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Inhibition of nucleotide excision repair by anti-XPA monoclonal antibodies which interfere with binding to RPA, ERCC1, and TFIIH
BBRC Biochemical and Biophysical Research Communications 321 (2004) 815–822 www.elsevier.com/locate/ybbrc
Inhibition of nucleotide excision repair by anti-XPA monoclonal antibodies which interfere with binding to RPA, ERCC1, and TFIIH Masafumi Saijo a,b, Toshiro Matsuda a, Isao Kuraoka a,b, Kiyoji Tanaka a,b,* a b
Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamada-oka, Suita, Osaka 565-0871, Japan Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, 1-3 Yamada-oka, Suita, Osaka 565-0871, Japan Received 2 July 2004 Available online 24 July 2004
Abstract The xeroderma pigmentosum group A protein (XPA) binds to three nucleotide excision repair (NER) factors: RPA, ERCC1, and TFIIH. XPA also binds preferentially to UV- or chemical carcinogen-damaged DNA. In this study, we prepared anti-XPA monoclonal antibodies and examined their effects on NER. Two clones inhibited cell-free NER reactions. The mode of inhibition appeared to differ; one clone inhibited both 5 0 and 3 0 incisions equally while the other inhibited the 5 0 incision more. The two clones inhibited the binding of XPA to RPA, ERCC1, and TFIIH. They did not inhibit the binding to damaged DNA either. These results suggest that the interaction of XPA with these NER factors is essential to the NER pathway. The epitopes of these antibodies were located outside of the binding regions for these NER factors. Steric hindrance or conformational changes of XPA brought about by the binding of anti-XPA IgG possibly cause the inhibitory effects. 2004 Elsevier Inc. All rights reserved. Keywords: Nucleotide excision repair; XPA; Monoclonal antibody; Protein interaction
Nucleotide excision repair (NER) is a versatile DNA repair system correcting a broad spectrum of DNA damage, including ultraviolet (UV)-induced cyclobutane pyrimidine dimers and (6–4) photoproducts as well as chemical carcinogen-induced lesions [1]. There are two subpathways in NER [2]. One is transcription-coupled repair (TCR), which efficiently removes the damage in the transcribed strand of transcriptionally active genes. The other is global genome repair (GGR), which occurs throughout the genome including the non-transcribed strand of active genes. Xeroderma pigmentosum (XP) is an autosomal recessive human disease characterized by hypersensitivity to sunlight and a high incidence of skin cancer on sun-exposed skin [1,3]. Cells from XP patients are hypersensitive *
Corresponding author. Fax: +81-6-6877-9136. E-mail address: [email protected] (K. Tanaka).
0006-291X/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.07.030
to killing by UV irradiation because of a defect in NER. XP is classified into seven complementation groups (XP-A to XP-G) and a variant form [1]. Except for the variant form, the primary defect of XP resides in the early steps of NER: damage recognition and dual incisions of the damaged strand on both sides of the DNA lesion [3]. Both TCR and GGR of NER are defective in groups A–G but not groups C and E, in which only GGR is impaired [4–6]. To date, all XP genes have been cloned. In addition, the ERCC1 protein, encoded by a human gene that can correct the repair deficiency in a UV-sensitive, NER-defective, rodent mutant cell line, is also involved in NER. The core reaction of GGR in humans has been reconstituted in vitro with purified XP and other proteins, and an outline of the GGR mechanism has been elucidated [7]. The XPA protein, consisting of 273 amino acid residues, binds to RPA, ERCC1, and TFIIH as well
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as UV- or chemical carcinogen-damaged DNA [8,9], and participates in the formation of an open complex [10]. We also identified two XPA-binding proteins, XAB1 and XAB2, by yeast two-hybrid screening [11,12]. Several distinct functional regions in XPA have been identified. The N-terminal region (residues 4–29) of the protein is responsible for binding to a 34 kDa subunit of RPA [13,14] and the basic amino acid region (residues 30–42) is important for the localization of XPA protein to the nucleus [15]. The region containing the glutamic acid-cluster (E-cluster) (residues 78– 84) is important for the interaction with ERCC1 [16,17]. The C-terminal region (residues 226–273) is shown to bind to TFIIH [18,19]. The central region is identified as the minimal polypeptide (residues 98– 219) for preferential binding to damaged DNA [20] and the region (residues 98–187) necessary for binding to 70 kDa subunit of RPA [13,14]. In addition, the binding of XPA to damaged DNA is markedly increased by the interaction of XPA with the NER factors [16,19,21,22]. These interactions should function to co-ordinate the early stages of NER [23]. Here, we obtained anti-XPA monoclonal antibodies that inhibit GGR reaction in vitro and analyzed the correlation between the inhibitory effects and molecular interactions of XPA.
Materials and methods Preparation of antibodies. His-tagged XPA protein and His-tagged XPA98–219 were produced in Escherichia coli, purified as described previously [8,20], and used as antigens. Four Balb/c mice were immunized with TiterMax (CytRx) as an adjuvant according to the procedure supplied by the manufacturer. The splenocytes from immunized mice were fused with mouse myeloma cells according to the standard procedure [24]. After selection in HAT medium, spent media from hybridomas were screened with the enzyme-linked immunosorbent assay (ELISA) on antigen-immobilized plates and Western blotting using the purified XPA protein. The limiting dilution method was used for the cloning of hybridomas producing anti-XPA antibodies and repeated twice. To purify anti-XPA IgGs, the hybridomas were cultured in a serum-free medium (S-Clone SF-O, Sankyo Junyaku). IgGs were precipitated with 50% saturated ammonium sulfate from the spent media and resuspended in a buffer containing 25 mM Hepes–KOH, pH 7.8. Then, the IgG fraction was purified with column chromatography, HiLoad 16/60 Superdex 200 pg (Amersham Biosciences), with a buffer containing 25 mM Hepes–KOH, pH 7.8, and 20 mM KCl, and Mono Q HR 5/5 (Amersham Biosciences) with a linear gradient of KCl from 20 to 500 mM in a buffer containing 25 mM Hepes–KOH, pH 7.8, successively. The eluted fractions were subjected to SDS–polyacrylamide gel electrophoresis and the gel was stained with Coomassie brilliant blue. The fractions containing IgG were pooled and dialyzed against a buffer containing 25 mM Hepes–KOH, pH 7.8, and 100 mM KCl. The isotype of the anti-XPA IgGs was determined using a Mouse monoclonal antibody isotyping kit (Amersham Biosciences). Immunoblotting. Cell lysates were prepared by adding NETN [50 mM Tris–HCl, pH 7.8, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, and complete protease inhibitor cocktail (Roche)] at a concen-
