©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Reconstitution of Human DNA Repair Excision Nuclease in a Highly Defined System (*)

(Received for publication, November 23, 1994; and in revised form, December 18, 1994)

David Mu (§) Chi-Hyun Park Tsukasa Matsunaga David S. Hsu Joyce T. Reardon Aziz Sancar (¶)

From the Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7260

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Xeroderma pigmentosum is a hereditary disease caused by defective DNA repair. Somatic cell genetics and biochemical studies with cell-free extracts indicate that at least 16 polypeptides are required to carry out the repair reaction proper, i.e. the removal of the lesion from the DNA by the dual incisions of the damaged strand. To find out if these proteins are necessary and sufficient for excision repair, they were obtained at a high level of purity in five fractions. The mixture of these five fractions reconstituted the excision nuclease (excinuclease) activity. Using the reconstituted excinuclease, we found that the excised fragment remains associated with the post-incision DNA-protein complex, suggesting that accessory proteins are needed to release the excised oligomer.


INTRODUCTION

Xeroderma pigmentosum (XP) (^1)is a hereditary disease characterized by photosensitivity, increased frequency of skin cancers, and, in some cases, neurological abnormalities(1) . The disease is caused by a defect in nucleotide excision repair (2) as a result of mutations in one of several genes, XPA through XPG. In addition to the proteins defined by the XP complementation groups, genetic and biochemical studies in mammalian systems indicate that the ERCC1 protein, the transcription factor TFIIH (which contains the XPB and XPD proteins and six other proteins), and the replication proteins PCNA (proliferating cell nuclear antigen) and HSSB (human single stranded DNA-binding protein, also known as RPA) are required for excision repair(3, 4) . These studies raised the possibility that additional proteins, not previously identified, may be involved in the initial reaction of excision repair which consists of the incisions of the 22nd through 24th phosphodiester bonds 5` and the 4th through 6th phosphodiester bonds 3` to the lesion(5, 6) .

In this report, we purified the components known to be required for the incision reaction and here we provide strong evidence that these components are sufficient for this reaction. Moreover, we show that the excised damage-containing fragment is not released by this reconstituted excinuclease system, suggesting that additional helicase activity is required to displace the excised oligomer before repair synthesis can take place.


EXPERIMENTAL PROCEDURES

Materials

HeLa S3 and mutant cells were from the stock of the Lineberger Cancer Center (University of North Carolina). Mutant cell lines used in this study are: XP-A, XP20S; XP-B, UV24 (rodent counterpart); XP-C, XP1BE; XP-D, UV5 (rodent counterpart); XP-F, UV47 (rodent counterpart); XP-G, UV135 (rodent counterpart); ERCC-1, UV20.

Purification of Reconstitution Fractions

Fraction I (recombinant XPA) in the form of MBP-XPA fusion protein was purified as described previously(7) .

Fraction II (TFIIH/XPG)

