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• W2070184329 abstract "The xeroderma pigmentosum group C protein complex XPC-HR23B was first isolated as a factor that complemented nucleotide excision repair defects of XP-C cell extracts in vitro.Recent studies have revealed that this protein complex plays an important role in the early steps of global genome nucleotide excision repair, especially in damage recognition, open complex formation, and repair protein complex formation. However, the precise function of XPC-HR23B in global genome repair is still unclear. Here we demonstrate that XPC-HR23B interacts with general transcription factor IIH (TFIIH) both in vivo and in vitro. This interaction is thought to be mediated through the specific affinity of XPC for the TFIIH subunits XPB and/or p62, which are essential for both basal transcription and nucleotide excision repair. Interestingly, association of TFIIH with DNA was observed in both wild-type and XP-A cell extracts but not in XP-C cell extracts, and XPC-HR23B could restore the association of TFIIH with DNA in XP-C cell extracts. Moreover, we found that XPC-HR23B was necessary for efficient association of TFIIH with damaged DNA in cell-free extracts. We conclude that the XPC-HR23B protein complex plays a crucial role in the recruitment of TFIIH to damaged DNA in global genome repair. The xeroderma pigmentosum group C protein complex XPC-HR23B was first isolated as a factor that complemented nucleotide excision repair defects of XP-C cell extracts in vitro.Recent studies have revealed that this protein complex plays an important role in the early steps of global genome nucleotide excision repair, especially in damage recognition, open complex formation, and repair protein complex formation. However, the precise function of XPC-HR23B in global genome repair is still unclear. Here we demonstrate that XPC-HR23B interacts with general transcription factor IIH (TFIIH) both in vivo and in vitro. This interaction is thought to be mediated through the specific affinity of XPC for the TFIIH subunits XPB and/or p62, which are essential for both basal transcription and nucleotide excision repair. Interestingly, association of TFIIH with DNA was observed in both wild-type and XP-A cell extracts but not in XP-C cell extracts, and XPC-HR23B could restore the association of TFIIH with DNA in XP-C cell extracts. Moreover, we found that XPC-HR23B was necessary for efficient association of TFIIH with damaged DNA in cell-free extracts. We conclude that the XPC-HR23B protein complex plays a crucial role in the recruitment of TFIIH to damaged DNA in global genome repair. nucleotide excision repair replication protein A transcription factor IIH polymerase chain reaction proliferating cell nuclear antigen polyacrylamide gel electrophoresis glutathione S-transferase single-stranded double-stranded N-acetoxy-2-acetyl-2-aminofluorene recombinant adenosine 5′-(β,γ-imino) triphosphate Nucleotide excision repair (NER)1 is the primary pathway for removal of lesions from DNA and is conserved across a wide range of species and from prokaryotes to eukaryotes. Studies of prokaryotic NER have shown that the NER pathway is controlled by phased enzymatic reactions (1.van Houten B. Microbiol. Rev. 1990; 54: 18-51Crossref PubMed Google Scholar, 2.Hoeijmakers J.H.J. Trends Genet. 