p52 Mediates XPB Function within the Transcription/Repair Factor TFIIH

Abstract

To further our understanding of the transcription/DNA repair factor TFIIH, we investigated the role of its p52 subunit in TFIIH function. Using a completely reconstitutedin vitro transcription or nucleotide excision repair (NER) system, we show that deletion of the C-terminal region of p52 results in a dramatic reduction of TFIIH NER and transcription activities. This mutation prevents promoter opening and has no effect on the other enzymatic activities of TFIIH. Moreover, we demonstrate that intact p52 is needed to anchor the XPB helicase within TFIIH, providing an explanation for the transcription and NER defects observed with the mutant p52. We show that these two subunits physically interact and map domains involved in the interface. Taken together, our results show that the p52/Tfb2 subunit of TFIIH regulates the function of XPB through pair-wise interactions as described previously for p44 and XPD. To further our understanding of the transcription/DNA repair factor TFIIH, we investigated the role of its p52 subunit in TFIIH function. Using a completely reconstitutedin vitro transcription or nucleotide excision repair (NER) system, we show that deletion of the C-terminal region of p52 results in a dramatic reduction of TFIIH NER and transcription activities. This mutation prevents promoter opening and has no effect on the other enzymatic activities of TFIIH. Moreover, we demonstrate that intact p52 is needed to anchor the XPB helicase within TFIIH, providing an explanation for the transcription and NER defects observed with the mutant p52. We show that these two subunits physically interact and map domains involved in the interface. Taken together, our results show that the p52/Tfb2 subunit of TFIIH regulates the function of XPB through pair-wise interactions as described previously for p44 and XPD. cdk-activating kinase cyclin-dependent kinase nucleotide excision repair xeroderma pigmentosum major late promoter C-terminal domain Human TFIIH is a multiprotein complex composed of nine subunits ranging from 89 to 32 kDa. These subunits are assembled into two subcomplexes: the core TFIIH, which is composed of six subunits (XPB, XPD, p62, p52, p44, and p34) and the cdk-activating kinase (CAK),1 which is composed of cdk7, cyclin H, and MAT1. Recently, the molecular structures of both human and yeast TFIIHs have been determined by electron microscopy and show similarities in size, shape, and architecture (1Schultz P. Fribourg S. Poterszman A. Mallouh V. Moras D. Egly J.M. Cell. 2000; 102: 599-607Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 2Chang W.H. Kornberg R.D. Cell. 2000; 102: 609-613Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Originally identified as a basal transcription factor, TFIIH was subsequently found to contain XPB (Rad 25) and XPD (Rad 3), two helicases involved in nucleotide excision repair (NER) (3Feaver W.J. Svejstrup J.Q. Bardwell L. Bardwell A.J. Buratowski S. Gulyas K.D. Donahue T.F. Friedberg E.C. Kornberg R.D. Cell. 1993; 75: 1379-1387Abstract Full Text PDF PubMed Scopus (283) Google Scholar, 4Schaeffer L. Moncollin V. Roy R. Staub A. Mezzina M. Sarasin A. Weeda G. Hoeijmakers J.H. Egly J.M. EMBO J. 1994; 13: 2388-2392Crossref PubMed Scopus (334) Google Scholar, 5Schaeffer L. Roy R. Humbert S. Moncollin V. Vermeulen W. Hoeijmakers J.H. Chambon P. Egly J.M. Science. 1993; 260: 58-63Crossref PubMed Scopus (666) Google Scholar, 6Guzder S.N. Qiu H. Sommers C.H. Sung P. Prakash L. Prakash S. Nature. 1994; 367: 91-94Crossref PubMed Scopus (122) Google Scholar, 7Guzder S.N. Sung P. Bailly V. Prakash L. Prakash S. Nature. 