XPG were immunopurified from appropriate
amounts of the extracts. These varied from 25-75 µg to 64-192 µg
of total protein, so a range of 25, 70, and 195 µg was chosen for the
control CSB lysate. The products of the endonuclease reactions were
analyzed on a denaturing 12% polyacrylamide gel. Maxam-Gilbert
sequencing ladders of the labeled strand are displayed as markers.
C, endonuclease activity of wild-type and A792V XPG proteins
in the presence of Mn2+. Equivalent amounts of XPG proteins
were immunopurified from 80-100-µg samples of total protein. The
reactions and gel conditions were as in B except that 2 mM MnCl2 was used instead of 2 mM
MgCl2 in reactions displayed in lanes 2,
4, and 6.
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Fig. 3.
Incision activity of mutant XPG proteins in
the reconstituted repair system. Reactions contained a DNA
substrate containing a single cisplatin lesion, XPA, RPA, XPC-hHR23B,
ERCC1-XPF, and TFIIH, and the XPG protein to test. DNA was recovered
from the repair reactions and was annealed with an oligonucleotide
complementary to the excised oligonucleotide and with a 5'-GGGG
overhang (32). Excised oligonucleotides carrying the platinum adduct
were labeled by a Sequenase reaction using [32P]dCTP and
then were separated on a denaturing 14% polyacrylamide gel and
visualized by autoradiography. Lane M, size markers
(pBR322-MspI digest); lane 1, reconstitution
omitting XPG; lanes 2-10, reconstitution with the indicated
vaccinia-expressed proteins; lane 11, reconstitution with
recombinant XPG from baculovirus; lane 12, HeLa whole cell
extract. Dual incision products are bracketed.
5,
4,
2, and 0, and at G(+1) as revealed by the BSA-only sample (Fig.
4A, lane 14). NER-related open
intermediates produced during the 1-min period of KMnO4
modification are detectable by intensification of sensitivity at these
positions, and the appearance of new diagnostic bands at positions
7,
9, and
10, and +5. Opening to positions
7,
9, and
10 can take
place in the absence of XPG protein (Fig. 4, lanes 3 and
6). This XPG-independent opening is probably equivalent to
the preincision complex that has been designated PIC2 (42).
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Fig. 4.
Opening and incision of DNA around the single
Pt-GTG lesion in the presence and absence of KMnO4.
A, open complex formation with purified incision components
and the indicated vaccinia-expressed XPG proteins. Except for
lane 1, all samples were treated with KMnO4 and
piperidine before gel analysis. B, as for A, but
samples were not treated with KMnO4 (except for lanes
2, 3, and 16). This specifically reveals sites of
enzymatic incision. All samples were treated with piperidine before gel
analysis. C, scheme showing opening around the Pt-GTG
lesion. Sensitive T residues are marked in boldface.
Arrows indicate the 5' and 3' incision sites. The
asterisk indicates the site of 3' 32P labeling
of the adducted strand at the AvaII site.
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Fig. 5.
Viability of transfected lymphoblastoid cells
after UV irradiation. A and B, LBL463 cells
from patient XP3BR were stably transfected with the indicated
XPG clones in the EBO-pLPP vector and were assayed by Alamar
blue fluorescence 48 h after irradiation. Results are expressed as
percentage of the fluorescence of nonirradiated cells. Error
bars show the standard deviations of groups of four measurements.
C and D, XPG83 cells from patient XP125LO were
stably transfected with the indicated XPG clones in the
vector EBO-pLPP and were pulse-labeled for 2 h with
[3H]thymidine 48 h after irradiation. Results are
expressed as percentage of thymidine incorporation of nonirradiated
cells. Error bars show the standard deviations of groups of
four measurements. E and F, Raji lymphoblasts
were stably transfected with the indicated XPG clones in the
vector EBS-PL and were assayed by Alamar blue fluorescence 48 h
after irradiation. Results are expressed as percentage of the
fluorescence of nonirradiated cells. Error bars show the
standard deviations of groups of four measurements. G, cells
from patients XP125LO and XP3BR were stably transfected with the
indicated XPG clones in the vector EBO-pLPP vector and were
pulse-labeled for 2 h with [3H]thymidine 48 h
after irradiation. Results are expressed as percentage of thymidine
incorporation of nonirradiated cells. Error bars show the
standard deviations of groups of four measurements.
promoter in the EBS-PL
episomal vector (34) and then were transfected into Raji cells derived
from a Burkitt lymphoma. These cells contain wild-type XP
genes but carry mutations in both alleles of the p53 gene
(44) that are expected to make them more resistant to UV cytotoxicity and to exhibit less UV-induced apoptosis than cells expressing wild-type p53 (45, 46). Untransfected Raji cells were indeed very
UV-resistant (Fig. 5E). This resistance was not impaired by
transfection of the EBS-PL vector alone or by overexpression of XPG
proteins having partial or complete complementation activity in the
XP-G cell lines (Fig. 5, E and F, see
A792V, wt, D77E, and E791D). However,
overexpression of the D77A and E791A XPG proteins made the Raji cells
significantly more UV-sensitive (Fig. 5F). These results are
thus consistent with the in vitro studies and suggest that
these alanine substituted proteins are capable of binding both to the
DNA substrate and to other components of the NER preincision complex.