1993; 9: 173-177Abstract Full Text PDF PubMed Scopus (109) Google Scholar). Recently, analyses of NER-deficient yeast, rodent, and human mutant cells have resulted in the identification and characterization of the eukaryotic proteins involved in NER. Xeroderma pigmentosum and Cockayne's syndrome are human autosomal recessive hereditary diseases that result from a deficiency in NER (3.Bootsma, D., Kreamer, K. H., Cleaver, J. E., and Hoeijmakers, J. H. J. (1997) in The Genetic Basis of Human Cancer. (Vogelstein, B., and Kinzler, K., eds) Chapter 9, McGraw-Hill Book Co., New YorkGoogle Scholar); to date, seven complementation groups (A to G) in xeroderma pigmentosum and two (A and B) in Cockayne's syndrome have been identified, and most of the corresponding NER genes have been cloned (4.Boulikas T. Anticancer Res. 1996; 16: 693-708PubMed Google Scholar). Additional factors involved in NER have also been identified (5.Coverley D. Kenny M.K. Lane D.P. Wood R.D. Nucleic Acids Res. 1992; 20: 3873-3880Crossref PubMed Scopus (137) Google Scholar, 6.Nichols A.F. Sancar A. Nucleic Acids Res. 1992; 20: 2441-2446Crossref PubMed Scopus (181) Google Scholar, 7.Shivji M.K.K. Kenny M.K. Wood R.D. Cell. 1992; 69: 367-374Abstract Full Text PDF PubMed Scopus (732) Google Scholar) using a cell-free NER assay (8.Wood R.D. Robins P. Lindahl T. Cell. 1988; 53: 97-106Abstract Full Text PDF PubMed Scopus (380) Google Scholar). Based on these studies, including studies on reconstitution of NER using purified proteins (9.Aboussekhra A. Biggerstaff M. Shivji M.K.K. Vilpo J.A. Moncollin V. Podust V.N. Protic M. Hübscher U. Egly J.-M. Wood R.D. Cell. 1995; 80: 859-868Abstract Full Text PDF PubMed Scopus (750) Google Scholar, 10.Guzder S.N. Habraken Y. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1995; 270: 12973-12976Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 11.Mu D. Park C.-H. Matsunaga T. Hsu D.S. Reardon J.T. Sancar A. J. Biol. Chem. 1995; 270: 2415-2418Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar), various models of the mechanism of eukaryotic NER have been proposed (see Refs. 12.Sancar A. Annu. Rev. Biochem. 1996; 65: 43-81Crossref PubMed Scopus (963) Google Scholar and 13.Wood R.D. Annu. Rev. Biochem. 1996; 65: 135-167Crossref PubMed Scopus (611) Google Scholar for review). The xeroderma pigmentosum group C protein complex XPC-HR23B is a tightly associated complex of the products of the XPC andHR23B genes and was initially purified from HeLa cell nuclear extracts as a protein factor that complemented the DNA repair defects of XP-C whole cell extracts in a cell-free NER reaction (14.Masutani C. Sugasawa K. Yanagisawa J. Sonoyama T. Ui M. Enomoto T. Takio K. Tanaka K. van der Spek P.J. Bootsma D. Hoeijmakers J.H.J. Hanaoka F. EMBO J. 1994; 13: 1831-1843Crossref PubMed Scopus (334) Google Scholar,15.Shivji M.K.K. Eker A.P.M. Wood R.D. J. Biol. Chem. 1994; 269: 22749-22757Abstract Full Text PDF PubMed Google Scholar). Although no enzymatic activity was observed in the purified complex, it has been reported that this complex is required for DNA repair prior to the excision step (15.Shivji M.K.K. Eker A.P.M. Wood R.D. J. Biol. Chem. 1994; 269: 22749-22757Abstract Full Text PDF PubMed Google Scholar, 16.Mu D. Sancar A. J. Biol. Chem. 1997; 272: 7570-7573Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). During purification, XPC-HR23B exhibited a high affinity for single-stranded DNA (14.Masutani C. Sugasawa K. Yanagisawa J. Sonoyama T. Ui M. Enomoto T. Takio K. Tanaka K. van der Spek P.J. Bootsma D. Hoeijmakers J.H.J. Hanaoka F. EMBO J. 1994; 13: 1831-1843Crossref PubMed Scopus (334) Google Scholar, 15.Shivji M.K.K. Eker A.P.M. Wood R.D. J. Biol. Chem. 1994; 269: 22749-22757Abstract Full Text PDF PubMed Google Scholar). Moreover, using recombinant human XPC and HR23B, the high affinity of XPC-HR23B for both double-stranded and UV-irradiated DNA has also been demonstrated (17.Reardon J.T. Mu D. Sancar A. J. Biol. Chem. 1996; 271: 19451-19456Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). 