(Received for publication, November 27, 1995)
From the
Nucleotide excision repair consists of removal of the damaged nucleotide(s) from DNA by dual incision of the damaged strand on both sides of the lesion, followed by filling of the resulting gap and ligation. In humans, 14-16 polypeptides are required for the dual incision step. We have purified the required proteins to homogeneity and reconstituted the dual incision activity (excision nuclease) in a defined enzyme/substrate system. The system was highly efficient, removing >30% of the thymine dimers under optimal conditions. All of the six fractions that constitute the excision nuclease were required for dual incision of the thymine dimer substrate. However, when a cholesterol-substituted oligonucleotide was used as substrate, excision occurred in the absence of the XPC-HHR23B complex, reminiscent of transcription-coupled repair in the XP-C mutant cell line. Replication protein A is absolutely required for both incisions. The XPG subunit is essential to the formation of the preincision complex, but the repair complex can assemble and produce normal levels of 3`-incision in the absence of XPF-ERCC1. Kinetic experiments revealed that the 3`-incision precedes the 5`-incision. Consistent with the kinetic data, uncoupled 5`-incision was never observed in the reconstituted system. Two forms of TFIIH were used in the reconstitution reaction, one containing the CDK7-cyclin H pair and one lacking it. Both forms were equally active in excision. The excised oligomer dissociated from the gapped DNA in a nucleoprotein complex. In total, these results provide a detailed account of the reactions occurring during damage removal by human excision nuclease.
DNA repair reactions play an important role in preventing cancer
development in humans. Individuals with defects in mismatch repair have
high incidence of internal cancers (Kolodner and Alani, 1994; Modrich
and Lahue, 1996). Similarly, individuals with defective nucleotide
excision repair specific for bulky DNA lesions suffer from xeroderma
pigmentosum (XP), ()which manifests itself by extremely high
incidence of actinic cancers, increased incidence of cancers of
internal organs, and mental and neurological abnormalities (Cleaver and
Kraemer, 1989; Friedberg et al., 1995). In humans, mutations
in seven genes, XPA through XPG, cause XP associated
with defective excision repair (Cleaver and Kraemer, 1989). In
addition, genetic and biochemical studies with rodent cell lines and
radiation-sensitive Saccharomyces cerevisiae mutants and
repair assays with cell-free extracts have implicated several other
proteins in the excision step of nucleotide excision repair. In
particular, it has been found that the general transcription factor
TFIIH (five to eight subunits) (Drapkin et al., 1994;
Schaeffer et al., 1994; Wang et al., 1994) and the
trimeric replication protein A (RPA or HSSB) are absolutely required
for the dual incision step of excision repair (Mu et al.,
1995).
Recently, excision repair has been reconstituted from highly purified repair factors in humans (Mu et al., 1995; Aboussekhra et al., 1995) and in the highly analogous S. cerevisiae system (Guzder et al., 1995). It has been reported that in both systems the following six fractions are necessary and sufficient for the dual incision activity (Mu et al., 1995; Guzder et al., 1995): XPA (Rad14), RPA, TFIIH (five to eight polypeptides including XPB (Rad25) and XPD (Rad3)), XPC-HHR23B (Rad4-Rad23), XPG (Rad2), and XPF-ERCC1 (Rad1-Rad10). In this study, we have obtained the six fractions of human excision nuclease (excinuclease) free of contaminants and have used synthetic substrates containing either a thymine dimer (T<>T) or a cholesterol molecule at a predetermined site to investigate the reaction mechanism of the excision nuclease. Our results reveal that XPC-HHR23B, in contrast with the other factors that are necessary for all excision reactions, is required for excision of the thymine dimer, but not for excision of a particular cholesterol lesion. In this study, it is also demonstrated that the 3`-incision made by XPG (Harrington and Lieber, 1994; O'Donovan et al., 1994; Matsunaga et al., 1995) precedes the 5`-incision made by XPF-ERCC1 (Matsunaga et al., 1995) and that the CDK-activating kinase constituents (CDK7 and cyclin H) that are present in TFIIH as integral subunits do not interfere with the repair activity of TFIIH and are not required for repair. Following the dual incision, the 27-29-nucleotide-long excised oligomers (Huang et al., 1992) are released in a complex with repair proteins, while the gapped DNA remains complexed with a separate set of repair proteins.