TFIIH was purified using a procedure modified from Drapkin et al. (8) and Flores et al.(9) ; HeLa whole cell extract (10) derived from approximately 1.25 times 10 cells (250 liters) was used as starting material. The multi-protein complex TFIIH was detected by Western blotting using polyclonal antibodies raised against a peptide sequence derived from its 89-kDa subunit (XPB), whereas XPC, XPG, and ERCC1 were traced with polyclonal antibodies raised against each recombinant fusion (MBP) protein expressed in Escherichia coli. The first DEAE-52 column (Whatman) and the second Mono S preparative FPLC column (Pharmacia, HR 16/10) were essentially as described by Nichols and Sancar (11) and Flores et al.(9) , respectively. ERCC1bulletXPF was in the flow-through (FT), whereas TFIIH was in the gradient-eluted fractions. The TFIIH-containing fractions (400 mg) were dialyzed in buffer C (20 mM Tris HCl, pH 7.9, 0.1 mM EDTA, 19% glycerol, 10 mM beta-mercaptoethanol) and 1.2 M (NH(4))(2)SO(4), loaded onto a phenyl-Superose column (Pharmacia Biotech Inc., HR 10/10), and eluted with a linear 160-ml gradient of 1.2-0 M (NH(4))(2)SO(4) in buffer C. Fractions containing TFIIH were combined and applied to a Sephacryl S-300 column (Pharmacia, HiLoad 26/60) equilibrated in buffer C with 0.8 M KCl. Fractions containing XPB as determined by Western analyses were pooled (0.15 mg) and loaded onto an analytical Mono S (HR 5/5) column and eluted with a 20-ml gradient of 0.1-0.4 M KCl. Finally, the TFIIH fractions were chromatographed on a phenyl-Superose HR 5/5 column using a gradient of 0.45-0 M (NH(4))(2)SO(4) in 15 ml. For storage, the TFIIH fractions (90 µg) were concentrated in an Amicon 8000 cell, dialyzed in storage buffer (25 mM Hepes, pH 7.9, 100 mM KCl, 12 mM MgCl(2), 0.5 mM EDTA, 2 mM dithiothreitol, 16-17% glycerol), and kept at -80 °C. A separate batch of highly purified TFIIH, which had been shown to contain XPC (8) was obtained from Dr. D. Reinberg (University of Medicine and Dentistry of New Jersey, Piscataway, NJ).

Fraction III (XPC)

Twenty percent of the TFIIH fractions after the Mono S column was chromatographed on an analytical phenylSuperose column (HR 5/5) and eluted with a 25-ml linear gradient of 0.42-0 M (NH(4))(2)SO(4). The presence of XPC protein and the absence of TFIIH in the eluted fractions of 0.3 M (NH(4))(2)SO(4) were confirmed by Western blotting. These fractions were then concentrated with a pressured stirred cell (Amicon, series 8000), dialyzed in storage buffer, and designated ``partially purified XPC/Fraction III.''

Fraction IV (ERCC1bulletXPF)

Subsequent to the Mono S preparative column, the flow-through was precipitated using 60% (NH(4))(2)SO(4), dialyzed in buffer C with 0.1 M KCl, and loaded onto a Mono Q HR 10/10 column. Elution was achieved with an initial gradient of 0.1-0.3 M KCl in 200 ml, followed by an 100-ml gradient of 0.3-0.6 M KCl. Fractions containing ERCC1 (0.18-0.23 M KCl) were pooled and fractionated on a Sephacryl S-300 column as described above. As a final step, a glutathione S-transferase-XPA affinity column was used to selectively retain ERCC1bulletXPF complex according to Park and Sancar(12) . The final pool of ERCC1bulletXPF complex (<1 µg) was concentrated using the pressured stirred cell, dialyzed in storage buffer, and stored at -80 °C .

Replication protein A (also known as HSSB) was a kind gift from Drs. B. Stillman (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) and J. Hurwitz (Sloan-Kettering Cancer Center, New York, NY).

Excision Assay

This assay measures the excision of radiolabeled fragments carrying the lesions. The substrate is an 140-mer DNA containing a cholesterol instead of a base in the center. It has been found that this ``lesion'' is one of the best substrates for human excinuclease. (^2)The P label is 6 nucleotides 5` to the lesion, and the substrate is assembled with 6 oligonucleotides as described by Huang et al.(13) . The appearance of the P-labeled fragments of 23-30-mers was taken as the measure of excinuclease activity. The complementation reaction (excision assay) mixtures contain: 40 mM Hepes, pH 7.9, 80 mM KCl, 8 mM MgCl(2), 2 mM ATP, 20 µM of each dNTP, 1 mM dithiothreitol, 7% (v/v) glycerol, 100 µg/ml bovine serum albumin, 50 µg of mutant cell-free extracts (CFE), the indicated amounts of purified fraction, and 1 nMP-labeled substrate. After a 60-min incubation at 30 °C, the reaction mixtures were deproteinized with proteinase K, phenol/chloroform-extracted, precipitated with ethanol, and resolved on 10% denaturing polyacrylamide gels. The reconstitution reaction mixture contained 50 ng of Fraction I, 5 ng of Fraction II, 20 ng of Fraction III, 1 µl (<1 ng) of Fraction IV, and 90 ng of RPA in the same reaction buffer, and the reaction product was processed similarly.