1994; 369: 578-581Crossref PubMed Scopus (160) Google Scholar, 8Drapkin R. Sancar A. Reinberg D. Cell. 1994; 77: 9-12Abstract Full Text PDF PubMed Scopus (143) Google Scholar). Mutations in one of these subunits induce UV sensitivity in both human and yeast and are responsible for three rare human genetic disorders, xeroderma pigmentosum (XP), Cockayne syndrome, and trichothiodystrophy (9Berneburg M. Lehmann A.R. Adv. Genet. 2001; 43: 71-102Crossref PubMed Google Scholar, 10Vermeulen W. Rademakers S. Jaspers N.G. Appeldoorn E. Raams A. Klein B. Kleijer W.J. Hansen L.K. Hoeijmakers J.H. Nat. Genet. 2001; 27: 299-303Crossref PubMed Scopus (84) Google Scholar). Many XP patients suffer from a high incidence of skin cancer due to their inability to remove lesions from their DNA. Further studies in yeast have shown that additional subunits of TFIIH, including p62 (Tfb1), p52 (Tfb2), and p44 (Ssl1) also have an essential role in DNA repair (11Prakash S. Prakash L. Mutat. Res. 2000; 451: 13-24Crossref PubMed Scopus (286) Google Scholar). In an effort to understand the function of TFIIH in the diverse fundamental cellular processes in which it participates (transcription and DNA repair but also cell cycle regulation through the CAK complex), efforts have been aimed at systematically characterizing all TFIIH subunits. Several functions have been determined for individual components: XPB and XPD are ATP-dependent helicases indispensable for opening the DNA around a promoter and/or a lesion (12Holstege F.C. Tantin D. Carey M. van der Vliet P.C. Timmers H.T. EMBO J. 1995; 14: 810-819Crossref PubMed Scopus (131) Google Scholar, 13Tirode F. Busso D. Coin F. Egly J.M. Mol. Cell. 1999; 3: 87-95Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, 14Dvir A. Conaway J.W. Conaway R.C. Curr. Opin. Genet. Dev. 2001; 11: 209-214Crossref PubMed Scopus (105) Google Scholar, 15Batty D.P. Wood R.D. Gene. 2000; 241: 193-204Crossref PubMed Scopus (252) Google Scholar, 16Kim T.K. Ebright R.H. Reinberg D. Science. 2000; 288: 1418-1422Crossref PubMed Scopus (215) Google Scholar). Cdk7 is a serine/threonine kinase, which is regulated by cyclin H and MATI and phosphorylates several substrates including the C-terminal domain (CTD) of RNA polymerase II (see Ref. 17Larochelle S. Chen J. Knights R. Pandur J. Morcillo P. Erdjument-Bromage H. Tempst P. Suter B. Fisher R.P. EMBO J. 2001; 20: 3749-3759Crossref PubMed Scopus (104) Google Scholar and references therein). Recently, the N-terminal part of p44, a subunit of core TFIIH, has been shown to positively regulate XPD helicase activity, whereas the C-terminal part is involved in promoter escape (18Tremeau-Bravard A. Perez C. Egly J.M. J. Biol. Chem. 2001; 276: 27693-27697Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). The XPD helicase regulation is lacking in a majority of XP-D patients because mutations in the C-terminal end of XPD abolish the XPD/p44 interaction (19Coin F. Marinoni J.C. Rodolfo C. Fribourg S. Pedrini A.M. Egly J.M. Nat. Genet. 1998; 20: 184-188Crossref PubMed Scopus (312) Google Scholar). This can explain the UV sensitivity and NER defect harbored by yeast deleted for Ssl1, the p44 yeast homolog (20Wang Z. Buratowski S. Svejstrup J.Q. Feaver W.J., Wu, X. Kornberg R.D. Donahue T.F. Friedberg E.C. Mol. Cell. Biol. 1995; 15: 2288-2293Crossref PubMed Scopus (75) Google Scholar, 21Sweder K.S. Chun R. Mori T. Hanawalt P.C. Nucleic Acids Res. 1996; 24: 1540-1546Crossref PubMed Scopus (18) Google Scholar, 22Maines S. Negritto M.C., Wu, X. Manthey G.M. Bailis A.M. Genetics. 