Figure 1: The six factors of human excision nuclease. A, silver-stained SDS-PAGE of the six factors. XPA and RPA are recombinant proteins purified from E. coli. The other factors were purified from HeLa cells. The amounts of individual factors analyzed by SDS-PAGE were as follows: XPA, 100 ng; TFIIH, 50 ng; XPC, 40 ng; XPG, 40 ng; XPF-ERCC1, 30 ng; and RPA, 800 ng. B, lack of XPC in the five fractions that excise cholesterol damage. Ten-fold the amount of the three HeLa cell-derived fractions used in the reconstitution assays was mixed and separated by SDS-PAGE (10%) and subjected to immunoblotting using the ECL detection system according to the manufacturer (Amersham Corp.) (lane 1). Lane 2 contained 1.5 ng of XPC protein as a positive control (<10% of this amount can be detected).
To further purify XPG, the 1.5 M NaSCN-eluted fractions
were loaded onto an SP-Sepharose column (2.5 30 cm). A 700-ml
gradient of 0.1-0.45 M KCl in storage buffer (25 mM Hepes/KOH, pH 7.9, 100 mM KCl, 12 mM MgCl
, 0.5 mM EDTA, 2 mM dithiothreitol, 17% glycerol) was used to elute this column at 1.3
ml/min. Unlike RPA and XPF-ERCC1, which came in the flow-through
fraction, XPG was found to elute at
0.4 M KCl by
immunoblotting. These XPG-containing fractions were subsequently
resolved to give homogeneous XPG using procedures modified from
Habraken et al.(1994).
As to TFIIH and XPC, the fractions
from the Affi-Gel blue column containing these factors were applied
onto the same SP-Sepharose column and eluted with a 1-liter gradient of
0.1-1.0 M KCl in storage buffer. XPC eluting at 0.3 M KCl was separated from TFIIH eluting at
0.16 M KCl. Subsequently, pure XPC as judged by silver-stained SDS-PAGE
was obtained using the procedures of Masutani et al.(1994).
Subsequent TFIIH purification was performed similarly to our previous
procedure (Mu et al., 1995), except that Mono S chromatography
(HR 5/5, Pharmacia Biotech Inc.) was added as the final step. This 1-ml
column was eluted at 0.5 ml/min using a 35-ml gradient of 50-300
mM KCl in buffer C (20 mM Tris-HCl, pH 7.9, 0.1
mM EDTA, 19% glycerol, 10 mM
-mercaptoethanol),
and 1-ml fractions were collected. Each fraction was concentrated using
an Amicon stirred cell, dialyzed in storage buffer, and stored at
-80 °C.
The XPA cDNA tagged with His using the
pRSET vector (Invitrogen) was overexpressed in Escherichia coli strain DR153 (Park and Sancar, 1993) and purified by a modified
procedure of Jones and Wood(1993). The DNA construct bearing the
three-subunit human RPA was obtained from Dr. M. Wold (University of
Iowa), and the recombinant RPA of apparent homogeneity was prepared
from E. coli according to Henricksen et al.(1994).
Figure 2: Substrates used in this study. A, and B, the two types of cholesterol substrates. C, thymine dimer. All of these lesions were incorporated into 140-base pair duplexes as described under ``Experimental Procedures.'' For convenience, lesion structures shown in A and B were termed cholesterol-A and cholesterol-B, respectively. D and E, schematic drawings of the full-length duplex substrates for the thymine dimer and cholesterol and the oligonucleotides (a-d) used for substrate construction.