tration of 2 · 107 cells/ml and incubated at 0 C for 30 min. The lysates were centrifuged for 10 min at 13,000g and the supernatant was recovered. Ten micrograms of protein was separated by SDS–PAGE and transferred to PVDF membrane. XPA protein was detected using the ECL plus Western blotting detection reagents (Amersham Biosciences). Truncated XPA proteins were produced in E. coli as GST fusion proteins and purified as described previously [13]. Dual incision assay. Effects of monoclonal antibodies on the NER reaction were examined by dual incision assay. Covalently closed circular DNA containing a single 1,3-intrastrand d(GpTpG)-cisplatin adduct (Pt-GTG) was prepared as described [25]. HeLa whole cell extract (100 lg of protein, containing about 5 pmol of XPA) was preincubated with purified anti-XPA antibody for 30 min at 30 C and an incision assay was carried out as described [10] using 150 ng PtGTG in a total volume of 25 ll. After the reaction, DNA was purified and digested with HindIII and XhoI in a total volume of 10 ll. The DNA was separated by denaturing 12% polyacrylamide gel electrophoresis and transferred by capillary action for 2 h from the gel to a positively charged nylon membrane (Immobilon-Ny+, Millipore) soaked in 0.9 M Tris–borate, 20 mM EDTA. DNA was fixed to the membrane with UV cross-linking according to the manufacturerÕs instructions. The blots were incubated with a 32P-labeled oligonucleotide (5 0 -GAAGAGTGCACAGAAGAAGAGGCCTGG-3 0 ) in hybridization buffer (7% SDS, 10% polyethylene glycol 8000, 250 mM NaCl, and 130 mM potassium phosphate, pH 7.0) for 16 h at 42 C and washed for 10 min in 2· SSC, 0.1% SDS before being exposed to X-ray film with an intensifying screen or an imaging plate for the Bio-imaging analyzer BAS-2500 (Fujifilm). DNA-binding assay. XPA protein was preincubated with the purified anti-XPA antibody for 4 h at 4 C. Nitrocellulose filter-binding assay was performed as described previously [20] with some modifications. The EcoRI-digested pUC19 was labeled with Klenow fragment in the presence of [a-32P]dATP and [a-32P]TTP and irradiated with 8 kJ/m2 of UV. The labeled DNA was added to the above protein mixture and incubated for 1 h at 4 C. DNA bound to XPA protein was collected by filtration on a nitrocellulose filter. Protein interaction assay. Recombinant RPA and 6· His-tagged ERCC1 protein were purified as described previously [13,26]. One hundred nanograms of purified RPA, 6· His-tagged ERCC1 protein, or TFIIH was incubated in 100 ll PBS for 1 h at 37 C to be immobilized on 96-well plates and unbound proteins were washed out with PBS. The wells were incubated with PBST (PBS–0.1% Tween 20) containing 3% skim milk for 1 h at room temperature and washed twice with PBS. XPA protein (10 ng) was preincubated with various amounts of purified anti-XPA antibody in 100 ll PBST containing 1% skim milk and 1 mM DTT for 4 h at 4 C. The mixture was then incubated in the RPA-, ERCC1-, or TFIIH-immobilized wells overnight at 4 C. Unbound proteins were removed by washing three times with PBST. The wells were then incubated with 1000-fold diluted anti-XPA polyclonal antibody (Santa Cruz Biotechnology) in PBST containing 1% skim milk for 1 h at room temperature and washed five times with PBST. Next, the wells were incubated with 1000-fold diluted alkaline phosphatase-conjugated anti-rabbit IgG (Cappel) in PBST containing 1% skim milk for 1 h at room temperature. Then the wells were washed five times with PBST and twice with AP buffer (100 mM Tris–HCl, pH 9.5, 100 mM NaCl, and 5 mM MgCl2), and incubated with 100 ll AP buffer containing 1 mg/ml p-nitrophenyl phosphate. The reaction was stopped with 100 ll of 1 N NaOH and the absorbance was measured at 405 nm. In the experiments of Fig. 4B, XPA protein was first incubated in the RPA-, ERCC1, or TFIIH-immobilized wells. Unbound XPA proteins were removed by washing three times with PBST. The wells were then incubated with various amounts of purified anti-XPA antibody in 100 ll PBST containing 1% skim milk and 1 mM DTT to examine the effects of anti-XPA antibodies on the interactions between these proteins.
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Results Preparation of anti-XPA monoclonal antibodies To investigate the functions of XPA protein, we prepared anti-XPA monoclonal antibodies. In the course of screening by ELISA, anti-gamma chain-specific antibody was used as secondary antibody to obtain hybridomas producing anti-XPA IgG and 11 ELISA-positive
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hybridoma clones were obtained. Next, we tested the specificity of the antibodies against the XPA protein by Western blotting using whole cell extracts from HeLa and XPA-deficient (XP12ROSV) cells. Three monoclonal antibodies (1H2, 5F12, and 10B4) showed good specificity for XPA (Fig. 1). These antibodies hardly reacted with other NER proteins in both extracts. We purified IgGs from serum-free spent media of the three hybridomas by ammonium sulfate-precipitation and column chromatography, and used them for subsequent experiments. Effects of anti-XPA antibodies on NER reactions in vitro
Fig. 1. Specificity of anti-XPA antibodies. Cell lysates (10 lg of protein) prepared from HeLa (lane 1) and XP12ROSV (lane 2) cells were subjected to immunoblotting with anti-XPA monoclonal antibodies.
To explore the effect of the antibodies on NER reactions in vitro, we applied the dual incision assay at a single 1,3-intrastrand d(GpTpG)-cisplatin lesion in a closed circular duplex DNA substrate (Fig. 2A). Platinated 23to 30-mer oligonucleotides were released by dual incision after the incubation of the DNA with HeLa whole cell extract (lane 2), but oligonucleotides with no damage were not (lane 1). The whole cell extract was preincubated with each anti-XPA antibody and then used for the incision assay. Two anti-XPA antibodies (1H2 and 5F12) inhibited the incisions around the lesion in a dose-dependent manner, but the other anti-XPA
Fig. 2. Effects of anti-XPA antibodies on dual incision. (A) HeLa whole cell extract (100 lg of protein) was preincubated with 0 (lane 2), 0.1 (lanes 3, 6, 9, and 12), 0.5 (lanes 4, 7, 10, and 13) or 1.0 lg (lanes 5, 8, 11, and 14) of purified anti-XPA antibody or control mouse IgG for 30 min at 30 C. Closed circular DNA containing a single 1,3-intrastrand d(GpTpG)-cisplatin cross-link was then added to give a total volume of 25 ll in the reaction mixture. After incubation for 30 min at 30 C, reaction products were purified, digested with HindIII and XhoI, separated in denaturing polyacrylamide gel, transferred to a charged nylon membrane, and hybridized with a 32P-labeled oligonucleotide probe complementary to the DNA sequence surrounding the cisplatin–DNA adduct. Lane 1, DNA without the cisplatin cross-link was used. (B) Quantification of (A). Values for the dual incisions (23- to 32-mers) and the uncoupled 3 0 incisions (48- and 49-mers) are displayed as a percentage of the value where no antibody was added. Closed circle, mouse IgG; open circle, 1H2; closed square, 5F12; open square, 10B4.