1998; 150: 963-976PubMed Google Scholar, 23Seroz T. Perez C. Bergmann E. Bradsher J. Egly J.M. J. Biol. Chem. 2000; 275: 33260-33266Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). The three remaining subunits, p62, p52, and p34, do not contain any known specific motif or enzymatic activities, and their function, as components of TFIIH structure, remains obscure. Concerning p52, in vitro transcription and DNA repair assays, as well as microinjection experiments using p52 antibody, have demonstrated that p52, the last subunit of TFIIH cloned (24Marinoni J.C. Roy R. Vermeulen W. Miniou P. Lutz Y. Weeda G. Seroz T. Gomez D.M. Hoeijmakers J.H. Egly J.M. EMBO J. 1997; 16: 1093-1102Crossref PubMed Scopus (61) Google Scholar), is involved in both transcription and NER. Moreover, deletion of the C-terminal region of Tfb2, the yeast counterpart of p52 (64% similar and 40% identical), is detrimental for NER activity of yeast TFIIHin vivo and in vitro (25Feaver W.J. Henry N.L. Wang Z., Wu, X. Svejstrup J.Q. Bushnell D.A. Friedberg E.C. Kornberg R.D. J. Biol. Chem. 1997; 272: 19319-19327Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). In the present paper we have introduced a similar mutation in the human p52 subunit. We demonstrate that the C-terminal region of p52 is required to anchor XPB (the helicase involved in the opening of the promoter) within the core TFIIH. This study defines p52 as a preferential and indispensable partner of XPB in the TFIIH complex and helps to define the function of the XPB helicase in class II genes transcription and NER. Baculoviruses expressing the TFIIH subunits XPB, His-XPB, XPD, p62, p52, His-p44, p44, p34, cdk7, His-cyclinH, and MAT1 were constructed as previously described (Ref. 13Tirode F. Busso D. Coin F. Egly J.M. Mol. Cell. 1999; 3: 87-95Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar and references therein). To obtain baculoviruses expressing p52(1–135), p52(137–296), p52(1–358), p52(381–462), XPB(44–782), and XPB(208–782), the corresponding cDNAs were amplified by PCR and inserted into a modified pVL1392 baculovirus transfer vector that adds a 6-histidine tag at the N terminus of the open reading frame. To obtain baculoviruses expressing p52, p52(1–304), and p52(305–462) with a FLAG peptide (MTKDDDDKH) fused to its N terminus, the corresponding cDNAs were cloned in the PSK277 vector using a NdeI and BamHI restricted site as described (26Jawhari A. Uhring M. Crucifix C. Fribourg S. Schultz P. Poterszman A. Egly J.M. Moras D. Protein Expression Purif. 2002; 24: 513-523Crossref PubMed Scopus (13) Google Scholar). The vectors were then recombined with baculovirus DNA (Baculogold, PharMingen), and the resulting viruses were plaque-purified and -amplified according to the manufacturer. Typically, 108 cells were infected with combinations of recombinant baculoviruses expressing combinations of XPB, XPD, p62, p52 or p52(1–358), His-p44, p34, cdk7, His-cyclinH, and MAT1 as indicated and were collected 48 h after infection. Cells were washed in 1× phosphate-buffered saline, 30% glycerol and dounced in 10 ml of buffer A (20 mm Tris-HCl, pH 7.5, 20% glycerol, 150 mm NaCl, 0.1% Nonidet P40, 5 mmβ-mercaptoethanol). After centrifugation at 14,000 ×g for 30 min at 4 °C, clarified lysates were loaded on a heparin-Ultrogel column (Sepracor) pre-equilibrated in buffer A. After extensive washing with buffer A containing 300 mmNaCl, the proteins were eluted with the same buffer containing 500 mm NaCl. The eluted fractions were dialyzed for 2 × 2 h against 50 mm Tris-HCl, pH 7.9, 20% glycerol, 50 mm KCl, 0.1 mm EDTA, and 0.5 mm dithiothreitol and immunopurified using the 1H5 anti-p44 antibody at different ionic strengths. Alternatively, to purify recombinant TFIIH by FLAG strategy, a virus derived from the pSK277 transfer vector expressing a p34 subunit with a FLAG peptide (MTKDDDDKH) fused at its N terminus was used. This virus was used together with viruses expressing p44, p52, p62, His-XPB, XPD, cdk7, His-cyclinH, and MAT1. 