To address the questions raised by these studies, we have purified all six repair factors extensively to obtain proteins of high purity as shown in Fig. 1A. The mixture of these fractions excised both thymine dimer and two types of cholesterol lesions (Fig. 2) from DNA in the form of 24-30-nucleotide-long damage-carrying oligomers with high efficiency. Both lesions were excised efficiently and to about the same extent (20-30%) in 5-7 h (Fig. 3). Thus, it appears that the efficiency of the defined excision repair system is comparable to the in vivo reaction, suggesting that it is unlikely that additional factors are required for optimal excision. The poor excision efficiency obtained in our previous reconstitution may be attributed to the disproportionate amount of the factors. For instance, we have found that increasing the amount of XPC-HHR23B in the reaction mixture from 15 to 60 ng can severely inhibit excision (data not shown).
Figure 3: Excision of cholesterol-A lesion and thymine dimer by reconstituted human excision nuclease. Upper panels, quantitative analyses of the data. The percent of the input substrate that was either nicked 3` only or excised is plotted. Lower panels, sequencing gels showing the excision of cholesterol-A and the thymine dimer. Cholesterol was excised mainly in the form of 26-28-nucleotide-long oligomers; the thymine dimer was excised in the form of 28-32-nucleotide-long oligomers. The reaction mixtures contained 1 nM DNA substrate and the following concentrations of the repair factors: 20 nM XPA, 0.2 nM TFIIH, 1.6 nM XPC, 0.5 nM XPG, 0.5 nM XPF-ERCC1, and 100 nM RPA.
Figure 4: Differential requirement for excision of thymine dimer (T<>T) and cholesterol-B lesions. A, excision of T<>T. The positions of the excision product and uncoupled 3`-incision are indicated. Substrate (3.5%) was excised, and 1.5% of the substrate contained uncoupled 3`-incision in the complete system (lane 3). In the reaction without XPF-ERCC1 (lane 8), 5% of the substrate contained an uncoupled 3`-nick. B, excision of cholesterol-B lesion. The positions of the main excision products are shown by a bracket. The level of excision in the area indicated by the bracket was 3.5% in the complete system (lane 4) and 2.7% in the reaction lacking XPC (lane 7). Lane 1, size markers (M) in nucleotides.
Since these results were quite unexpected, we considered the possibility that low level contaminant XPC in our other factors may be responsible for the excision nuclease activity reconstituted without XPC-HHR23B. The mixture of other HeLa cell-derived factors (TFIIH, XPG, and XPF-ERCC1) was tested for XPC contamination by Western analysis. With an assay capable of detecting 10 pg of XPC in our reaction mixture, no XPC was detectable (Fig. 1B). This enables us to rule out the presence of contaminating XPC that would support the excision of cholesterol-B lesion by XPC-omitted reconstitution reaction. An implication emerging from these observations is that with certain substrates or with all substrates in special structures as exist during transcription, XPC is not needed for excision nuclease activity.
In contrast, no 5`-incision was observed in the absence of XPG (Fig. 4A, lane 7). Two explanations were considered for this finding. (i) XPG must be present to form the preincision complex; and (ii) the 5`-incision made by XPF-ERCC1 depends on the presence of the 3`-incision made by XPG, but not on the actual existence of XPG in the preincision complex. Previous studies have shown that inhibition of 3`-incision by anti-XPG antibodies does not interfere with the 5`-incision, indicating that the formation of 5`-incision is not dependent on the production of the 3`-nick (Matsunaga et al., 1995). Hence, the failure to observe 5`-incision in the absence of XPG might be due to the fact that XPG must be present in the preincision complex to enable XPF-ERCC1 to bind and carry out the 5`-incision. To test this model, we conducted the following experiment. CFE from an XP-F or XP-G mutant cell line was mixed with substrate that contained biotinylated nucleotides at the 3`-termini. Following incubation, the substrate and preincision complexes on the substrate were removed from the unbound cellular proteins using streptavidin-attached magnetic beads. An XP-G or XP-F cell-free extract was then added to the immobilized complexes isolated from XP-F or XP-G CFE, respectively. The results reveal that addition of the XP-G extract (which contains XPF-ERCC1) or purified XPF-ERCC1 to the DNA-protein complex isolated from the XP-F extract produced excision (Fig. 5, lane 2). In contrast, addition of XP-F CFE to the complexes pulled down from the XP-G extract in the same manner did not result in excision (lane 4). From these data, we conclude that XPG enters the preincision complex before XPF and that XPG is required for positioning of the XPF-ERCC1 nuclease in nicking at the 5`-side of damage.