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antibody (10B4) and control mouse IgG did not, indicating that the inhibitory effects were specific to these two antibodies. The effect of 5F12 on the dual incisions was greater than that of 1H2 (Fig. 2B). Uncoupled 3 0 incision products were also decreased in correlation with dual incision products by 1H2. But, the amounts of some uncoupled 3 0 incision products remained constant even at higher doses of 5F12 where the dual incision products almost disappeared (Figs. 2A and B). It is considered that 1H2 inhibits both 5 0 and 3 0 incisions equally while 5F12 inhibits the 5 0 incision more than the 3 0 incision. Effects on the binding of XPA protein to damaged DNA To determine the molecular mechanism by which the incision reactions in vitro were inhibited by these antiXPA antibodies, the DNA-binding activity of XPA protein was examined in the presence of anti-XPA antibodies. XPA protein was preincubated with each antibody, then the binding was assayed with a nitrocellulose filter (Fig. 3). XPA protein incubated with antiRPA34 monoclonal antibody (as an unrelated control) or buffer alone preferentially bound to UV-irradiated DNA. In the two experimental conditions used, where IgG doses were relatively high, the three anti-XPA antibodies showed stimulatory rather than inhibitory effects on the binding activity. The antibodies did not affect the activity to bind undamaged DNA. Effects on NER protein interactions XPA protein binds to three NER factors, ERCC1 protein, RPA, and TFIIH. To analyze the effects of the anti-XPA antibodies on the interactions of these proteins, we employed an ELISA and determined the conditions for the assay. Equal amounts of ERCC1 protein, RPA, or TFIIH were immobilized on a 96-well plate and then various amounts of XPA protein were incubated with the immobilized protein. The bound XPA
protein was detected with anti-XPA polyclonal antibody. The absorbance linearly correlated with the amount of XPA up to 0.75 pmol (data not shown), indicating that the binding of XPA to ERCC1, RPA, or TFIIH was not saturated. Thereafter, we used 0.3 pmol of XPA protein for the binding assay. As shown in Fig. 4A, the binding of XPA to RPA was inhibited by the preincubation of XPA with 1H2 and 5F12, but not 10B4, which did not inhibit repair reactions in vitro. We also observed that 1H2 and 5F12 inhibited the binding of XPA to ERCC1 and TFIIH. Inhibitory effects on the binding of XPA to ERCC1 protein by the antibodies appeared stronger than those on the binding to RPA. 1H2 and 5F12 inhibited the binding of XPA to TFIIH to a lesser extent than the binding to ERCC1 and RPA and the inhibition was about 50% at the high IgG dose. 10B4 did not inhibit the binding of XPA to ERCC1or TFIIH. When the antibodies were added after XPA protein first bound to RPA, ERCC1 protein, or TFIIH, bound XPA protein was not released by the addition of each antibody (Fig. 4B). These results indicate that anti-XPA antibodies 1H2 and 5F12 could interfere with the association of XPA with these three NER factors but could not dissociate XPA from the NER factors once the binding had occurred. Epitope mapping and subclasses of the antibodies Binding regions of the monoclonal antibodies were determined by Western blotting and ELISA using recombinant truncated XPA proteins. Representative results of Western blotting are shown in Fig. 5A. 1H2 and 5F12 showed the same pattern. These antibodies reacted to amino acid residues 1–137 (lane 1), 1–97 (lane 8), 1–57 (lane 9), 25–47 (lane 11), and 30–273 (data by ELISA) of XPA protein. 10B4 reacted to residues 1– 137 (lane 1), 98–219 (lane 3), and 98–115 (lane 4). The results are summarized in Fig. 5B and indicated that 1H2 and 5F12 bind to the region from the amino acid residue 30 to 47 of XPA protein and that 10B4 binds to the region from 98 to 115. The subclasses of the IgGs were determined by ELISA; 1H2 and 10B4 were IgG1, and 5F12 was IgG2b. 1H2 and 5F12 recognized the same region in XPA protein, but were different subclasses of IgG. We therefore deduced that the hybridomas producing 1H2 and 5F12 were different clones.
Discussion Fig. 3. Effects of anti-XPA antibodies on the binding of damaged DNA by XPA. (A) Two picomoles of XPA protein was preincubated with 2 lg of purified anti-XPA antibody for 6 h at 4 C. UV-irradiated (8000 J/m2) or unirradiated DNA labeled with 32P (2.5 ng) was then added. After incubation for 1 h at 4 C, DNA bound to XPA protein was collected by filtration on nitrocellulose filters. (B) One picomole of XPA, 5 lg IgG, and 5 ng DNA were used.
NER is a pathway in which more than 25 polypeptides are involved. At least in mammalian cells, NER factors are considered to assemble sequentially at DNA damage sites [27–30]. It has been reported that some antibodies against NER factors inhibit NER
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Fig. 4. Effects of anti-XPA antibodies on protein interactions. (A) XPA protein (0.3 pmol) was preincubated with 0, 0.05, 0.1, 0.5, or 1.0 lg of each purified anti-XPA monoclonal antibody for 4 h at 4 C. The mixture was then incubated in RPA-, ERCC1-, or TFIIH-coated wells. (B) XPA protein (0.3 pmol) was incubated in RPA-, ERCC1-, or TFIIH-coated wells. After unbound XPA protein was washed out, the wells were incubated with 0, 0.5, 1.0, or 2.0 mg of each purified anti-XPA monoclonal antibody. Bound XPA protein was detected by ELISA using anti-XPA polyclonal antibody. Closed circle, mouse IgG; open circle, 1H2; closed square, 5F12; open square, 10B4.