2C. Masutani, K. Sugasawa, and F. Hanaoka, unpublished observations. Several lines of evidence have been accumulated that indicate that this complex plays a role in NER before and after damage recognition. We recently demonstrated, using a novel DNA damage recognition-competition assay, that XPC-HR23B is the earliest acting of the damage detectors that initiate NER (18.Sugasawa K. Ng J.M.Y. Masutani C. Iwai S. van der Spek P.J. Eker A.P.M. Hanaoka F. Bootsma D. Hoeijmakers J.H.J. Mol. Cell. 1998; 2: 223-232Abstract Full Text Full Text PDF PubMed Scopus (743) Google Scholar). XPC, together with TFIIH, is necessary for the initial opening reaction immediately around the lesion (19.Evans E. Moggs J.G. Hwang J.R. Egly J.-M. Wood R.D. EMBO J. 1997; 16: 6559-6573Crossref PubMed Scopus (401) Google Scholar). The XPC-HR23B complex was characterized as a molecular matchmaker that participates in the assembly of the NER proteins on damaged DNA but is not present in the ultimate dual incision complex (20.Wakasugi M. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6669-6674Crossref PubMed Scopus (150) Google Scholar). These data prompted us to hypothesize that XPC-HR23B plays important roles in the assembly of NER proteins on the damaged DNA and in initiation of NER. However, the precise molecular mechanisms that follow damage detection by XPC remain unclear. Here we report that XPC-HR23B interacts directly with TFIIH in vitro and that XPC-HR23B is absolutely required for association of TFIIH with damaged DNA in cell extracts. We conclude that XPC-HR23B is necessary for recruitment of TFIIH to damaged DNA. Human 293, XP7CASV (group A), and XP4PASV (group C) cells were grown at 37 °C in Dulbecco's modified Eagle's medium (Nissui) supplemented with 10% fetal bovine serum. HeLa cells were grown in suspension at 37 °C in Dulbecco's modified Eagle's medium supplemented with 10% bovine serum. Whole cell extracts were prepared as described previously (21.Manley J.L. Fire A. Cano A. Sharp P.A. Gefter M.L. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3855-3859Crossref PubMed Scopus (735) Google Scholar). Untagged (rHR23B), His6-tagged (rHR23B-His6), and GST-tagged (GST-rHR23B) HR23B constructs were expressed in Escherichia coli and purified as described previously (22.Masutani C. Araki M. Sugasawa K. van der Spek P.J. Yamada A. Uchida A. Maekawa T. Bootsma D. Hoeijmakers J.H.J. Hanaoka F. Mol. Cell. Biol. 1997; 17: 6915-6923Crossref PubMed Scopus (96) Google Scholar, 23.Sugasawa K. Masutani C. Uchida A. Maekawa T. van der Spek P.J. Bootsma D. Hoeijmakers J.H.J. Hanaoka F. Mol. Cell. Biol. 1996; 16: 4852-4861Crossref PubMed Scopus (144) Google Scholar). Recombinant XPC (rXPC) was expressed using a baculovirus expression system and purified as described previously (23.Sugasawa K. Masutani C. Uchida A. Maekawa T. van der Spek P.J. Bootsma D. Hoeijmakers J.H.J. Hanaoka F. Mol. Cell. Biol. 1996; 16: 4852-4861Crossref PubMed Scopus (144) Google Scholar). Eight subunits of TFIIH (XPB, XPD, p62, p44, p34, MO15, cyclin H, and MAT1) were expressed inE. coli as GST fusion proteins. The coding regions of these subunits were cloned using PCR technique. PCR primers designed to introduce an NdeI restriction site at the first methionine codon of XPB, XPD, p62, p44, p34, MO15, and cyclin H or anNcoI site at the first methionine codon of MAT1 were used in conjunction with antisense primers containing an appropriate restriction site: a BamHI restriction site for XPB, p62, p44, MO15, cyclin H, and MAT1, a HindIII restriction site for XPD, and an EcoRI restriction site for p34. PCR products were digested with the appropriate restriction enzymes, subcloned into