Analyses of Excision Reaction Products by Non-denaturing Polyacrylamide Gels

Excision assays were performed using the reconstituted excinuclease of Fractions I-V as described above. Following a 60-min incubation at 30 °C, M13 single-stranded DNA (0.5 µg) and/or SDS (0.4%) were added to the reaction mixtures, which were incubated at 30 °C for additional 10 min and then subjected to gel electrophoresis on a 12% nondenaturing polyacrylamide gel containing 5% glycerol, at a constant voltage of 150 V in 0.5 times TBE (45 mM Tris borate, 1 mM EDTA).


RESULTS AND DISCUSSION

Purified Components

The damage recognition factor (3) XPA protein (Fraction I) was purified from an E. coli strain expressing it as a fusion protein in the form of MBP-XPA by affinity chromatography on amylose, followed by further purification on heparin-agarose(7) . The biological activity of MBP-XPA was detected by its ability to restore the excinuclease activity in XP-A mutant cell-free extract(7) .

The XPB (p89) and XPD (p80) proteins make a tight complex (14) within the transcription factor TFIIH, which contains six other subunits (p62, p50, p44, p41, p38, and p34) of which p62, p44, and p34 are also known to be required for excision repair(15, 16, 17) . Therefore, in order to purify TFIIH and other repair proteins, we followed the purification scheme outlined in Fig. 1. The purified TFIIH (Fraction II) contained the p89 (XPB), p80 (XPD), p62, p50, p44, p41, p38, and p34 subunits visible in silver-stained SDS-PAGE (Fig. 2B) as well as some higher molecular weight proteins also seen in other TFIIH preparations(8, 18, 19) , one of which we identified as the XPG protein (see below). The identities of the p89, p80, and p62 subunits were confirmed by Western blotting using a mixture of antibodies made against the XPB, XPD, and p62 proteins (Fig. 2B, lane 3). Furthermore, the TFIIH preparation complements the excinuclease activity in the CFEs of XP-B and XP-D mutants as well as the CFE of an XP-G mutant (Fig. 3). This is reminiscent of the findings in yeast, as it is known that the yeast homologs of XPC (Rad 4) and XPG (Rad 2) are loosely associated with the yeast TFIIH counterpart, Factor b(20) .


Figure 1: Purification scheme for the reconstitution fractions of the human excinuclease.




Figure 2: Analyses of the five reconstitution fractions by silver staining and/or immunoblotting. Approximately 1-5 ng of each reconstitution fraction was subjected to 10% SDS-PAGE analyses. A, SDS-PAGE of Fraction I (MBP-XPA). B, lane 1 shows SDS-PAGE of Fraction II (TFIIH/XPG) (the TFIIH subunits are indicated by arrows); lane2 shows the immunoblotting with anti-XPG antibodies; lane3 shows immunoblotting with anti-XPB, anti-XPD and anti-p62 antibodies. C, immunoblotting of Fraction III (partially purified XPC) with anti-XPC polyclonal antibodies. D, lanes1-3, SDS-PAGE of load (L), flow-through (FT), and elution (E) of fractions of ERCC1bulletXPF complex on a glutathione S-transferase-XPA affinity column. Lanes 4-6, Western blot of the same fraction. Lanes 3 and 6 contained 30 µl of the eluted (E) fraction (<1 ng of protein). E, SDS-PAGE of Fraction V (HSSB). F, SDS-PAGE of a highly purified TFIIH preparation using a seven-column purification protocol(8) . This purification procedure yields TFIIH complementing XP-B, XP-D, and XP-C but not any of the other XP complementation groups(8) . Lane 2 shows the immunoblotting using anti-XPC polyclonal antibodies.