reactions in vitro. Anti-RPA monoclonal antibodies caused inhibition of the repair reaction in whole cell extracts [31,32]. Anti-XPG and anti-ERCC1 polyclonal antibodies inhibited the 3 0 incision and both the 5 0 and 3 0 incisions, respectively [33]. Addition of anti-XPA polyclonal antibody to whole cell extracts inhibited the formation of dual incision products [34], but the mechanism of the inhibition was not elucidated. In the present study, we prepared and characterized anti-XPA monoclonal antibodies. Two antibodies (clones 1H2 and 5F12) inhibited NER reactions in vitro. They also inhibited the binding of XPA protein to RPA, ERCC1 protein, and TFIIH, but not to damaged DNA. These results indicate that the binding of XPA protein to RPA, ERCC1 protein, and/or TFIIH is important for the NER reactions. Consistent with our results, XP-A cells expressing XPA with a deletion of the binding region for ERCC1 [35], RPA [14], or TFIIH [15] have been shown to be UV-sensitive. Both 1H2 and 5F12 bind to the region spanning amino acid residues 30–47 of XPA protein. This area contains several basic residues and is considered a putative nuclear localization signal [15], but is outside of the regions required for binding to RPA, ERCC1 protein, and TFIIH (Fig. 5B). The XP-A cells expressing XPA with a deletion of the epitope region showed the same level of UV-resistance as normal cells [15], indicat-
ing that this region is not essential for the repair function. As the IgG molecule is larger than XPA protein, binding of the IgGs to XPA protein even outside of the binding regions could hinder the bindings. Alternatively, binding of the IgGs could cause some structural change in XPA protein that inhibits the binding to RPA, ERCC1 protein, and TFIIH. Although the whole structure of XPA protein has not been determined, it was shown that the ERCC1-binding domain containing a negatively charged glutamic acid-cluster might associate with the positively charged surface in the RPA70/ DNA-binding domain [36]. In addition, it was suggested that conformational changes of XPA protein could be related to its binding to TFIIH [19]. These structural changes could affect the protein interactions. We detected some differences in the inhibitory effects of the antibodies on incisions. The effects on protein interactions were similar, but inhibition of the dual incisions by 5F12 was stronger than that by 1H2. In addition, the efficacy of the antibodies to the 5 0 and 3 0 incision sides appeared to be different. XPA participates in the formation of an unwound open DNA intermediate around a lesion [10]. We speculate that the spatial position of XPA in the open complex is affected differently by the addition of each antibody, which could cause the differences in the inhibition of incision reactions.
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Fig. 5. Epitope mapping of the antibodies. (A) Truncated XPA proteins were subjected to immunoblotting. The regions of XPA protein used are amino acid residues 1–137 (lane 1), 138–273 (lane 2), 98–219 (lane 3), 98–115 (lane 4), 116–219 (lane 5), 116–273 (lane 6), 98–273 (lane 7), 1–97 (lane 8), 1–57 (lane 9), 53–137 (lane 10), and 25–47 (lane 11). (B) Schematic presentation of the functional regions in XPA proteins and the results of immunoblotting and ELISA. NLS, putative nuclear localization signal; E-cluster, the glutamic acid residue cluster; and Zn-finger, C4-type zinc finger motif.
XPA protein binds damaged DNA [8,9], and functional roles for XPA protein in damage verification during NER are proposed [37–39]. The minimal DNAbinding domain has been identified to be a 122 amino acid region between M98 and F219 that contains a C4type zinc finger motif [20] and overlaps with the RPA70-binding region [13]. The structure of the DNA/ RPA70-binding domain was determined by nuclear magnetic resonance spectroscopy [40,41]. The domain consists of a zinc-containing subdomain and a C-terminal subdomain. The epitope of 10B4 was located within amino acid residues 98–115. These residues form an antiparallel b-sheet that encompasses two cysteine residues, Cys105 and Cys108, whose side chains are co-ordinated by a zinc ion. This region is supposed to be a binding surface for RPA and becomes more mobile in the presence of DNA [42]. We expected the antibody 10B4 to affect the binding of XPA protein to damaged DNA. But it did not hinder XPA protein from binding
to either damaged DNA or NER proteins. No other clones we obtained inhibited the binding of damaged DNA either. Therefore, we could not clarify the significance of the binding activity of XPA protein in the NER reactions. Many proteins are involved in NER, and interact to co-ordinate the reaction. XPA is a critical factor in NER, because a deficiency of XPA causes severe sensitivity to UV. Although XPA protein has no enzymatic activity, it would function as a core component in NER reactions by interacting with damaged DNA, RPA, ERCC1 protein, and TFIIH. The present results indicate the significance of the interactions between XPA protein and the NER factors, but we could not show the role of individual interactions using monoclonal antibodies. Detailed analyses should be performed using XPA proteins with single amino acid substitutions that affect the interaction with each NER factor.
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Acknowledgments We thank Drs. R.D. Wood and J.G. Moggs for instruction in the cell-free dual incision assay, Dr. J.-M. Egly for TFIIH and Dr. Y. Nakatsu for helpful suggestions. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, and CREST of Japan Science and Technology (JST).
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[13] M. Saijo, I. Kuraoka, C. Masutani, F. Hanaoka, K. Tanaka, Sequential binding of DNA repair proteins RPA and ERCC1 to XPA in vitro, Nucleic Acids Res. 24 (1996) 4719–4724. [14] L. Li, X. Lu, C.A. Peterson, R.J. Legerski, An interaction between the DNA repair factor XPA and replication protein A appears essential for nucleotide excision repair, Mol. Cell. Biol. 15 (1995) 5396–5402. [15] I. Miyamoto, N. Miura, H. Niwa, J. Miyazaki, K. Tanaka, Mutational analysis of the structure and function of the xeroderma pigmentosum group A complementing protein. Identification of essential domains for nuclear localization and DNA excision repair, J. Biol. Chem. 267 (1992) 12182–12187. [16] A. Nagai, M. Saijo, I. Kuraoka, T. Matsuda, N. Kodo, Y. Nakatsu, T. Mimaki, M. Mino, M. Biggerstaff, R.D. Wood, A. Sijbers, J.H.J. Hoeijmakers, K. Tanaka, Enhancement of damage-specific DNA binding of XPA by interaction with the ERCC1 DNA repair protein, Biochem. Biophys. Res. Commun. 211 (1995) 960–966. [17] L. Li, S.J. Elledge, C.A. Peterson, E.S. Bales, R.J. Legerski, Specific association between the human DNA repair proteins XPA and ERCC1, Proc. Natl. Acad. Sci. USA 91 (1994) 5012– 5016. [18] C.H. Park, D. Mu, J.T. Reardon, A. Sancar, The general transcription-repair factor TFIIH is recruited to the excision repair complex by the XPA protein independent of the TFIIE transcription factor, J. Biol. Chem. 270 (1995) 4896–4902. [19] S. Nocentini, F. Coin, M. Saijo, K. Tanaka, J.-M. Egly, DNA damage recognition by XPA protein promotes efficient recruitment of transcription factor II H, J. Biol. Chem. 272 (1997) 22991–22994. [20] I. Kuraoka, E.H. Morita, M. Saijo, T. Matsuda, K. Morikawa, M. Shirakawa, K. Tanaka, Identification of a damaged-DNA binding domain of the XPA protein, Mutat. Res. 362 (1996) 87– 95. [21] Z. He, L.A. Henricksen, M.S. Wold, C.J. Ingles, RPA involvement in the damage-recognition and incision steps of nucleotide excision repair, Nature 374 (1995) 566–569. [22] S.M. Patrick, J.J. Turchi, Xeroderma pigmentosum complementation group A protein (XPA) modulates RPA-DNA interactions via enhanced complex stability and inhibition of strand separation activity, J. Biol. Chem. 277 (2002) 16096–16101. [23] S.J. Araujo, R.D. Wood, Protein complexes in nucleotide excision repair, Mutat. Res. 435 (1999) 23–33. [24] E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1988. [25] J.G. Moggs, K.J. Yarema, J.M. Essigmann, R.D. Wood, Analysis of incision sites produced by human cell extracts and purified proteins during nucleotide excision repair of a 1,3-intrastrand d(GpTpG)-cisplatin adduct, J. Biol. Chem. 271 (1996) 7177– 7186. [26] L.A. Henricksen, C.B. Umbrichit, M.S. Wold, Recombinant replication protein A: expression, complex formation, and functional characterization, J. Biol. Chem. 269 (1994) 11121– 11132. [27] M. Volker, M.J. Mone, P. Karmakar, A. van Hoffen, W. Schul, W. Vermeulen, J.H.J. Hoeijmakers, R. van Driel, A.A. van Zeeland, L.H.F. Mullenders, Sequential assembly of the nucleotide excision repair factors in vivo, Mol. Cell 8 (2001) 213–224. [28] J.T. Reardon, A. Sancar, Molecular anatomy of the human excision nuclease assembled at sites of DNA damage, Mol. Cell. Biol. 22 (2002) 5938–5945. [29] J.-S. You, M. Wang, S.-H. Lee, Biochemical analysis of the damage recognition process in nucleotide excision repair, J. Biol. Chem. 27 (2003) 7476–7485. [30] T. Riedl, F. Hanaoka, J.-M. Egly, The comings and goings of nucleotide excision repair factors on damaged DNA, EMBO J. 22 (2003) 5293–5303.