the multicloning site of the E. coli expression vector pGEX-2TL(+) (24.Hoffmann A. Roeder R.G. J. Biol. Chem. 1996; 271: 18194-18202Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), and expressed as described previously (25.Okamoto T. Yamamoto S. Watanabe Y. Ohta T. Hanaoka F. Roeder R.G. Ohkuma Y. J. Biol. Chem. 1998; 273: 19866-19876Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Recombinant PCNA (rPCNA) and recombinant RPA (rRPA) were expressed and purified as described previously (26.Sugasawa K. Ng J.M.Y. Masutani C. Maekawa T. Uchida A. van der Spek P.J. Eker A.P.M. Rademakers S. Visser C. Aboussekhra A. Wood R.D. Hanaoka F. Bootsma D. Hoeijmakers J.H.J. Mol. Cell. Biol. 1997; 17: 6924-6931Crossref PubMed Scopus (110) Google Scholar). Purification of the XPC-HR23B complex (14.Masutani C. Sugasawa K. Yanagisawa J. Sonoyama T. Ui M. Enomoto T. Takio K. Tanaka K. van der Spek P.J. Bootsma D. Hoeijmakers J.H.J. Hanaoka F. EMBO J. 1994; 13: 1831-1843Crossref PubMed Scopus (334) Google Scholar) and TFIIH (27.Ohkuma Y. Roeder R.G. Nature. 1994; 368: 160-163Crossref PubMed Scopus (138) Google Scholar) from HeLa cells was also carried out as described previously. Anti-cyclin H antibody was conjugated to Immunopure Immobilized protein G beads (Pierce) in 100 μl of buffer C (20 mm Tris-HCl (pH 7.9), 200 μm EDTA, 20% glycerol, 0.2 m KCl, 200 μg/ml bovine serum albumin, 2 mm ATP, 5 mmMgCl2, 0.1% Nonidet P-40, 0.25 mmphenylmethylsulfonyl fluoride, 10 mm 2-mercaptoethanol) at 4 °C for 1 h with rotation. After washing three times with buffer C, the antibody-bound Sepharose beads (5 μl) were incubated with 80 μg of whole cell extracts prepared from 293 human fibroblasts in 80 μl of buffer C at 4 °C for 1 h with gentle rotation. After washing three times with buffer C, the bound proteins were extracted by boiling in SDS sample buffer (1× concentration is 50 mm Tris-HCl (pH 6.8), 1.5% SDS, 0.02% bromphenol blue, 10% glycerol), separated by 10% SDS-PAGE, and detected by immunoblotting with antibodies raised against XPC, XPB, p62, XPA, or RPA32. rHR23B-His6 (2.2 mg) was dialyzed against buffer containing 20 mm sodium phosphate (pH 6.8) and 0.3m NaCl and then incubated with activated CH-Sepharose 4B beads (Amersham Pharmacia Biotech) at 4 °C overnight in the same buffer. Unreacted groups were quenched by washing with excess buffer containing 0.1 m Tris-HCl (pH 8.0) and 0.5 mNaCl. The rHR23B-His6-Sepharose beads (10 μl) were preincubated with or without rXPC (90 ng) in buffer C for 30 min on ice, mixed with purified TFIIH (200 ng), and further incubated at 4 °C for 1 h with gentle rotation. After washing three times with buffer C, bound proteins were extracted by boiling in SDS sample buffer, separated by 10% SDS-PAGE, and analyzed by immunoblotting with anti-p62 antibody. Alternatively, GST-tagged HR23B, which had been incubated with or without rXPC for 30 min on ice, was incubated with purified TFIIH (200 ng), and incubated at 4 °C for 1 h with gentle rotation. The mixture was adsorbed to glutathione-Sepharose beads (Amersham Pharmacia Biotech) and then washed three times with buffer C. The bound proteins were extracted, separated, and analyzed as described above. GST-tagged TFIIH subunits (100 ng) were adsorbed to glutathione-Sepharose beads in 100 μl of buffer D (20 mmTris-HCl (pH 7.5), 10% glycerol, 0.5 m NaCl, 2 mm dithiothrietol, 0.5 mm phenylmethylsulfonyl fluoride, 0.6 μg/ml antipain, 0.6 μg/ml aprotinin, 0.3 μg/ml leupeptin, 0.24 μg/ml pepstatin, 150 μm EGTA) at 4 °C for 1 h with rotation. After washing three times with buffer C, the beads were incubated in the same buffer at 4 °C for 1 h with rotation in the presence of purified XPC-HR23B