Figure 5: Preincision complex formation with extract lacking XPF, but not with extract deficient in XPG. Cell-free extracts from XP-F (GM08437A) or a Chinese hamster ovary cell line defective in XPG (UV135) were mixed with internally radiolabeled cholesterol-A substrate with a 3`-biotin tag. The preincision DNA-protein complex was isolated by the pull-down method as described under ``Experimental Procedures'' and complemented with either a second mutant extract or purified repair factor. Lane 1, the XP-F plus XP-G cell-free extract was used as a positive control. Lane 2, the substrate pulled down in XP-F CFE with streptavidin beads was incubated in XP-G CFE. Lane 3, same as lane 2, except that purified XPF-ERCC1 (2 ng), instead of the XP-G cell-free extract, was added to the pull-down DNA-protein complex. Lane 4, the substrate pulled down following incubation with XP-G CFE by beads was then incubated with XP-F CFE. The positions of the major excision products are marked to the left of the lane 1.
Figure 6: Order of incision. Upper panel, shown are quantitative analyses of the data in the lower panel. Lower panel, internally radiolabeled thymine dimer substrate was incubated with the reconstituted excision nuclease. Aliquots were taken at the indicated time points, quenched with 20 µg of proteinase K, and analyzed on an 8% sequencing gel. The uncoupled 3`-incision appeared before the excision product, which required both 3`- and 5`-incisions. Uncoupled 5`-incision (which is not observed) would have produced fragments of 88-92 nucleotides. Lane 1, size markers (M) in nucleotides.
Figure 7:
ATP hydrolysis is required for both 3`-
and 5`-incisions. To the excision assay containing 2 mM ATP
were added the indicated amounts of ATPS (Pharmacia Biotech Inc.),
and the reaction was carried out for 60 min at 30 °C with
5`-terminally radiolabeled cholesterol-A substrate. Lane 1,
size markers (M) in nucleotides.
Recently, the CDK7-cyclin H pair was found to be a constituent of TFIIH and to be responsible for its kinase activity (Roy et al., 1994; Serizawa et al., 1995; Shiekhattar et al., 1995). Furthermore, it was reported that microinjection of antibodies to CDK7 inhibited excision repair in vivo (Roy et al., 1994), leading to the conclusion that CDK7 was essential for excision repair. Our purest TFIIH fraction (fraction 9 of the last Mono S column chromatography step) contained stoichiometric CDK7 and cyclin H subunits. However, in the last purification step, a second form of TFIIH devoid of CDK7 and cyclin H was also obtained (fraction 10) (Fig. 8, A and B), as evidenced by immunoblottings using anti-CDK7 and anti-cyclin H antibodies and a kinase assay to detect the phosphorylation of a tetraheptapeptide repeat (YSPTSPS) of the carboxyl-terminal domain (CTD) of RNA polymerase II. Fig. 8C shows that the CDK-activating kinase-lacking TFIIH fraction 10 has <2% of the CTD peptide kinase activity of the TFIIH holoenzyme (faction 9), consistent with the notion that CDK7-cyclin H is responsible for the kinase activity of TFIIH. To ensure that CDK7-cyclin H was not in our other HeLa cell-purified basal repair factors (XPC, XPG, and XPF-ERCC1), we performed the same kinase assay on both individual factors and a combination of the entire set and found no indication of CTD peptide kinase activity (data not shown). When the two forms were tested for reconstitution of excision nuclease activity, no difference was detected between the form containing CDK7-cyclin H and the fraction without it (Fig. 8D). Thus, we conclude that the CDK7-cyclin H pair neither is necessary for nor interferes with the repair function of TFIIH.