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[31] D. Coverley, M.K. Kenny, M. Munn, W.D. Rupp, D.P. Lane, R.D. Wood, Requirement for the replication protein SSB in human DNA excision repair, Nature 349 (1991) 538–541. [32] D. Coverley, M.K. Kenny, D.P. Lane, R.D. Wood, A role for the human single-stranded DNA binding protein HSSB/RPA in an early stage of nucleotide excision repair, Nucleic Acids Res. 20 (1992) 3873–3880. [33] T. Matsunaga, D. Mu, C.-H. Park, J.T. Reardon, A. Sancar, Human DNA repair excision nuclease. Analysis of the roles of the subunits involved in dual incisions by using anti-XPG and antiERCC1 antibodies, J. Biol. Chem. 270 (1995) 20862–20869. [34] I. Kuraoka, C. Bender, A. Romieu, J. Cadet, W.D. Wood, T. Lindahl, Removal of oxygen free-radical-induced 5 0 ,8-purine cyclodeoxynucleosides from DNA by the nucleotide excisionrepair pathway in human cells, Proc. Natl. Acad. Sci. USA 97 (2000) 3832–3837. [35] L. Li, C.A. Peterson, X. Lu, R.J. Legerski, Mutations in XPA that prevent association with ERCC1 are defective in nucleotide excision repair, Mol. Cell. Biol. 15 (1995) 1993–1998. [36] G.W. Buchko, N.G. Isern, L.D. Spicer, M.A. Kennedy, Human nucleotide excision repair protein XPA: NMR spectroscopic studies of an XPA fragment containing the ERCC1-binding region and the minimal DNA-binding domain (M59-F219), Mutat. Res. 486 (2001) 1–10. [37] K. Sugasawa, J.M.Y. Ng, C. Masutani, S. Iwai, P.J. van der Spek, A.P.M. Eker, F. Hanaoka, D. Bootsma, J.H.J. Hoeijmak-
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Inhibition of nucleotide excision repair by anti-XPA monoclonal antibodies which interfere with binding to RPA, ERCC1, and TFIIH Masafumi Saijo a,b, Toshiro Matsuda a, Isao Kuraoka a,b, Kiyoji Tanaka a,b,* a b
Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamada-oka, Suita, Osaka 565-0871, Japan Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, 1-3 Yamada-oka, Suita, Osaka 565-0871, Japan Received 2 July 2004 Available online 24 July 2004
Abstract The xeroderma pigmentosum group A protein (XPA) binds to three nucleotide excision repair (NER) factors: RPA, ERCC1, and TFIIH. XPA also binds preferentially to UV- or chemical carcinogen-damaged DNA. In this study, we prepared anti-XPA monoclonal antibodies and examined their effects on NER. Two clones inhibited cell-free NER reactions. The mode of inhibition appeared to differ; one clone inhibited both 5 0 and 3 0 incisions equally while the other inhibited the 5 0 incision more. The two clones inhibited the binding of XPA to RPA, ERCC1, and TFIIH. They did not inhibit the binding to damaged DNA either. These results suggest that the interaction of XPA with these NER factors is essential to the NER pathway. The epitopes of these antibodies were located outside of the binding regions for these NER factors. Steric hindrance or conformational changes of XPA brought about by the binding of anti-XPA IgG possibly cause the inhibitory effects. 2004 Elsevier Inc. All rights reserved. Keywords: Nucleotide excision repair; XPA; Monoclonal antibody; Protein interaction
Nucleotide excision repair (NER) is a versatile DNA repair system correcting a broad spectrum of DNA damage, including ultraviolet (UV)-induced cyclobutane pyrimidine dimers and (6–4) photoproducts as well as chemical carcinogen-induced lesions [1]. There are two subpathways in NER [2]. One is transcription-coupled repair (TCR), which efficiently removes the damage in the transcribed strand of transcriptionally active genes. The other is global genome repair (GGR), which occurs throughout the genome including the non-transcribed strand of active genes. Xeroderma pigmentosum (XP) is an autosomal recessive human disease characterized by hypersensitivity to sunlight and a high incidence of skin cancer on sun-exposed skin [1,3]. Cells from XP patients are hypersensitive *
Corresponding author. Fax: +81-6-6877-9136. E-mail address: [email protected] (K. Tanaka).
0006-291X/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.07.030
to killing by UV irradiation because of a defect in NER. XP is classified into seven complementation groups (XP-A to XP-G) and a variant form [1]. Except for the variant form, the primary defect of XP resides in the early steps of NER: damage recognition and dual incisions of the damaged strand on both sides of the DNA lesion [3]. Both TCR and GGR of NER are defective in groups A–G but not groups C and E, in which only GGR is impaired [4–6]. To date, all XP genes have been cloned. In addition, the ERCC1 protein, encoded by a human gene that can correct the repair deficiency in a UV-sensitive, NER-defective, rodent mutant cell line, is also involved in NER. The core reaction of GGR in humans has been reconstituted in vitro with purified XP and other proteins, and an outline of the GGR mechanism has been elucidated [7]. The XPA protein, consisting of 273 amino acid residues, binds to RPA, ERCC1, and TFIIH as well
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as UV- or chemical carcinogen-damaged DNA [8,9], and participates in the formation of an open complex [10]. We also identified two XPA-binding proteins, XAB1 and XAB2, by yeast two-hybrid screening [11,12]. Several distinct functional regions in XPA have been identified. The N-terminal region (residues 4–29) of the protein is responsible for binding to a 34 kDa subunit of RPA [13,14] and the basic amino acid region (residues 30–42) is important for the localization of XPA protein to the nucleus [15]. The region containing the glutamic acid-cluster (E-cluster) (residues 78– 84) is important for the interaction with ERCC1 [16,17]. The C-terminal region (residues 226–273) is shown to bind to TFIIH [18,19]. The central region is identified as the minimal polypeptide (residues 98– 219) for preferential binding to damaged DNA [20] and the region (residues 98–187) necessary for binding to 70 kDa subunit of RPA [13,14]. In addition, the binding of XPA to damaged DNA is markedly increased by the interaction of XPA with the NER factors [16,19,21,22]. These interactions should function to co-ordinate the early stages of NER [23]. Here, we obtained anti-XPA monoclonal antibodies that inhibit GGR reaction in vitro and analyzed the correlation between the inhibitory effects and molecular interactions of XPA.