complex (50 ng), rXPC (60 ng), rHR23B (60 ng), rRPA (200 ng), or rPCNA (200 ng). After washing three times with buffer C, bound proteins were eluted by boiling in SDS sample buffer, separated by 8% (for detection of XPC and HR23B) or 12% (for detection of RPA and PCNA) SDS-PAGE, and analyzed by immunoblotting with anti-XPC, HR23B, RPA32, or PCNA antibodies. In advance, we confirmed that full-length GST-tagged TFIIH subunits were expressed in E. coli by immunoblotting with anti-GST antibody (data not shown). Antibodies raised against XPC and HR23B were obtained as described previously (23.Sugasawa K. Masutani C. Uchida A. Maekawa T. van der Spek P.J. Bootsma D. Hoeijmakers J.H.J. Hanaoka F. Mol. Cell. Biol. 1996; 16: 4852-4861Crossref PubMed Scopus (144) Google Scholar). Antibodies raised against XPB, p62, cyclin H, RPA32, PCNA, and GST were prepared by Medical and Biological Laboratories Co., Ltd. Anti-XPA polyclonal antibody was kindly provided by Kiyoji Tanaka. To prepare three kinds of cellulose solution for the precipitation experiments, ssDNA-cellulose and dsDNA-cellulose (both from Sigma-Aldrich) or cellulose CF11 (Whatman) was mixed with equal volumes of Sepharose CL4B (Amersham Pharmacia Biotech) and suspended (50% v/v) in buffer C. The dsDNA cellulose-Sepharose solution contained 7 μg of DNA in 10 μl. Whole cell extracts (125 μg) from 293 or xeroderma pigmentosum cells were incubated with 10 μl of the cellulose-Sepharose suspensions in buffer C at 4 °C for 1 h with gentle rotation. After washing three times with buffer C, the bound proteins were extracted, separated by 10–14% gradient SDS-PAGE, and analyzed by immunoblotting with anti-XPC, p62, XPA, or RPA32 antibodies. Buffer C without ATP or with 2 mm AMP-PNP was used in separate experiments designed to test the ATP dependence of binding. PCR reactions were performed with T3 and biotin-labeled T7 primers, using a partial (nucleotides 8330–9079) human Rev3 cDNA (28.Gibbs P.E. McGregor W.G. Maher V.M. Nisson P. Lawrence C.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6876-6880Crossref PubMed Scopus (295) Google Scholar) subcloned into the EcoRI site of pBS as template. PCR reactions (35 cycles) were carried out in a total volume of 50 μl containing 10 ng of the template DNA, 10 mm KCl, 20 mm Tris-HCl (pH 8.8), 2 mmMgSO4, 10 mm(NH4)2SO4, 0.1% Triton X-100, 100 μg/ml bovine serum albumin, 200 μm dNTPs, 20 pmol of each oligonucleotide primer, and 2.5 units of Pfu DNA polymerase (Stratagene), using a GeneAmp PCR System 9700 thermal cycler (Perkin-Elmer Applied Biosystems). Each cycle consisted of 30 s at 94 °C, 30 s at 55 °C, and 1 min at 72 °C. After removal of excess primer by ethanol precipitation, the PCR products were adsorbed to Streptavidin-agarose (Life Technologies, Inc.) in TE (pH 8.0) containing 0.1 m NaCl at room temperature for 1 h with gentle agitation. Damaged DNA substrate was prepared by treating PCR products in TE (10 mm Tris-HCl, 1 mm EDTA) (pH 7.5) containing 20% ethanol (0.2 μg/ml) with 0.15 mm N-AAAF at 37 °C for varying times followed by di-ethyl-ether extraction, chloroform extraction, and ethanol precipitation. Damaged or undamaged DNA substrate (2 μg) dissolved in TE (pH 8.0) was incubated with 20 μl of a 2-fold suspension of Streptavidin-agarose in TE (pH 8.0) containing 0.1 m NaCl at room temperature for 1 h with gentle agitation. After washing with the same buffer, whole cell extracts (125 μg) was incubated with DNA bound-agarose in 100 μl of buffer C at 4 °C for 1 h. After washing three times with buffer C, the bound proteins were extracted, separated by 10–14% gradient SDS-PAGE, and analyzed by immunoblotting with anti-XPC, p62, XPA, or RPA32 antibodies. The ATP dependence was examined as described above. SDS-PAGE were performed as described previously (29.