48h hours after infection, 109 cells were collected, washed in 1× phosphate-buffered saline, 30% glycerol and dounced in buffer B (20 mm Tris-HCl, pH 7.8, 10% glycerol, NaCl 250 mm, 2 mm β-mercaptoethanol). DNA and cell were by centrifugation at 14,000 ×g 30 The were for h at 4 with of After a with buffer B containing 5 mm TFIIH were eluted with the same buffer containing 250 The fractions from the which TFIIH were for 4 h at 4 with with After three with buffer B containing 0.1% Nonidet P40, proteins were eluted in one of buffer B containing peptide for interactions were by in cells × with the corresponding recombinant baculoviruses at a of of collected 48 h after infection, washed in 1× phosphate-buffered saline, 30% glycerol and dounced in ml of buffer A. lysates were by centrifugation at 14,000 × for 30 min at 4 50 of clarified was on of with the antibody which the site of XPB, and which the N terminus of in buffer (20 pH 7.5, 10% glycerol, 50 mm KCl, 0.1 mm After h of at 4 °C, the were washed in buffer containing 150 and in The proteins were by and by using the DNA containing a was as described Wood R.D. Mol. Biol. 1999; Google Scholar). were in buffer containing mm at 7.8, mm KCl, 5 mm mm mm EDTA, 10% glycerol, of and 2 mm 50 of of 10 of 50 of of NER or of TFIIH for 10 min at 30 °C, 50 of was and were for min at 30 were by of an to the DNA containing at the was to the excision polymerase and of were used to to excision The were by on and by The helicase was by 5 of an a to of DNA to of The resulting was for h at and then to and with the in the of 50 mm and of The helicase was then as described (24Marinoni J.C. Roy R. Vermeulen W. Miniou P. Lutz Y. Weeda G. Seroz T. Gomez D.M. Hoeijmakers J.H. Egly J.M. EMBO J. 1997; 16: 1093-1102Crossref PubMed Scopus (61) Google Scholar). was as previously fractions were for 2 h at 30 in the of of in a containing mm pH 7.9, 4 mm and 50 were by to 50 The were then in 0.5 and (25Feaver W.J. Henry N.L. Wang Z., Wu, X. Svejstrup J.Q. Bushnell D.A. Friedberg E.C. Kornberg R.D. J. Biol. Chem. 1997; 272: 19319-19327Abstract Full Text Full Text PDF PubMed Scopus (62) Google were in a containing mm pH 7.9, mm pH 7.9, mm 0.5 30 mm KCl, of of and of were 30 min at °C, and were by the of 5 of After the was and on The was by (20 was at for 30 min with recombinant and TFIIH as indicated in a that 50 mm pH 7.9, 10% glycerol, mm EDTA, 0.5 mm and 5 mm and were for the last 5 of was for 2 after which the was by of 2 of After DNA was by in and to 30 of using an After and the was loaded a The was and (12Holstege F.C. Tantin D. Carey M. van der Vliet P.C. Timmers H.T. EMBO J. 1995; 14: 810-819Crossref PubMed Scopus (131) Google Scholar). the 30 of recombinant TFIIH by the FLAG were with 10 of at and h at The was by of 5 of mm and the were by by using against the N-terminal or the C-terminal part of p52. of recombinant we have TFIIH in of a p52. and were in cells with baculoviruses expressing the or of p52 as well as the other TFIIH subunits F. Busso D. Coin F. Egly J.M. Mol. Cell. 1999; 3: 87-95Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). The cell were then loaded on a heparin-Ultrogel and the was eluted with 0.5 including and which were a After extensive washing of the column mm both were eluted with the same buffer containing 50 mm by the of both are The TFIIH from the complex by the of the p52 with p44 recombinant TFIIHs were then for their DNA repair and transcription activities. (25Feaver W.J. Henry N.L. Wang Z., Wu, X. Svejstrup J.Q. Bushnell D.A. Friedberg E.C. Kornberg R.D. J. Biol. Chem. 1997; 272: 19319-19327Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), the recombinant human TFIIH was in of the of of DNA to an in vitro F. Coin F. H. M. Egly J.M. Wood R.D. Dev. 2000; 14: PubMed Google containing recombinant and In the transcription activity of was to in vitro transcription containing the major late RNA and basal transcription TFIIH. CAK is to the core TFIIH, we the p52 C-terminal deletion have the effect of has shown that for as on the CAK subunits is not a function of the kinase activity of but is due to the