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Fig. 6.
Hydrodynamic properties of XPG protein.
A, determination of Stokes radius by gel filtration
chromatography. Purified XPG protein was applied to a fast protein
liquid chromatography Superose 6 column in buffer containing 1 M KCl. The elution position of both XPG protein and
structure-specific endonuclease activity is indicated by the
dashed lines. This was compared with the elution positions
of the indicated markers with Stokes radii as follows: myoglobin
(Myo), 19 Å; ovalbumin (Oval), 30 Å; bovine
-globulin (B
G), 51 Å; thyroglobulin (Tg),
81 Å. B, determination of S values by sedimentation
velocity centrifugation. Purified XPG protein and an aliquot of HeLa
cell extract containing overexpressed XPG (5 µg of XPG/ml) were
applied to identical 10-30% glycerol gradients in buffer containing
0.4 M KCl. In each case, the peak of XPG protein sedimented
as indicated by the dashed line. The S value was calculated
by comparison with the sedimentation positions of the following markers
in an identical gradient run in parallel: carbonic anhydrase, 2.8 S;
bovine serum albumin, 4.4 S; alcohol dehydrogenase, 7.6 S;
-amylase,
8.9 S; apoferritin, 16.6 S. C, immunoblot of fractions from the 0.4 M KCl glycerol
gradient (B) probed with the 8H7 monoclonal antibody. Highly
purified XPG and the protein present in a crude cell extract sedimented
to the same position (peak fraction 8, 1.4 ml).
N136 XPG proteins were stable, easily distinguishable major
labeled species, whether expressed singly or together (Fig.
7A).
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Fig. 7.
Co-immunoprecipitation of wild-type and
N-terminally deleted XPG proteins. A, HeLa cells
infected with the vTF7-3 vaccinia virus recombinant were transfected
with cDNAs for wild-type XPG (lane 1) or N136XPG
(lanes 7 and 9), or were co-transfected with the two
cDNAs at different ratios (lanes 2-6 and 8).
After overnight incubation followed by 4-h label with
[35S]methionine and [35S]cysteine, crude
cell lysates were electrophoresed on 5% acrylamide gels, and XPG
proteins were revealed by autoradiography. Lysates containing the two
separately expressed proteins were also mixed before electrophoresis
(lane 10). B, the lysates were challenged with a
polyclonal antibody raised against the conserved N region of XPG and
immunoprecipitated proteins were analyzed by gel electrophoresis and
autoradiography as in A. C, HeLa cells infected with the
vTF7-3 vaccinia virus recombinant were transfected with cDNAs for
wild-type XPG (lanes 1 and 4),
N136XPG
(lanes 2 and 5), and the two cDNAs together
(lanes 3 and 6). After overnight incubation,
crude cell lysates were prepared and challenged with the anti-N region
of XPG polyclonal antibody. Proteins in the crude lysates (lanes
1-3) and the immunoprecipitates (IP) (lanes
4-6) were electrophoresed on a 5% acrylamide gel and then
immunoblotted with the anti-XPG 8H7 monoclonal antibody.
N136
version without the epitope, was immunoprecipitated by the polyclonal
antibody in a buffer containing 0.15 M KCl and 0.4%
Nonidet P-40 (Fig. 7B, compare lane 1 with
lanes 7 and 9). However, the truncated protein
was found in the immunoprecipitate after the two proteins were
co-expressed in the same cells (Fig. 7B, lanes
2-6 and 8). The same result was obtained when the
buffer contained 50 µg/ml ethidium bromide to abolish nonspecific
interactions mediated via DNA (50), when MgCl2 was omitted
from the buffer, or when the salt concentration was raised to 1 M KCl (data not shown). However,
N136 XPG was not found
in the immunoprecipitate when the two proteins were expressed
separately and then mixed (Fig. 7B, lane 10). To confirm the
identities of the protein bands, unlabeled extracts were
immunoprecipitated with the anti-N region polyclonal antibody and then
immunoblotted with the anti-XPG 8H7 monoclonal antibody. The
N136
XPG protein was again found in the immunoprecipitate with co-expressed
wild-type XPG (Fig. 7C, lanes 5 and 6). We
conclude that when co-expressed, the full-length and truncated XPG
proteins are able to interact with one another and that this
interaction does not require the conserved N region.
