(Received for publication, February 28, 1997)
From the Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7260
XPG is a member of the FEN-1 structure-specific
endonuclease family. It has 3-junction cutting activity on bubble
substrates and makes the 3
-incision in the human dual incision
(excision nuclease) repair system. To investigate the precise role of
XPG in nucleotide excision repair, we mutagenized two amino acid
residues thought to be involved in DNA binding and catalysis,
overproduced the mutant proteins using a baculovirus/insect cell
system, and purified and characterized the mutant proteins. The
mutation D77A had a modest effect on junction cutting and excision
activity and gave rise to uncoupled 5
-incision by mammalian cell-free extracts. The D812A mutation completely abolished the junction cutting
and 3
-incision activities of XPG, but the excision nuclease reconstituted with XPG (D812A) carried out normal 5
-incision at the
23rd-24th phosphodiester bonds 5
to a (6-4) photoproduct without
producing any 3
-incision. It is concluded that Asp-812 is an active
site residue of XPG and that in addition to making the 3
-incision, the
physical presence of XPG in the protein-DNA complex is required
non-catalytically for subsequent 5
-incision by XPF-ERCC1.
Xeroderma pigmentosum (XP)1 is an autosomal recessive disease caused by defective excision repair (1, 2). XP patients are hypersensitive to sunlight and develop actinic keratoses and skin cancer at a high incidence and frequency. In addition, XP individuals with severe repair defects develop sensorineural and motor neurological symptoms. It has been shown that mutations in seven genes (XPA through XPG) cause XP. Recently, the proteins encoded by these genes (with the exception of XPE) have been purified, and it has been established that six repair factors that include these gene products are necessary and sufficient for carrying out dual incisions that remove damage such as cyclobutane pyrimidine dimers and (6-4) photoproducts from DNA. These six repair factors are XPA, RPA, TFIIH (five polypeptides including XPB and XPD), XPC-HHR23B, XPF-ERCC1, and XPG (3, 4).
A second disease associated with defective DNA repair is Cockayne's syndrome (CS). CS individuals are characterized by photosensitivity, arrested growth and development, and neurological abnormalities. Mutations in five genes cause Cockayne's syndrome: CSA, CSB, XPB, XPD, and XPG (5). Of these, the proteins encoded by CSA and CSB are required for coupling transcription to repair (6), and the XPB and XPD proteins are subunits of the general transcription factor TFIIH (7, 8). The only gene associated with Cockayne's syndrome but with no known function in transcription is XPG, and for this reason, characterization of the XPG protein is of considerable interest.
The XPG protein is a member of the FEN-1 family of structure-specific
endonucleases (9). These enzymes incise DNA at a junction of
single-stranded to double-stranded DNA such as those found in
"flap," bubble, and loop structures (9-12), and XPG makes the
3-incision in human nucleotide excision repair (13). Sequence comparison of FEN-1 family nucleases and procaryotic nucleases including the 5
to 3
- exonuclease domain of Escherichia
coli DNA polymerase I revealed two regions of high homology (14), and within these two regions, eight amino acids were conserved in all
members of the family. Site-directed mutagenesis of FEN-1 revealed that
three aspartate residues (Asp-34, Asp-86, and Asp-181) were essential
for catalysis (14). To find out if the corresponding residues perform a
similar function in XPG, we mutagenized Asp-77 (Asp-86 of FEN-1) and
Asp-812 (Asp-181 of FEN-1) and characterized the mutants. We found that
the D77A mutation has only a modest effect on catalysis but that the
D812A mutation abolishes the nuclease activity of XPG and has a
dominant effect when used in competition-type experiments with
wild-type XPG. Furthermore, the availability of an XPG active site
mutant enabled us to investigate its additional roles in the excision
nuclease system. We found that the presence of XPG in the preincision
complex is absolutely required for the generation of the 5
incision by
the XPF-ERCC1 junction-specific endonuclease (12, 15). Thus, XPG plays
a direct role in 3
incision and an indirect role in 5
incision activities of the human excision nuclease complex.
Mutations were generated in the
plasmid pVL1392-XPG (12). The following oligonucleotides were used to
construct the mutants by double-stranded DNA mutagenesis: D77A,
5-CGTCCTATTTTTGTGTTTGCTGGGGATGCTCC (32-mer); D812A,
ACCATCACTGATGACAGTGCCATCTGGCTGTTTGGAGCG (39-mer).