of CAK to an of TFIIH within the transcription complex H. L. Fisher L. Egly J.M. Reinberg D. Nature. PubMed Scopus Google Scholar, J. M. Cell. Full Text PDF PubMed Scopus Google Scholar). and were in cells and as described CAK was then to the transcription containing the or the In both CAK and transcription activity by 2 and 4 with and These results that the defect with is of CAK transcription and is to the core of TFIIH. We then p52 mutations the enzymatic activities of TFIIH that in transcription and DNA repair We then investigated the by the subunit of TFIIH of peptide that of the found in the C-terminal domain of the RNA II subunit. TFIIHs that the activity of CAK is not by the p52 In of we the activity of both and the XPB the XPD activities were by the p52 mutation we mutation of p52 the XPD or XPB activities. We found that the ATP-dependent XPD helicase activity of is similar to the XPB helicase activity using a F. Bergmann E. A. Egly J.M. EMBO J. 1999; PubMed Scopus Google and XPB has been shown to the helicase involved in promoter we investigated its to promoter opening using a Wood R.D. Mol. Biol. 1999; Google Scholar). In the of of basal transcription including TFIIH and RNA of both and the of the to an of the sensitivity of the around the promoter and and promoter opening around the transcription is of promoter opening is 2 and This with the in that of transcription can to an of promoter promoter opening is and XPB has been shown to the helicase involved in the transcription F. Bergmann E. A. Egly J.M. EMBO J. 1999; PubMed Scopus Google Scholar), we then p52 mutation of XPB with the TFIIH multiprotein against p44 were used to TFIIHs in cells with baculoviruses p52 or together with all of the TFIIH subunits or with XPB, p62, p44, and at XPB not with other TFIIH subunits is present with 250 mm not TFIIH we that prevents of XPB in the core TFIIH In of we demonstrate that p52 is required for the of XPB in the core TFIIH. XPB from infected cells with p44, p62, and p34 in the of p52 2 and these results show that the C-terminal domain of p52 the of the of XPB within TFIIH or not is The a role for p52 in promoter opening and for the of XPB into TFIIH. We these two subunits cells were with two baculoviruses expressing p52 and one of the subunits of the core TFIIH (XPB, XPD, p62, p44, and proteins of the core TFIIH in cell lysates were then with the corresponding After extensive washing at 250 mm KCl, the were by these XPB and to a p62 not were to interact with p52. These results that XPB preferential to p52 within TFIIH. To map the region of p52 that is involved in the interaction with XPB, we a of baculoviruses expressing of p52 corresponding to and on (see at We then of the with XPB a we that all proteins were is to with XPB, p52(1–135), p52(1–304), and as well as p52(305–462) but not and The results show that p52 two XPB that to and and the of the C-terminal end of p52 in XPB To recombinant TFIIH containing the nine subunits in cells was to a to at that are to the is that are involved in the of TFIIH was with of and by by Using an antibody against the N terminus of p52, we that p52 was to in a of that to these the C-terminal part of p52 is to as the antibody against the terminus of p52 not any molecular This that the terminus of the is to the and is in with the that not interact with To map in the region of XPB involved in the interaction with p52, we several XPB including and and as described and but not not p52 with is also that together with p34, p44, p52, and p62 subunits, is to into core TFIIH Taken together, the demonstrate that the of p52 with XPB which the DNA motif is regulated by the transcription that are involved in RNA of these transcription enzymatic activities that the of the transcription as promoter and RNA II has been known for that the enzymatic activities of the and XPD subunits of TFIIH are The activity of kinase on the of and two other TFIIH subunits, as well as on the of its S. Chen J. Knights R. Pandur J. Morcillo P. Erdjument-Bromage H. Tempst P. Suter B. Fisher R.P. EMBO J. 2001; 20: 3749-3759Crossref PubMed Scopus (104) Google Scholar). interaction with p44, XPD helicase activity (19Coin F. Marinoni J.C. Rodolfo C. Fribourg S. Pedrini A.M. Egly J.M. Nat. Genet. 