Figure 3: Complementation of mutant CFEs by the purified fractions. Top panel, schematic drawing of the incision pattern of human excinuclease on the P-labeled 140-mer DNA-containing a cholesterol adduct in the center. The arrows indicate the major incision sites, which generate a 28-nt-long oligomer. Bottom panel, A, complementation of XP-A mutant CFE (50 µg) by Fraction I (MBP-XPA, 50 ng); B, complementation of rodent mutant CFE defective in XPB, XPD, and XPG by Fraction II (2 ng); C, complementation of XP-C mutant CFE by fraction III (60 ng); D, complementation of ERCC-1 and ERCC-4 mutant CFEs by Fraction IV (<1 ng). Comparable complementation of XP-F mutant CFE by Fraction IV was observed (not shown).



In fact, a different purification scheme yielded TFIIH associated with XPC protein(8) . In this current purification scheme, XPC was separated from TFIIH on the third column and designated reconstitution Fraction III (see below).

The ERCC1 and XPF proteins are in a complex (14, 21, 22) and can be purified by following ERCC1. The complex was purified as outlined in Fig. 1. After chromatography on Sephacryl S-300, the fractions eluting at approximately 200 kDa were found to contain ERCC1 by Western analysis. Highly purified ERCC1bulletXPF complex was obtained by passing the sample through a glutathione S-transferase-XPA affinity column. This procedure removed all of the HeLa cell derived contaminants from the preparation (12) (Fig. 2D, lanes 1-3). Protein bands were not visible by silver staining using 3% of our last purification fraction (Fig. 2D, lane 3), but immunoblotting (lanes 4-6) using anti-ERCC1 indicated quantitative recovery of ERCC1 in this fraction (lane 6). Furthermore, this fraction complemented both ERCC-1 and XP-F mutant cell-free extracts (Fig. 3D). As shown in Fig. 2D (lane 6), this is our reconstitution Fraction IV.

Fraction V is HSSB. This three-subunit protein was purified from E. coli cells co-expressing the three subunits(23) .

Reconstitution

Fig. 4shows the results of the reconstitution experiments. Surprisingly, the mixture of Fractions I-IV, which contains XPA, XPB, XPC, XPD, XPF, XPG, ERCC1, and all of the subunits of repair factor TFIIH, failed to reconstitute the excision nuclease activity (lane 1). A previous study had implicated HSSB (RPA) in the early steps of excision repair(24) . In that study the repair synthesis assay was used, and thus it was uncertain whether HSSB was required for incision. However, the study did raise the possibility that HSSB might be needed for steps prior to repair synthesis. Hence, we decided to add homogeneous HSSB (Fraction V) to our reaction mixture (Fig. 4, lane 2). This addition resulted in excinuclease activity at a level comparable to that obtained with HeLa cell-free extract (lane 3), providing the first direct evidence that HSSB is required for the dual incision reactions, in addition to its potential function in repair synthesis.


Figure 4: Reconstitution of human excinuclease. Lane 1, Fractions I-IV; lane 2, Fractions I-V; lane 3, HeLa CFE; lane 4, Fractions I, II, IV, V, and TFIIH/XPC (TFIIH/XPC was from Dr. D. Reinberg); lane 5, Fractions I, II, IV, and V. The excision assays were carried out as described under ``Experimental Procedures'' except that dNTPs were omitted. The total amount of proteins present in each reaction from lane1 to lane 5 is approximately 75 ng, 165 ng, 50 µg, 147 ng, and 145 ng, respectively.