Figure 8:
Roles of TFIIH subunits in excision. A, column profile of the final Mono S column chromatography
step visualized by silver-stained SDS-PAGE (10%). B, analysis
of the same fractions by immunoblotting using a mixture of anti-XPB,
anti-XPD, anti-p62, anti-CDK7, and anti-cyclin H (CycH)
antibodies. C, CTD peptide kinase assay with holo-TFIIH and
core TFIIH using increasing amounts of fractions 9 and 10. Each
microliter of TFIIH fractions 9 and 10 contained 1 ng of protein
as determined by the dye binding protein assay (Bio-Rad). Kinase assay
was conducted as described by Aprelikova et al. (1995). D, excision assays with holo-TFIIH and core TFIIH. The
positions of the major excision products are indicated. M,
size markers in nucleotides; FT, flow-through
fractions.
Figure 9: Human excision nuclease releases excised oligomers from gapped duplex. The 3`-terminally biotinylated, internally radiolabeled cholesterol-A substrate 156 nucleotides in length (Huang and Sancar, 1994; Shi et al., 1987) was incubated with E. coli or human excision nuclease. The DNA was then pulled down with streptavidin beads, and the streptavidin bead-bound (B) and -unbound (U) fractions were analyzed on a sequencing gel. Lane 1, size markers (M) in nucleotides; lanes 2 and 3, substrate DNA incubated only in reaction buffer and then separated by streptavidin beads; lanes 4 and 5, reaction with the bacterial enzyme; lanes 6 and 7, reaction with the human excision nuclease. The locations of bacterial (12-13 nucleotides) and human (26-28 nucleotides) excision products are indicated. The excision reaction with the E. coli UvrA, UvrB, and UvrC proteins was carried out as described by Thomas et al.(1985).
To investigate whether or not the excised oligomer and the gapped DNA were also released from the excision nuclease or its subunits or subassemblies, we conducted an excision assay using a mixture of internally and 5`-terminally labeled substrates. The reaction products were analyzed on nondenaturing polyacrylamide gels. As shown in Fig. 10A, three major bands (bands a-c) were observed in addition to free substrate (lane 2). The DNA within these bands was analyzed on sequencing gels. Fig. 10B (lane 5) shows that the slowest migrating band (band a) contained unrepaired DNA and DNA with an uncoupled 3`-nick. The middle band (band b) contained the excised oligomer, but not gapped DNA, as evidenced by the lack of 50-51-nucleotide-long fragments generated from the 5`-labeled DNA by 5`-incision (lane 6 versus 7). Finally, the fastest migrating DNA-protein complex (band c) contained the gapped DNA and no excised oligomer (lane 7). To ensure that band c was not protein-free gapped DNA with anomalous migration due to its unusual structure, the same reaction mixture was treated with proteinase K prior to analysis by native gels. As seen in Fig. 10A (lane 3), under these conditions, only two closely migrating bands were observed: the unrepaired DNA and a second, fainter band (band e). Analysis of band e on a sequencing gel revealed that it contained the gapped DNA, as evidenced by the presence of 50-51-nucleotide-long labeled oligomers (Fig. 10B, lane 9). Therefore, the retarded band c in lane 2 represents a protein-bound form of gapped DNA.
Figure 10: Analysis of postexcision complexes. A, analysis of reaction products using nondenaturing PAGE. Excision reaction with a mixture of internally and terminally labeled T<>T substrates was performed, and the products were separated on a 4% nondenaturing polyacrylamide gel. Lane 1, DNA alone; lane 2, total excision reaction mixture; lane 3, total excision reaction mixture treated with proteinase K. Nondenaturing gel electrophoresis conditions were as described by Mu et al.(1995). B, analysis of the DNA in the DNA-protein complexes on sequencing gels. Lane 4, size markers (M) in nucleotides; lanes 5-9, bands a-e, respectively, from the gels shown in A. The positions of the excision product at 29-30 nucleotides and of the 5`-incision at 50-51 nucleotides are indicated. Note that due to the large amount of unrepaired DNA, all retarded bands contained some full-length substrate as a contaminant.
In conclusion, these experiments reveal that even though the excised oligomer is released from the gapped DNA, both the excised oligomer and the gapped DNA are complexed with proteins. Currently, we do not know the identities of the proteins in these complexes. However, it is important that the excision gap is protected by repair proteins from nonspecific nucleases until repair synthesis takes place.