1998; 20: 184-188Crossref PubMed Scopus (312) Google Scholar). Mutations in the C-terminal domain of XPD the with p44, the NER defect with of the XP-D patients E. M. Sarasin A. Jaspers N.G. H. Lehmann A.R. S. A. 1997; PubMed Scopus Google Scholar). In mutations in XPD or in p44 that the interaction the of TFIIH by the of XPD and CAK subunits with the core and/or the of CAK to the core TFIIH T. Perez C. Bergmann E. Bradsher J. Egly J.M. J. Biol. Chem. 2000; 275: 33260-33266Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, F. Bergmann E. A. Egly J.M. EMBO J. 1999; PubMed Scopus Google with a in the of genes A. A. Sarasin A. C. Egly J.M. Cell. 2002; Full Text Full Text PDF Scopus Google Scholar). In the present we show that the p52 subunit is the partner of the XPB of the enzymatic activities found in TFIIH its partner within the Using a recombinant TFIIH, with which the C-terminal domain of p52 was we have demonstrated that the p52 mutation is detrimental for both repair and transcription activities. several including to defect to an in the opening of the DNA around the previously demonstrated that the XPB helicase was in the promoter opening (12Holstege F.C. Tantin D. Carey M. van der Vliet P.C. Timmers H.T. EMBO J. 1995; 14: 810-819Crossref PubMed Scopus (131) Google Scholar, F. Bergmann E. A. Egly J.M. EMBO J. 1999; PubMed Scopus Google Scholar), we further investigated the role of p52 in of XPB to core TFIIH. We show that the of p52 is a for the of XPB in core TFIIH. the complex not interact with XPB in the of p52. In XPB is by p52 as also observed in yeast W.J. W. L. Kornberg R.D. Friedberg E.C. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar), the two proteins interact XPB two of the is within and a p52 The region as with XPB, whereas This region is from human to yeast 4 and at The that not interact with XPB with the that the terminus of p52 is in a that domain is for the architecture and/or the of TFIIH, as a deletion of the of XPB within the core TFIIH. This can explain the NER defect and as well as the observed in yeast cells containing a similar We that the C-terminal domain of p52 a that XPB TFIIH. The of the p52 deletion a and/or of the XPB helicase within TFIIH and within the transcription and/or DNA repair This of XPB with TFIIH in a defect in one of the essential function of TFIIH, DNA the XPB and XPD no human genetic have been with the p52. a in the last of XPB from patients results in a similar defect in promoter opening and RNA F. Bergmann E. A. Egly J.M. EMBO J. 1999; PubMed Scopus Google Scholar). Moreover, using we that the of the XPB helicase of TFIIH promoter DNA was M. Coin J.M. Y. Y. Egly J.M. B. Mol. Cell. Biol. 2000; 20: PubMed Scopus Google Scholar). as well as deletion experiments that XPB is composed of three a N-terminal domain to as DNA the core which the helicase and the C-terminal domain A. V. Egly J.M. Moras D. J.C. 1997; Full Text PDF PubMed Scopus Google Scholar). We have shown that the region of XPB that p52 to the N-terminal DNA domain of helicase G. J. van der van van der A.J. Hoeijmakers J.H. PubMed Scopus Google Scholar, L. van der A.J. Nucleic Acids Res. 1994; PubMed Scopus Google Scholar). is that the C-terminal of p52 and of XPB within TFIIH. deletion of the C-terminal region of p52 the of and/or the of TFIIH within the transcription and NER remains to further We are to P. for reading A. is for and and J. L. for and of TFIIH. with