The altered nucleotides are indicated in italics. Nucleotide changes were introduced using the Chameleon double-stranded site-directed mutagenesis kit (Stratagene). The mutations were identified by restriction enzyme analysis and confirmed by double-stranded DNA sequencing using the Sequenase version 2 kit from U. S. Biochemical Corp.
Expression and Purification of Mutant ProteinsThe
wild-type and mutant XPG proteins were expressed in a
baculovirus/insect cell system. Mutant proteins were purified by sequential chromatography on phosphocellulose P11 (Whatman) and Affi-Gel blue (Bio-Rad) columns, and the XPG protein was located by
SDS-polyacrylamide gel electrophoresis followed by silver staining and
immunoblotting. The purified proteins were dialyzed against storage
buffer (25 mM Hepes-KOH, pH 7.9, 100 mM KCl, 12 mM MgCl2, 0.5 mM EDTA, 2 mM dithiothreitol, 17% glycerol) and stored at 80 °C
until use.
5-labeled 90-base pair duplex and
"bubble-30" substrates (12) were used to test the endonuclease
activities associated with XPG. The reaction mixture (7.5 µl)
contained 2.5 fmol of DNA in 25 mM Hepes-KOH, pH 7.9, 25 mM KCl, 3 mM MgCl2, 1 mM dithiothreitol, and 6.5% glycerol. The reaction was at
37 °C for 90 min, and the reaction products were separated on 10%
denaturing polyacrylamide gels. Quantitation was done using a
PhosphorImager (Molecular Dynamics).
A 136-base pair duplex containing a (6-4)
photoproduct (T[6-4]T) at nt 68-69 of one strand and
32P label at the 5th phosphodiester bond 5 to the
photolesion was used as substrate (16, 17). Complementation assays with
cell-free extracts were performed as described previously (18).
Reconstitution of the excision nuclease with wild-type or mutant XPG
was done using highly purified repair factors and under conditions
described elsewhere (3, 4); all of the repair factors with the
exception of TFIIH and XPC-HHR23B were recombinant proteins produced
either in E. coli (XPA, RPA) or Sf21 insect cells
(XPF-ERCC1 and XPG). Repair reactions were performed at 30 °C for 45 min (complementation assay) or for 3 h (reconstitution assay). The
reaction products were separated on 8% denaturing polyacrylamide gels
and visualized by autoradiography.
Analyses of eucaryotic nucleases
belonging to the FEN-1/XPG family and the exonuclease domains of
procaryotic DNA polymerases led to the identification of eight amino
acid residues that are conserved in the entire set (14). Based on this
sequence conservation as well as on analysis of the exonuclease domains
of the polymerases, it was predicted that these eight residues may be
essential for substrate binding or catalysis, and an elegant study with
human FEN-1 endonuclease revealed that the Asp-34, Asp-86, and Asp-181 residues of this 380-amino acid-long polypeptide contribute to the
active site of the enzyme (14). We wished to know if the corresponding
residues in XPG are involved in forming the active site of the protein.
Toward this goal we made alanine substitutions at positions Asp-77 and
Asp-812 of the XPG protein, corresponding to Asp-86 and Asp-181 of
FEN-1, two of the three residues most critical for FEN-1 catalytic
activity. Fig. 1 shows the wild-type and mutant proteins
analyzed by SDS-polyacrylamide gel electrophoresis. All three proteins
are overproduced to comparable levels and behave similarly on
chromatographic resins. Hence, it is reasonable to assume that the
mutant proteins folded properly and that any change in activity would
reflect the function of those specifically altered amino acids.
Nuclease Activities of Mutant XPG Proteins
Since XPG is a
3-junction cutting nuclease (11, 12), the D77A and D812A proteins were
tested for junction cutting activity using a substrate with a 30-nt
bubble. Neither the wild-type nor the mutant XPG proteins have
endonuclease activity when double stranded DNA is the substrate (Fig.