Materials and methods Preparation of antibodies. His-tagged XPA protein and His-tagged XPA98–219 were produced in Escherichia coli, purified as described previously [8,20], and used as antigens. Four Balb/c mice were immunized with TiterMax (CytRx) as an adjuvant according to the procedure supplied by the manufacturer. The splenocytes from immunized mice were fused with mouse myeloma cells according to the standard procedure [24]. After selection in HAT medium, spent media from hybridomas were screened with the enzyme-linked immunosorbent assay (ELISA) on antigen-immobilized plates and Western blotting using the purified XPA protein. The limiting dilution method was used for the cloning of hybridomas producing anti-XPA antibodies and repeated twice. To purify anti-XPA IgGs, the hybridomas were cultured in a serum-free medium (S-Clone SF-O, Sankyo Junyaku). IgGs were precipitated with 50% saturated ammonium sulfate from the spent media and resuspended in a buffer containing 25 mM Hepes–KOH, pH 7.8. Then, the IgG fraction was purified with column chromatography, HiLoad 16/60 Superdex 200 pg (Amersham Biosciences), with a buffer containing 25 mM Hepes–KOH, pH 7.8, and 20 mM KCl, and Mono Q HR 5/5 (Amersham Biosciences) with a linear gradient of KCl from 20 to 500 mM in a buffer containing 25 mM Hepes–KOH, pH 7.8, successively. The eluted fractions were subjected to SDS–polyacrylamide gel electrophoresis and the gel was stained with Coomassie brilliant blue. The fractions containing IgG were pooled and dialyzed against a buffer containing 25 mM Hepes–KOH, pH 7.8, and 100 mM KCl. The isotype of the anti-XPA IgGs was determined using a Mouse monoclonal antibody isotyping kit (Amersham Biosciences). Immunoblotting. Cell lysates were prepared by adding NETN [50 mM Tris–HCl, pH 7.8, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, and complete protease inhibitor cocktail (Roche)] at a concen-
tration of 2 · 107 cells/ml and incubated at 0 C for 30 min. The lysates were centrifuged for 10 min at 13,000g and the supernatant was recovered. Ten micrograms of protein was separated by SDS–PAGE and transferred to PVDF membrane. XPA protein was detected using the ECL plus Western blotting detection reagents (Amersham Biosciences). Truncated XPA proteins were produced in E. coli as GST fusion proteins and purified as described previously [13]. Dual incision assay. Effects of monoclonal antibodies on the NER reaction were examined by dual incision assay. Covalently closed circular DNA containing a single 1,3-intrastrand d(GpTpG)-cisplatin adduct (Pt-GTG) was prepared as described [25]. HeLa whole cell extract (100 lg of protein, containing about 5 pmol of XPA) was preincubated with purified anti-XPA antibody for 30 min at 30 C and an incision assay was carried out as described [10] using 150 ng PtGTG in a total volume of 25 ll. After the reaction, DNA was purified and digested with HindIII and XhoI in a total volume of 10 ll. The DNA was separated by denaturing 12% polyacrylamide gel electrophoresis and transferred by capillary action for 2 h from the gel to a positively charged nylon membrane (Immobilon-Ny+, Millipore) soaked in 0.9 M Tris–borate, 20 mM EDTA. DNA was fixed to the membrane with UV cross-linking according to the manufacturerÕs instructions. The blots were incubated with a 32P-labeled oligonucleotide (5 0 -GAAGAGTGCACAGAAGAAGAGGCCTGG-3 0 ) in hybridization buffer (7% SDS, 10% polyethylene glycol 8000, 250 mM NaCl, and 130 mM potassium phosphate, pH 7.0) for 16 h at 42 C and washed for 10 min in 2· SSC, 0.1% SDS before being exposed to X-ray film with an intensifying screen or an imaging plate for the Bio-imaging analyzer BAS-2500 (Fujifilm). DNA-binding assay. XPA protein was preincubated with the purified anti-XPA antibody for 4 h at 4 C. Nitrocellulose filter-binding assay was performed as described previously [20] with some modifications. The EcoRI-digested pUC19 was labeled with Klenow fragment in the presence of [a-32P]dATP and [a-32P]TTP and irradiated with 8 kJ/m2 of UV. The labeled DNA was added to the above protein mixture and incubated for 1 h at 4 C. DNA bound to XPA protein was collected by filtration on a nitrocellulose filter. Protein interaction assay. Recombinant RPA and 6· His-tagged ERCC1 protein were purified as described previously [13,26]. One hundred nanograms of purified RPA, 6· His-tagged ERCC1 protein, or TFIIH was incubated in 100 ll PBS for 1 h at 37 C to be immobilized on 96-well plates and unbound proteins were washed out with PBS. The wells were incubated with PBST (PBS–0.1% Tween 20) containing 3% skim milk for 1 h at room temperature and washed twice with PBS. XPA protein (10 ng) was preincubated with various amounts of purified anti-XPA antibody in 100 ll PBST containing 1% skim milk and 1 mM DTT for 4 h at 4 C. The mixture was then incubated in the RPA-, ERCC1-, or TFIIH-immobilized wells overnight at 4 C. Unbound proteins were removed by washing three times with PBST. The wells were then incubated with 1000-fold diluted anti-XPA polyclonal antibody (Santa Cruz Biotechnology) in PBST containing 1% skim milk for 1 h at room temperature and washed five times with PBST. Next, the wells were incubated with 1000-fold diluted alkaline phosphatase-conjugated anti-rabbit IgG (Cappel) in PBST containing 1% skim milk for 1 h at room temperature. Then the wells were washed five times with PBST and twice with AP buffer (100 mM Tris–HCl, pH 9.5, 100 mM NaCl, and 5 mM MgCl2), and incubated with 100 ll AP buffer containing 1 mg/ml p-nitrophenyl phosphate. The reaction was stopped with 100 ll of 1 N NaOH and the absorbance was measured at 405 nm. In the experiments of Fig. 4B, XPA protein was first incubated in the RPA-, ERCC1, or TFIIH-immobilized wells. Unbound XPA proteins were removed by washing three times with PBST. The wells were then incubated with various amounts of purified anti-XPA antibody in 100 ll PBST containing 1% skim milk and 1 mM DTT to examine the effects of anti-XPA antibodies on the interactions between these proteins.