Since the XPC protein (Fraction III) used in reconstitution (Fig. 4, lanes 1 and 2) was an early purification fraction, we tested another XPC preparation purified as part of the TFIIH factor to a high level of purity by a different scheme (8) to ascertain that only XPC in Fraction III was needed for reconstitution. Fig. 4(lane 4) shows that this substitution also led to reconstitution of the excinuclease activity, whereas no signal was detected with the mixture lacking the highly pure TFIIH/XPC fraction (lane 5). In addition, we have obtained highly purified XPC free of any of the other XP proteins by the method of Masutani et al. (25) and have reconstituted the excinuclease activity with this protein (data not shown). The results in Fig. 4thus lead us to propose that these five fractions are necessary and sufficient for the excision step of human nucleotide excision repair. Of special note, none of these five fractions complemented an XP-E mutant cell extract (which has residual excision activity), suggesting that the XPE protein is not necessary for making the dual incisions. (^3)Thus, we hypothesize that XPE and the proteins defined by the moderately UV-sensitive rodent complementation groups 6-11 (see (26, 27, 28) ) are accessory factors, which may stimulate excision repair but are not required for the basal reaction.

Release of the Excised Fragment

Both XPB (8, 29) and XPD (30) , the subunits of TFIIH, as well as TFIIH (15) have helicase activities. Thus, it has been postulated that TFIIH is responsible for unwinding DNA and releasing the excised fragment from the complementary strand(31) . Given the highly defined system, we wished to test this proposal by analyzing the excision assay mixture using non-denaturing polyacrylamide gels. Under our experimental conditions, all of the DNA including the excised fragment was retained in the origin, presumably bound to HSSB and other DNA-binding proteins in a nonspecific manner (Fig. 5, lane 2). Addition of competitor M13 single-stranded DNA dissociated most of the substrate from these nonspecific complexes, but only a very small fraction of the excision product was released (lane 3). In contrast, treating the reaction mixture with SDS quantitatively released the excised oligomer (lane4). Heat denaturation at 95 °C for 2 min did not increase the level of released excision product (lane5), suggesting that the oligomer does not remain bound to the duplex following the SDS-induced dissociation of the excinuclease. Taken together, these results indicate that the excised oligomer remains in the post-incision complex and that additional proteins are required to release the damage-containing oligomers following dual incisions.


Figure 5: The excised oligomer remains in the post-incision complex. Excision reaction was carried out with the reconstituted system, and the reaction mixture was loaded onto 12% polyacrylamide native gel following the indicated additions/treatments. The positions of the markers (24- and 27-mers), the substrate (140-mer), and the excision product (28-mer) are indicated by arrows. Heating was achieved at 95 °C for 2 min.



In conclusion, we have reconstituted the human excinuclease in a highly defined system. We believe that the proteins known to exist in our system (XPA, TFIIH (XPB and XPD), XPC, XPF, XPG, ERCC1, and HSSB) constitute a minimal set necessary for dual incision. The possibility that some of the other proteins that exist in our system as ``contaminants'' participate in excision cannot be eliminated until the entire system is reconstituted with recombinant proteins. However, the availability of this highly defined system now makes it possible to investigate the roles of individual XP proteins in excision and to dissect the individual steps of human excision repair. Indeed, this system has enabled us to determine that proteins in addition to those known to exist in our system are required to dissociate the post-incision complex and release the excised oligomer.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant GM32833 and a grant from the Human Frontier Science Program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by Grant DRG-1319 from the Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation.

To whom correspondence should be addressed. Tel.: 919-962-0115; Fax: 919-966-2852.

(^1)
The abbreviations used are: XP, xeroderma pigmentosum; CFE, cell-free extract; ERCC, excision repair cross-complementing; HSSB, human single-stranded DNA-binding protein; MBP, maltose-binding protein; RPA, replication protein A; PAGE, polyacrylamide gel electrophoresis.

(^2)
J. Reardon and A. Sancar, unpublished results.

(^3)
A. Kazantsev and A. Sancar, unpublished results.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.