2, lanes 1-4). D77A retains about 75% of
the wild-type activity while D812A is totally defective in junction
cutting with the 30-nt bubble substrate (Fig. 2, lanes 5-8). These results suggest that Asp-77 is not essential for
catalysis but are uninformative with regard to the function of Asp-812
since lack of activity could be due to misfolding of the protein,
failure to bind to DNA, or absence of catalytic activity. Although
these issues can be addressed by a variety of means, we reasoned that the most direct test would be to assay the mutant proteins for DNA
repair activity.
Excision Nuclease Complementing Activity with Mutant XPG Proteins
The effect of mutations at the presumptive active site
residues on excision nuclease activity was tested by a complementation assay. Mutant proteins were added to XP-G cell-free extracts prepared from the Chinese hamster ovary (CHO) cell line UV135 or the human lymphoblastoid line AG08802. Fig. 3 shows the results
obtained with XPG-D77A. As is apparent, this mutant protein complements the excision activity of the XP-G cell-free extract to wild-type level
as indicated by the presence of excision products 24-28 nt in length.
Thus, it appears that the D77A mutation has near-normal functions with
regard both to interactions with other general repair factors and 3
incision activity as evidenced by complementation of excision nuclease
activity in XP-G but not XP-B extracts (Fig. 3B). In
addition, we observe 91-92-nt-long fragments corresponding to
incisions at the 23rd and 24th phosphodiester bonds 5
to the (6-4)
photoproduct (17). In a small fraction of the excision complexes mutant
XPG assembles with the other repair factors and apparently fails to
make the 3
incision but enables XPF-ERCC1 to make the 5
incision,
thus generating the DNA fragments that migrate at 91-92 nt (4, 12).
This uncoupled 5
incision is specific to XPG CFE supplemented with
XPG-D77A as the 91-92 mers are not seen when XPG-D77A is added to XPB
CFE (Fig. 3A, lane 5; Fig 3B, compare
lanes 2 and 4). These results suggest that while
the Asp-77 residue may be involved in 3
incision, the D77A mutation
does not have a significant effect on this catalytic activity of
XPG.
In contrast, the D812A mutation completely abolished the
excision-complementing activity of the XPG protein (Fig.
4A, lane 5; Fig 4B,
lanes 3 and 6). Addition of mutant protein to the
CHO-UV135 (XP-G) extract gave rise to 5-uncoupled incision products
(91-92 mers) equal in intensity to the level of excision (coupled
incisions, 24-28 mers) achieved with addition of the wild-type protein
(Fig. 4A, lane 5 versus lane 4; Fig.
4B, lane 3 versus lane 2). Qualitatively similar
results were obtained with the extract prepared from the human XP-G
cell line AG08802 (Fig. 4B, lane 6 versus 5);
however, both the excision signal obtained with wild-type XPG
(lane 5) and the band arising from the 5
-uncoupled incision
(lane 6) were weak due to poor quality of CFE from this
human lymphoblastoid cell line. The high level of uncoupled 5
-incision
events is unique to complementation experiments with XP-G mutant CFE
and D812A mutant protein as no such uncoupled incision is observed when XPG-D812A is added to a CFE from an XP-B mutant (compare Fig. 4C, lanes 2 and 4). These data show
that D812A is in the nuclease active site of XPG. Of greatest
importance, our data demonstrate that XPG (in addition to making the
3
-incision) is necessary for assembly of a preincision complex to
enable XPF-ERCC1 to make the 5
-incision. Furthermore, these data show
that it is the physical presence of XPG and not the 3
-incision that is
required for the XPF-ERCC1 complex to make the 5
-incision.
Uncoupled 5
The data
presented above are consistent with XPG being involved in the formation
of a preincision complex that recruits XPF-ERCC1 to the 5 incision
site. However, experiments with CFE do not exclude the requirement for
other proteins for priming the preincision complex for XPF-ERCC1
action. Hence, we decided to carry out reconstitution experiments with
the active site mutant XPG protein. Fig. 5 shows that
the excision nuclease reconstituted with wild-type XPG makes dual nicks
almost exclusively (lane 1, oligonucleotides in the 24-28
nt range); in contrast, dual incisions do not occur when the enzyme
system is reconstituted with XPG-D812A (lane 2), but a 5
incision (dependent on XPF-ERCC1; compare lane 3 with
lane 2) equal in intensity to the excision seen with
wild-type protein is observed. The data are in agreement with that
obtained with CFE and support the conclusion that XPG plays at least
two roles in excision repair: making the 3
incision and forming the
proper protein-protein interactions to enable XPF-ERCC1 to make the 5
incision.