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Results Preparation of anti-XPA monoclonal antibodies To investigate the functions of XPA protein, we prepared anti-XPA monoclonal antibodies. In the course of screening by ELISA, anti-gamma chain-specific antibody was used as secondary antibody to obtain hybridomas producing anti-XPA IgG and 11 ELISA-positive
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hybridoma clones were obtained. Next, we tested the specificity of the antibodies against the XPA protein by Western blotting using whole cell extracts from HeLa and XPA-deficient (XP12ROSV) cells. Three monoclonal antibodies (1H2, 5F12, and 10B4) showed good specificity for XPA (Fig. 1). These antibodies hardly reacted with other NER proteins in both extracts. We purified IgGs from serum-free spent media of the three hybridomas by ammonium sulfate-precipitation and column chromatography, and used them for subsequent experiments. Effects of anti-XPA antibodies on NER reactions in vitro
Fig. 1. Specificity of anti-XPA antibodies. Cell lysates (10 lg of protein) prepared from HeLa (lane 1) and XP12ROSV (lane 2) cells were subjected to immunoblotting with anti-XPA monoclonal antibodies.
To explore the effect of the antibodies on NER reactions in vitro, we applied the dual incision assay at a single 1,3-intrastrand d(GpTpG)-cisplatin lesion in a closed circular duplex DNA substrate (Fig. 2A). Platinated 23to 30-mer oligonucleotides were released by dual incision after the incubation of the DNA with HeLa whole cell extract (lane 2), but oligonucleotides with no damage were not (lane 1). The whole cell extract was preincubated with each anti-XPA antibody and then used for the incision assay. Two anti-XPA antibodies (1H2 and 5F12) inhibited the incisions around the lesion in a dose-dependent manner, but the other anti-XPA
Fig. 2. Effects of anti-XPA antibodies on dual incision. (A) HeLa whole cell extract (100 lg of protein) was preincubated with 0 (lane 2), 0.1 (lanes 3, 6, 9, and 12), 0.5 (lanes 4, 7, 10, and 13) or 1.0 lg (lanes 5, 8, 11, and 14) of purified anti-XPA antibody or control mouse IgG for 30 min at 30 C. Closed circular DNA containing a single 1,3-intrastrand d(GpTpG)-cisplatin cross-link was then added to give a total volume of 25 ll in the reaction mixture. After incubation for 30 min at 30 C, reaction products were purified, digested with HindIII and XhoI, separated in denaturing polyacrylamide gel, transferred to a charged nylon membrane, and hybridized with a 32P-labeled oligonucleotide probe complementary to the DNA sequence surrounding the cisplatin–DNA adduct. Lane 1, DNA without the cisplatin cross-link was used. (B) Quantification of (A). Values for the dual incisions (23- to 32-mers) and the uncoupled 3 0 incisions (48- and 49-mers) are displayed as a percentage of the value where no antibody was added. Closed circle, mouse IgG; open circle, 1H2; closed square, 5F12; open square, 10B4.
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antibody (10B4) and control mouse IgG did not, indicating that the inhibitory effects were specific to these two antibodies. The effect of 5F12 on the dual incisions was greater than that of 1H2 (Fig. 2B). Uncoupled 3 0 incision products were also decreased in correlation with dual incision products by 1H2. But, the amounts of some uncoupled 3 0 incision products remained constant even at higher doses of 5F12 where the dual incision products almost disappeared (Figs. 2A and B). It is considered that 1H2 inhibits both 5 0 and 3 0 incisions equally while 5F12 inhibits the 5 0 incision more than the 3 0 incision. Effects on the binding of XPA protein to damaged DNA To determine the molecular mechanism by which the incision reactions in vitro were inhibited by these antiXPA antibodies, the DNA-binding activity of XPA protein was examined in the presence of anti-XPA antibodies. XPA protein was preincubated with each antibody, then the binding was assayed with a nitrocellulose filter (Fig. 3). XPA protein incubated with antiRPA34 monoclonal antibody (as an unrelated control) or buffer alone preferentially bound to UV-irradiated DNA. In the two experimental conditions used, where IgG doses were relatively high, the three anti-XPA antibodies showed stimulatory rather than inhibitory effects on the binding activity. The antibodies did not affect the activity to bind undamaged DNA. Effects on NER protein interactions XPA protein binds to three NER factors, ERCC1 protein, RPA, and TFIIH. To analyze the effects of the anti-XPA antibodies on the interactions of these proteins, we employed an ELISA and determined the conditions for the assay. Equal amounts of ERCC1 protein, RPA, or TFIIH were immobilized on a 96-well plate and then various amounts of XPA protein were incubated with the immobilized protein. The bound XPA
protein was detected with anti-XPA polyclonal antibody. The absorbance linearly correlated with the amount of XPA up to 0.75 pmol (data not shown), indicating that the binding of XPA to ERCC1, RPA, or TFIIH was not saturated. Thereafter, we used 0.3 pmol of XPA protein for the binding assay. As shown in Fig. 4A, the binding of XPA to RPA was inhibited by the preincubation of XPA with 1H2 and 5F12, but not 10B4, which did not inhibit repair reactions in vitro. We also observed that 1H2 and 5F12 inhibited the binding of XPA to ERCC1 and TFIIH. Inhibitory effects on the binding of XPA to ERCC1 protein by the antibodies appeared stronger than those on the binding to RPA. 1H2 and 5F12 inhibited the binding of XPA to TFIIH to a lesser extent than the binding to ERCC1 and RPA and the inhibition was about 50% at the high IgG dose. 10B4 did not inhibit the binding of XPA to ERCC1or TFIIH. When the antibodies were added after XPA protein first bound to RPA, ERCC1 protein, or TFIIH, bound XPA protein was not released by the addition of each antibody (Fig. 4B). These results indicate that anti-XPA antibodies 1H2 and 5F12 could interfere with the association of XPA with these three NER factors but could not dissociate XPA from the NER factors once the binding had occurred. Epitope mapping and subclasses of the antibodies Binding regions of the monoclonal antibodies were determined by Western blotting and ELISA using recombinant truncated XPA proteins. Representative results of Western blotting are shown in Fig. 5A. 1H2 and 5F12 showed the same pattern. These antibodies reacted to amino acid residues 1–137 (lane 1), 1–97 (lane 8), 1–57 (lane 9), 25–47 (lane 11), and 30–273 (data by ELISA) of XPA protein. 10B4 reacted to residues 1– 137 (lane 1), 98–219 (lane 3), and 98–115 (lane 4). The results are summarized in Fig. 5B and indicated that 1H2 and 5F12 bind to the region from the amino acid residue 30 to 47 of XPA protein and that 10B4 binds to the region from 98 to 115. The subclasses of the IgGs were determined by ELISA; 1H2 and 10B4 were IgG1, and 5F12 was IgG2b. 1H2 and 5F12 recognized the same region in XPA protein, but were different subclasses of IgG. We therefore deduced that the hybridomas producing 1H2 and 5F12 were different clones.