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207218) Google Scholar). For immunoblotting, proteins separated on SDS gels were electrotransferred onto polyvinylidene difluoride membranes (Immobilon-P; Millipore) at 8 V/cm 15 h in ice-cold transfer buffer (50 mm Tris, 38.4 mm glycine, 0.01% SDS, and 15% methanol). The membranes were successively incubated in blocking buffer (1% Blocking reagent (Roche Molecular Biochemicals) in 0.1 m maleic acid (pH 7.5), 150 mm NaCl), antibody in blocking buffer, and finally anti-rabbit or anti-mouse F(ab′)2 antibody conjugated with horseradish peroxidase (Amersham Pharmacia Biotech). Detection was carried out with SuperSignal Substrate (Pierce) according to the manufacturer's instructions. Protein concentrations were measured as described previously (30.Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216412) Google Scholar) using the Bio-Rad Protein Assay reagent and bovine serum albumin as a standard. Of the mammalian NER factors identified to date, only TFIIH has been suggested to interact with XPC (31.Drapkin R. Reardon J.T. Ansari A. Huang J.-C. Zawel L. Ahn K. Sancar A. Reinberg D. Nature. 1994; 368: 769-772Crossref PubMed Scopus (408) Google Scholar, 32.Maldonado E. Shiekhattar R. Sheldon M. Cho H. Drapkin R. Rickert P. Lees E. Anderson C.W. Linn S. Reinberg D. Nature. 1996; 381: 86-89Crossref PubMed Scopus (306) Google Scholar, 33.Svejstrup J.Q. Wang Z. Feaver W.J. Wu X. Bushnell D.A. Donahue T.F. Friedberg E.C. Kornberg R.D. Cell. 1995; 80: 21-28Abstract Full Text PDF PubMed Scopus (239) Google Scholar), although conclusive evidence of such interaction has been lacking. We performed co-immunoprecipitation experiments with NER-proficient human cell extracts and an antibody raised against cyclin H, which is one of the components of TFIIH. As shown in Fig.1, not only XPB and p62, two other subunits of TFIIH, but also XPC were detected in the fraction precipitated by anti-cyclin H antibody (lane 3) but not with the control antibody (lane 2). On the other hand, no clear evidence to imply the interaction between TFIIH and XPA nor RPA was obtained. This result indicated that XPC interacted with TFIIH in the whole cell extracts; to examine whether the interaction was direct or mediated by other proteins or DNA present in the cell extracts, co-precipitation experiments were performed using purified proteins. TFIIH purified from HeLa nuclear extracts (Fig.2 C) was incubated with glutathione-Sepharose beads bound to GST-rHR23B fusion protein that had been preincubated with or without rXPC. Co-precipitation of TFIIH with the Sepharose beads was assessed by immunoblotting with anti-p62 antibody. As shown in Fig. 2 A, p62 was detected in the precipitate fraction in an XPC-dependent manner (comparelane 4 with lanes 2 and 3), suggesting that XPC alone or the XPC-HR23B complex interacted directly with purified TFIIH. This XPC-dependent interaction was also observed using rHR23B-conjugated Sepharose (Fig. 2 B,lane 3). Therefore, we conclude that the XPC-HR23B complex interacts directly with TFIIH in vitro and that XPC is indispensable for this specific interaction. Next, a series of pull-down experiments were performed using the GST-tagged TFIIH subunits to assess which subunit of TFIIH interacted with the XPC-HR23B complex and whether HR23B was necessary for the interaction. As shown in Fig. 3, both the purified XPC-HR23B complex (A) and free rXPC (B) were found to bind XPB and p62. In contrast, rHR23B, rRPA, and rPCNA were not co-precipitated with any of the TFIIH subunits (C–E). These results indicate