N136 XPG
proteins in a ~3:1 molar ratio (Fig. 7B, lane 4). Because this method detects wild-type/wild-type and wild-type/
N136
interactions, but not
N136/
N136 interactions, this result implies
an interaction between an even number of molecules, most simply that
XPG can dimerize.
DISCUSSION
-sheets superimpose extremely well and more closely than the surrounding
-helices. The
metal coordinating side chains in the active sites are all clustered at
the top of the
-sheets and are highly conserved both in sequence
(Fig. 1) and in position.
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Fig. 8.
Model for the active site of XPG.
A, superposition of three nucleotide cleaving enzymes with
significant sequence similarity to XPG: T5 D15exonuclease
(magenta), Taq DNA polymerase (green),
and T4 RNase H (cyan). The two Mg2+ cations
present in the T4 structure are represented by two yellow
spheres. B, model for the XPG active site. The model
includes all residues that could be equivalenced to residues
superimposed to under 1.0 Å used in A. Only an area from
domain I is shown. Residues from the N region are shown in
green and those from the I region in cyan. The
numbered yellow spheres represent the two Mg2+
ions that are expected to be located in positions equivalent to those
found in T4 RNase H. Conserved side chains that coordinate these
cations are shown in red. Residues mutated in this and
another study (56) are labeled.
attacks
the scissile phosphodiester bond (51-55). For the XPG nuclease family,
there is an extensive network of charged side chain to metal
interactions that link together the two metal cations (Fig. 8). The two
acidic residues mutated in this study, Asp-77 and Glu-791, are both
involved in this network. In this regard, the report of nuclease
activity in a D77A XPG mutant (56) is very surprising because
substitution of this neutral side chain would be expected to disrupt
this electrostatic network. We found instead that changing either
Asp-77 or Glu-791 to Ala completely inactivated the nuclease function
of XPG (Figs. 2-4) and abolished the ability of XPG to restore UV
resistance to XP-G cell lines (Fig. 5). Another highly conserved acidic
residue, Asp-812, is also expected to be part of this electrostatic
network (Fig. 8B). Conversion of Asp-812 to Ala also
inactivated the nuclease function of XPG (56). These results are thus
consistent with a role for all three acidic residues in catalysis.
-helix away from the
catalytic center (Fig. 8B). It thus may have a subtle role
in conformation rather than being directly involved in catalysis.
-helix and just after the presumed metal
coordinating side chain Glu-791 (Fig. 8B). Valine side
chains are
-branched and are less favored within
-helices than
non-
-branched side chains (71). Hence, this particular
-helix may
unwind slightly in the A792V protein, and this in turn may interfere
with the ability of Glu-791 to accurately coordinate a metal cation.
The 3' endonuclease activity in the presence of Mn2+ may
reflect partial correction of such a distortion in the metal binding
site. This missense mutation appears to have additional effects,
however, because the A792V protein exhibited the lowest level of
uncoupled 5' incisions (Fig. 4). This indicates that it does not stay
firmly bound in the repair complex, because an altered conformation
either reduces its ability to bind to DNA or disrupts its interaction
with another protein. These interesting properties of this naturally
occurring mutation indicate that it would be very valuable to identify
the XPG defects in other mildly affected XP-G patients.
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ACKNOWLEDGEMENTS |
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We thank C. Arlett and J. Cole for helping to establish the LBL463 cell line from patient XP3BR and G. Mottet and J.-B. Marq for help with the vaccinia expression system.
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FOOTNOTES |
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* This work was supported by postdoctoral fellowships from the Swiss National Science Foundation (to D. G.) and from l'Association pour la Recherche sur le Cancer (to P. L.), by the Human Frontiers of Science Program (to E. E. and R. D. W.), by Grants 31-36481.92 and 31-52777.97 from the Swiss National Science Foundation (to S. G. C.), and by the Imperial Cancer Research Fund.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.
¶ Present address: Section on Genetics and Development, 457 Biotechnology Bldg., Cornell University, Ithaca, NY 14853-2703.
Present address: Institut de Biologie et Chimie des
Protéines, UPR412 CNRS, 7 passage du Vercors, 69007 Lyon, France.
To whom correspondence should be addressed. Tel.:
41-22-702-5664; Fax: 41-22-702-5702; E-mail:
Stuart.Clarkson{at}medecine.unige.ch.
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ABBREVIATIONS |
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The abbreviations used are: N region, N-terminal region; I region, internal region; NER, nucleotide excision repair; CS, Cockayne syndrome; XP, xeroderma pigmentosum; XP-G, XP complementation group G; RPA, replication protein A; TFIIH, transcription factor IIH; BSA, bovine serum albumin.
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REFERENCES |
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