Using the excision repair factors purified to homogeneity, we have reconstituted human excision repair nuclease and investigated the roles of the individual components and the sequence of events in the excision reaction. We have used synthetic substrates with either a natural (thymine dimer) or an artificial (cholesterol) lesion at a unique site for these studies. Thus, the combination of repair factors of high purity and of uniform substrate has enabled us to probe the reaction mechanism in considerable detail. In previous reconstitution studies of both human (Mu et al., 1995; Aboussekhra et al., 1995) and yeast (Guzder et al., 1995) excision nucleases, the efficiency of the reconstituted systems was very low. Typically, <1% of the substrate was repaired. Such low efficiency raised the possibility that the reconstituted systems lacked some additional factors required for optimal activity. The present study shows that, in fact, no additional factors are required for the basal excision reaction. By having sufficient quantities of all of the repair factors, we were able to optimize the concentration of each in the reaction mixture so that the excision rate and extent were comparable to the in vivo values. Thus, we consider the results obtained with this system to be applicable to excision repair in vivo. In the following text, we attempt to address the roles of the various components in light of results presented in this paper and previous studies and present a reaction scheme (Fig. 11) consistent with existing data on human nucleotide excision repair.
Figure 11: Model for mechanism of human excision nuclease. Damage recognition is achieved by the combination of RPA-XPA (step 1), followed by the recruitment of TFIIH and XPC-HHR23B to locally perturb the DNA structure around the lesion in an ATP-dependent manner (step 2). The arrival of XPG through its interaction with TFIIH and RPA stabilizes the preincision complex and results in the 3`-incision (step 3). Subsequent recruitment of XPF-ERCC1 by XPA to the incision complex produces the 5`-incision (step 4). Upon the cutting by XPF-ERCC1 at the 5`-side, the excised lesion-containing oligomer is dissociated from the gapped duplex DNA and bound by protein (possibly XPC), leaving behind the gapped DNA protected by RPA from nucleases (step 5).
In light of the documented interactions between XPA and XPF-ERCC1 (Li et al., 1994; Park and Sancar, 1994) and data presented herein, it is tempting to speculate that XPA not only participates in damage recognition, but also acts as an anchor to recruit XPF-ERCC1 to the proper 5`-incision sites. In other words, this model predicts that XPA must be an integral part of the excision nuclease-DNA complex from the first step of damage recognition to the step prior to 5`-incision.
Human XP-F mutants exhibit a unique phenotype: shortly after UV irradiation, these cells repair damage at 10-15% of the normal level, but excision increases up to 60% with further incubation. Unlike most other XP mutants (with the exception of XP-E), XP-F cells are only moderately UV-sensitive (Cleaver and Kraemer, 1989). Surprisingly, however, CFEs from these mutants are totally defective in excision nuclease activity as determined by an assay capable of detecting 0.1% of normal excision activity (Reardon et al., 1993). In view of the results in this paper that XPF-ERCC1 is not required for 3`-incision, an explanation can be offered for this abnormal phenotype of XP-F cells: the other five factors of the excision nuclease assemble and make the 3`-incision, which ultimately leads to damage removal by nonspecific 3` to 5` acting exonuclease(s). Hence, the reaction occurs slowly compared with the normal excision. Such an explanation, in turn, raises an important question: is the uncoupled 3`-incision a normal event during the course of action of human excinuclease, and does it persist for a significant length of time until the XPF-ERCC1 complex diffuses to the lesion site and makes the 5`-incision? When excision reaction was carried out in HeLa CFE, futile 3`-nicking was a rare event, implicating the presence of ``a mechanism'' in CFE to maintain efficient coupling of the two nicks. This mechanism of keeping tightly coupled dual incision may be accomplished by several means. (i) The entire set of excision repair proteins may exist in a large complex termed ``repairosome,'' as has been suggested for yeast (Svejstrup et al., 1995). (ii) XPF-ERCC1 is actually recruited to the damage site early on, despite the failure to obtain supporting evidence in this study. (iii) An as yet unidentified protein can suppress 3`-incision by XPG. Upon displacement of this protein by XPF-ERCC1, both 3`- and 5`-incisions occur in a concerted but nonsynchronous manner. More experiments are needed to differentiate these models.