that the interaction between the XPC-HR23B complex and TFIIH is mediated through specific protein-protein binding (XPC-XPB and/or XPC-p62) and that HR23B is not essential for this interaction.Figure 2Interaction of purified TFIIH with XPC. A, purified TFIIH was incubated with glutathione-Sepharose beads prebound to no protein (lane 2), GST-rHR23B (lane 3), or GST-rHR23B plus rXPC (lane 4).Lane 1 contains 10% of the input TFIIH. B, Sepharose beads covalently coupled with rHR23B were incubated with purified TFIIH in the absence (lane 2) or presence (lane 3) of rXPC. Panels A and B, bound proteins were subjected to SDS-PAGE, and the presence of TFIIH was subsequently assessed by immunoblotting with anti-p62 antibody.Lane 1 contains 10% of the input TFIIH. C, the purified TFIIH used for these experiments was subjected to 10% SDS-PAGE followed by silver staining. Bands corresponding to known TFIIH subunits are indicated by arrows.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3XPB and p62 subunits are responsible for interaction of TFIIH with XPC. Glutathione-Sepharose beads prebound to the indicated GST-tagged TFIIH subunits were incubated with purified HeLa XPC-HR23B complex (A), rXPC (B), rHR23B (C), rPCNA (D), or heterotrimeric rRPA complex (E). Co-precipitation of each protein was examined by immunoblotting with antibody raised against XPC (A andB), HR23B (C), PCNA (D), or the p32 subunit of RPA (E). Lane 1, 10% of each protein input: lane 2, bound fractions without any GST-tagged TFIIH subunits.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To examine the relevance of the interaction between XPC-HR23B and TFIIH, several precipitation experiments were performed. The XPC-HR23B complex has a high affinity for both ssDNA and dsDNA (14.Masutani C. Sugasawa K. Yanagisawa J. Sonoyama T. Ui M. Enomoto T. Takio K. Tanaka K. van der Spek P.J. Bootsma D. Hoeijmakers J.H.J. Hanaoka F. EMBO J. 1994; 13: 1831-1843Crossref PubMed Scopus (334) Google Scholar,17.Reardon J.T. Mu D. Sancar A. J. Biol. Chem. 1996; 271: 19451-19456Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 23.Sugasawa K. Masutani C. Uchida A. Maekawa T. van der Spek P.J. Bootsma D. Hoeijmakers J.H.J. Hanaoka F. Mol. Cell. Biol. 1996; 16: 4852-4861Crossref PubMed Scopus (144) Google Scholar). Thus, not only XPC-HR23B but also those proteins that interact with this complex or which associate with DNA might be expected to be precipitated from whole cell extracts by DNA-cellulose. We therefore examined the presence of not only XPC and TFIIH but also XPA and RPA, which have been characterized as DNA binding NER factors, in DNA-cellulose bound fractions. Because it has been reported that ATP is necessary to form the repair protein complex on DNA (19.Evans E. Moggs J.G. Hwang J.R. Egly J.-M. Wood R.D. EMBO J. 1997; 16: 6559-6573Crossref PubMed Scopus (401) Google Scholar, 20.Wakasugi M. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6669-6674Crossref PubMed Scopus (150) Google Scholar, 34.Mu D. Wakasugi M. Hsu D.S. Sancar A. J. Biol. Chem. 1997; 272: 28971-28979Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar), we also examined the ATP dependence of the precipitation of TFIIH by DNA-cellulose. XPC, p62, XPA, and RPA32 were detected in the dsDNA-cellulose bound fraction of NER-proficient 293 whole cell extracts, as expected (Fig. 4,lanes 3 and 5). Interestingly, ATP was not required for efficient precipitation of TFIIH by DNA-cellulose from whole cell extracts (lane 3). Moreover, the presence of the nonhydrolyzable ATP analog AMP-PNP did not alter the amount of any precipitates (lane 6). Evans et al. (19.Evans E. Moggs J.G. Hwang J.R. Egly J.