Dominant-negative Effect of D812A Mutation
When normal XPG
protein was added to HeLa cell-free extract and incubated with
substrate DNA, there was no effect on excision activity.2 However, when XPG-D812A was
added to the HeLa CFE/substrate mixture, we observed a decrease in the
level of excision (dual incision) activity. Concomitant with this
decrease in excision was the appearance of and XPG
concentration-dependent increase in 5-uncoupled incision (Fig. 6). Apparently, the mutant protein effectively
competes with the wild-type protein in HeLa cell-free extract and
replaces the normal XPG protein in a fraction of the preincision
complexes with the net effect of reduced dual incisions (excision) and
increased uncoupled 5
incisions by XPF-ERCC1.
Nucleotide excision repair in humans is rather complex with 13 polypeptides in six repair factors required for the dual incision event
preceding the resynthesis step (3, 4, 15, 19). Significant progress has
been made toward defining the roles of the individual polypeptides in
damage recognition and formation of the preincision complex (20, 21).
Work with model substrates (9-12) and with anti-XPG antibodies (13)
implicated XPG in 3 incision. Addition of anti-XPG antibodies to HeLa
CFE and the reconstituted excision repair system inhibited excision and
perhaps gave rise to uncoupled 5
incision (13). Furthermore, in the reconstituted excision nuclease system it was found that omission of
XPF-ERCC1 from the reaction resulted in uncoupled 3
incision, that
omission of XPG generated no incisions, and that in the complete reaction, 3
incision preceded the 5
incision (4). In addition, it was
shown that XPG is an essential constituent of the pre-3
-incision nucleoprotein complex (4). In these reconstitution experiments it was
not possible to unambiguously distinguish between two scenarios: 1) 3
incision by XPG is a prerequisite for 5
incision by XPF-ERCC1 or 2)
that the presence of XPG in the preincision complex enables XPF-ERCC1
to make the 5
incision.
In the E. coli (A)BC excinuclease system, not only the
presence of UvrB but also the 3 incision by this subunit (perhaps aided by UvrC (22, 23)) is absolutely required for the 5
incision by
the UvrC subunit (24). Here we demonstrate that the human excinuclease
has a different mechanism. The presence of XPG in the preincision
complex (but not the 3
nick by this subunit) is a prerequisite for 5
incision. In fact, because of the absolute requirement for XPG in the
dual incision complex for the 5
incision to occur it is conceivable
that XPG is directly involved in both incisions. Although the facts
that the active site mutant D812A can promote 5
incision and that the
XPF-ERCC1 complex has 5
junction cutting activity (12) strongly
suggest that XPG plays an indirect role in 5
incision, a direct role in catalysis of 5
incision by XPG cannot be formally eliminated.
Since xeroderma pigmentosum is well characterized as an autosomal recessive disease (1, 2), it was of special interest to find that under our conditions the XPG-D812A mutation acted as a dominant-negative mutation with regard to damage removal. A similar dominant-negative effect was observed in this in vitro system with a special XPA mutant protein as well (25). Furthermore, it has been found that overexpression of an ATPase active site mutant of XPD in a wild-type cell also results in dominant-negative phenotype as evidenced by increased sensitivity to UV irradiation.3 These data raise the possibility that certain mutations in some of the XP genes would have a co-dominant effect in heterozygotes, assuming that the wild-type and mutant protein are expressed to the same levels. It is possible that careful screening of the XP kindreds would reveal partial repair defects in heterozygotes in a subset of XP patients.
Finally, the isolation of an XPG active site mutant described in this study makes it now possible to isolate preincision complexes containing only those polypeptides necessary for making dual incisions upon addition of XPF-ERCC1. Such a complex should be useful in identifying the polypeptides present during the dual incision events and the structure of the DNA prior to the dual incision reaction.
We thank Drs. T. Matsunaga and T. Bessho for help in the initial stages of this project and Dr. D. Mu for providing the repair factors and substrate used in the reconstitution experiment. We also thank Drs. T. Bessho, C. P. Selby, and D. Mu for critical comments on the manuscript.