Discussion Fig. 3. Effects of anti-XPA antibodies on the binding of damaged DNA by XPA. (A) Two picomoles of XPA protein was preincubated with 2 lg of purified anti-XPA antibody for 6 h at 4 C. UV-irradiated (8000 J/m2) or unirradiated DNA labeled with 32P (2.5 ng) was then added. After incubation for 1 h at 4 C, DNA bound to XPA protein was collected by filtration on nitrocellulose filters. (B) One picomole of XPA, 5 lg IgG, and 5 ng DNA were used.
NER is a pathway in which more than 25 polypeptides are involved. At least in mammalian cells, NER factors are considered to assemble sequentially at DNA damage sites [27–30]. It has been reported that some antibodies against NER factors inhibit NER
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Fig. 4. Effects of anti-XPA antibodies on protein interactions. (A) XPA protein (0.3 pmol) was preincubated with 0, 0.05, 0.1, 0.5, or 1.0 lg of each purified anti-XPA monoclonal antibody for 4 h at 4 C. The mixture was then incubated in RPA-, ERCC1-, or TFIIH-coated wells. (B) XPA protein (0.3 pmol) was incubated in RPA-, ERCC1-, or TFIIH-coated wells. After unbound XPA protein was washed out, the wells were incubated with 0, 0.5, 1.0, or 2.0 mg of each purified anti-XPA monoclonal antibody. Bound XPA protein was detected by ELISA using anti-XPA polyclonal antibody. Closed circle, mouse IgG; open circle, 1H2; closed square, 5F12; open square, 10B4.
reactions in vitro. Anti-RPA monoclonal antibodies caused inhibition of the repair reaction in whole cell extracts [31,32]. Anti-XPG and anti-ERCC1 polyclonal antibodies inhibited the 3 0 incision and both the 5 0 and 3 0 incisions, respectively [33]. Addition of anti-XPA polyclonal antibody to whole cell extracts inhibited the formation of dual incision products [34], but the mechanism of the inhibition was not elucidated. In the present study, we prepared and characterized anti-XPA monoclonal antibodies. Two antibodies (clones 1H2 and 5F12) inhibited NER reactions in vitro. They also inhibited the binding of XPA protein to RPA, ERCC1 protein, and TFIIH, but not to damaged DNA. These results indicate that the binding of XPA protein to RPA, ERCC1 protein, and/or TFIIH is important for the NER reactions. Consistent with our results, XP-A cells expressing XPA with a deletion of the binding region for ERCC1 [35], RPA [14], or TFIIH [15] have been shown to be UV-sensitive. Both 1H2 and 5F12 bind to the region spanning amino acid residues 30–47 of XPA protein. This area contains several basic residues and is considered a putative nuclear localization signal [15], but is outside of the regions required for binding to RPA, ERCC1 protein, and TFIIH (Fig. 5B). The XP-A cells expressing XPA with a deletion of the epitope region showed the same level of UV-resistance as normal cells [15], indicat-
ing that this region is not essential for the repair function. As the IgG molecule is larger than XPA protein, binding of the IgGs to XPA protein even outside of the binding regions could hinder the bindings. Alternatively, binding of the IgGs could cause some structural change in XPA protein that inhibits the binding to RPA, ERCC1 protein, and TFIIH. Although the whole structure of XPA protein has not been determined, it was shown that the ERCC1-binding domain containing a negatively charged glutamic acid-cluster might associate with the positively charged surface in the RPA70/ DNA-binding domain [36]. In addition, it was suggested that conformational changes of XPA protein could be related to its binding to TFIIH [19]. These structural changes could affect the protein interactions. We detected some differences in the inhibitory effects of the antibodies on incisions. The effects on protein interactions were similar, but inhibition of the dual incisions by 5F12 was stronger than that by 1H2. In addition, the efficacy of the antibodies to the 5 0 and 3 0 incision sides appeared to be different. XPA participates in the formation of an unwound open DNA intermediate around a lesion [10]. We speculate that the spatial position of XPA in the open complex is affected differently by the addition of each antibody, which could cause the differences in the inhibition of incision reactions.
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Fig. 5. Epitope mapping of the antibodies. (A) Truncated XPA proteins were subjected to immunoblotting. The regions of XPA protein used are amino acid residues 1–137 (lane 1), 138–273 (lane 2), 98–219 (lane 3), 98–115 (lane 4), 116–219 (lane 5), 116–273 (lane 6), 98–273 (lane 7), 1–97 (lane 8), 1–57 (lane 9), 53–137 (lane 10), and 25–47 (lane 11). (B) Schematic presentation of the functional regions in XPA proteins and the results of immunoblotting and ELISA. NLS, putative nuclear localization signal; E-cluster, the glutamic acid residue cluster; and Zn-finger, C4-type zinc finger motif.
XPA protein binds damaged DNA [8,9], and functional roles for XPA protein in damage verification during NER are proposed [37–39]. The minimal DNAbinding domain has been identified to be a 122 amino acid region between M98 and F219 that contains a C4type zinc finger motif [20] and overlaps with the RPA70-binding region [13]. The structure of the DNA/ RPA70-binding domain was determined by nuclear magnetic resonance spectroscopy [40,41]. The domain consists of a zinc-containing subdomain and a C-terminal subdomain. The epitope of 10B4 was located within amino acid residues 98–115. These residues form an antiparallel b-sheet that encompasses two cysteine residues, Cys105 and Cys108, whose side chains are co-ordinated by a zinc ion. This region is supposed to be a binding surface for RPA and becomes more mobile in the presence of DNA [42]. We expected the antibody 10B4 to affect the binding of XPA protein to damaged DNA. But it did not hinder XPA protein from binding
to either damaged DNA or NER proteins. No other clones we obtained inhibited the binding of damaged DNA either. Therefore, we could not clarify the significance of the binding activity of XPA protein in the NER reactions. Many proteins are involved in NER, and interact to co-ordinate the reaction. XPA is a critical factor in NER, because a deficiency of XPA causes severe sensitivity to UV. Although XPA protein has no enzymatic activity, it would function as a core component in NER reactions by interacting with damaged DNA, RPA, ERCC1 protein, and TFIIH. The present results indicate the significance of the interactions between XPA protein and the NER factors, but we could not show the role of individual interactions using monoclonal antibodies. Detailed analyses should be performed using XPA proteins with single amino acid substitutions that affect the interaction with each NER factor.
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Acknowledgments We thank Drs. R.D. Wood and J.G. Moggs for instruction in the cell-free dual incision assay, Dr. J.-M. Egly for TFIIH and Dr. Y. Nakatsu for helpful suggestions. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, and CREST of Japan Science and Technology (JST).
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