-M. Wood R.D. EMBO J. 1997; 16: 6559-6573Crossref PubMed Scopus (401) Google Scholar) reported that ATP hydrolysis is necessary for the open complex formation immediately around the lesion. We therefore propose that the initial NER protein complex on DNA may be formed without reliance on ATP, but the initial opening reaction requires ATP. TFIIH has been shown to have little affinity for DNA by itself (20.Wakasugi M. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6669-6674Crossref PubMed Scopus (150) Google Scholar, 35.Nocentini S. Coin F. Saijo M. Tanaka K. Egly J.-M. J. Biol. Chem. 1997; 272: 22991-22994Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Therefore, the observed precipitation of TFIIH by dsDNA may be due to specific interaction with other protein(s) bound to DNA or dependent upon conformational changes in DNA induced by DNA-binding proteins. To test the possibility that other NER proteins mediate the interaction between TFIIH and DNA, several precipitation experiments were performed using whole cell extracts derived from NER-deficient cell lines (Fig.5). XPC, p62, and RPA32 were precipitated from XP-A and 293 whole cell extracts (A) with ssDNA-cellulose (lanes 3 and 11) or dsDNA-cellulose (lanes 4 and 12). In contrast, p62 was not recovered in the precipitates from XP-C whole cell extracts, whereas binding of XPA and RPA32 to both ssDNA- and dsDNA-cellulose were unaffected (compare lanes 3 and4 with 7 and 8, respectively). The same results were obtained with extracts from XP-B (GM2252A), XP-D (XP6BESV), XP-G (XP3BRSV), CS-A (CS2OSSV), and CS-B (GM1629SV) cells, all of which express the XPC protein, as with 293 whole cell extracts (data not shown). Intriguingly, ssDNA was insufficient to associate with TFIIH even though XPC binds to ssDNA as well as to dsDNA (Fig.5 A, compare lanes 3, 4, 11, and 12). Furthermore, when the XP-C cell extracts were supplemented with purified XPC-HR23B complex, TFIIH was detected in the precipitate fraction of dsDNA-cellulose (Fig. 5 B). Thus TFIIH stably associated with dsDNA, either directly or indirectly, only in the presence of XPC. It should be noted that extra signals were observed above the bands corresponding to XPA. These extra bands probably represent modified forms of the target proteins, because specific antibodies were used. We should also mention here that the amounts of precipitated proteins by ssDNA- or dsDNA-cellulose in each experiments could be altered when the different batch, but the same cell line, of cell extracts was employed (data not shown).Figure 5XPC-dependent precipitation of TFIIH with DNA-cellulose. A, whole cell extracts from 293, XP4PASV (group C), or XP7CASV (group A) cells were incubated with cellulose (lanes 2, 6, and 10), ssDNA-cellulose (lanes 3, 7, and 11), or dsDNA-cellulose (lanes 4, 8, and12) beads. The bound protein fractions were examined for the presence of XPC, TFIIH (p62), XPA, and RPA (p32) by immunoblotting with specific antibodies. Lanes 1, 5, and 9contain 10% of the extracts included in the binding reactions.B, similar binding experiments were carried out with the XP-C cell extracts supplemented with purified XPC-HR23B complex (lanes 7–9). Lanes 1, 4, and7, 10% of the input extracts; lanes 2,5, and 8, cellulose-bound fractions: lanes 3, 6, and 9, dsDNA-cellulose-bound fractions.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To assess the relevance of the XPC-dependent asso" @default.
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• W2070184329 title "The Xeroderma Pigmentosum Group C Protein Complex XPC-HR23B Plays an Important Role in the Recruitment of Transcription Factor IIH to Damaged DNA" @default.
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