Yeast DNA Repair Protein RAD23 Promotes Complex Formation between Transcription Factor TFIIH and DNA Damage Recognition Factor RAD14

In Saccharomyces cerevisiae, the multisubunit RNA polymerase II general transcription factor TFIIH is indispensable for transcription initiation and some of its subunits are known to be required for nucleotide excision repair (NER) of DNA damaged by ultraviolet light. RAD3, a subunit of TFIIH, binds UV-damaged DNA in an ATP-dependent manner. It has, however, remained unclear how TFIIH is assembled with the other damage recognition component RAD14. Here, we demonstrate a higher order complex consisting of TFIIH, RAD14, and another NER protein RAD23, and complex formation between TFIIH and RAD14 is facilitated by the RAD23 protein. In Saccharomyces cerevisiae, the multisubunit RNA polymerase II general transcription factor TFIIH is indispensable for transcription initiation and some of its subunits are known to be required for nucleotide excision repair (NER) of DNA damaged by ultraviolet light. RAD3, a subunit of TFIIH, binds UV-damaged DNA in an ATP-dependent manner. It has, however, remained unclear how TFIIH is assembled with the other damage recognition component RAD14. Here, we demonstrate a higher order complex consisting of TFIIH, RAD14, and another NER protein RAD23, and complex formation between TFIIH and RAD14 is facilitated by the RAD23 protein. Extensive genetic studies in Saccharomyces cerevisiae have indicated the requirement of 11 genes, RAD1, RAD2, RAD3, RAD4, RAD7, RAD10, RAD14, RAD16, RAD23, RAD25, and MMS19 in nucleotide excision repair (NER). 1The abbreviations used are:NERnucleotide excision repairMOPS3-(N-morpholino)propanesulfonic acidBSAbovine serum albuminPAGEpolyacrylamide gel electrophoresis. 1The abbreviations used are:NERnucleotide excision repairMOPS3-(N-morpholino)propanesulfonic acidBSAbovine serum albuminPAGEpolyacrylamide gel electrophoresis. Among these genes, RAD3 and RAD25 are of particular interest because in addition to their role in NER, they are essential for cell viability (1Prakash S. Sung P. Prakash L. Annu. Rev. Genet. 1993; 27: 33-70Crossref PubMed Scopus (256) Google Scholar). Studies with temperature-sensitive conditional lethal mutations have indicated a direct and essential role of RAD3 and RAD25 in RNA polymerase II transcription (2Guzder S.N. Qiu H. Sommers C.H. Sung P. Prakash L. Prakash S. Nature. 1994; 367: 91-94Crossref PubMed Scopus (121) Google Scholar, 3Guzder S. Sung P. Bailly V. Prakash L. Prakash S. Nature. 1994; 369: 578-581Crossref PubMed Scopus (159) Google Scholar, 4Qiu H. Park E. Prakash L. Prakash S. Genes & Dev. 1993; 7: 2161-2171Crossref PubMed Scopus (73) Google Scholar). Both RAD3 and RAD25 proteins contain single-stranded DNA-dependent ATPase and DNA helicase activities (3Guzder S. Sung P. Bailly V. Prakash L. Prakash S. Nature. 1994; 369: 578-581Crossref PubMed Scopus (159) Google Scholar, 5Sung P. Prakash L. Matson S.W. Prakash S. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8951-8955Crossref PubMed Scopus (167) Google Scholar, 6Sung P. Prakash L. Weber S. Prakash S. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6045-6049Crossref PubMed Scopus (90) Google Scholar). The DNA helicase activity of RAD3 is required for NER but is dispensable for polymerase II transcription (7Sung P. Higgins D. Prakash L. Prakash S. EMBO J. 1988; 7: 3263-3269Crossref PubMed Scopus (221) Google Scholar). In contrast, the DNA helicase activity of RAD25 is essential for both transcription and DNA repair (3Guzder S. Sung P. Bailly V. Prakash L. Prakash S. Nature. 1994; 369: 578-581Crossref PubMed Scopus (159) Google Scholar, 4Qiu H. Park E. Prakash L. Prakash S. Genes & Dev. 1993; 7: 2161-2171Crossref PubMed Scopus (73) Google Scholar). RAD3 and RAD25 are components of the yeast polymerase II general transcription factor TFIIH. In addition, TFIIH contains four other subunits of 75, 55, 50, and 38 kDa (8Feaver 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 (281) Google Scholar). TFB1 and SSL1 encode the 75- and 50-kDa subunits, whereas the genes for the 55- and 38-kDa subunits have not yet been identified. A role of TFIIH in NER has been inferred from the observation that TFIIH corrects the NER defect in rad3 and rad25 mutant extracts (9Wang Z. Svejstrup J.Q. Feaver W.J. Wu X. Kornberg R.D. Friedberg E.C. Nature. 1994; 368: 74-75Crossref PubMed Scopus (137) Google Scholar). nucleotide excision repair 3-(N-morpholino)propanesulfonic acid bovine serum albumin polyacrylamide gel electrophoresis. nucleotide excision repair 3-(N-morpholino)propanesulfonic acid bovine serum albumin polyacrylamide gel electrophoresis. Recognition of DNA damage represents the first crucial step in NER. We have previously shown that RAD14, a zinc metalloprotein, binds specifically to ultraviolet-damaged DNA (10Guzder S.N. Sung P. Prakash L. Prakash S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5433-5437Crossref PubMed Scopus (88) Google Scholar). Interestingly, RAD3 also binds preferentially to UV-damaged DNA in a manner dependent upon ATP and negative superhelicity (11Sung P. Watkins J.F. Prakash L. Prakash S. J. Biol. Chem. 1994; 269: 8303-8308Abstract Full Text PDF PubMed Google Scholar). The rad3 Arg-48 mutant protein defective in DNA helicase activity also binds UV damaged DNA like the wild type RAD3 protein, indicating that DNA helicase activity and damage binding are two distinct and separable functions in RAD3 (11Sung P. Watkins J.F. Prakash L. Prakash S. J. Biol. Chem. 1994; 269: 8303-8308Abstract Full Text PDF PubMed Google Scholar). Because RAD3 is a damage recognition protein, it is important to determine how TFIIH is assembled with RAD14 2We have observed that the RAD14 gene contains an intron, representing the first example of an intron in an S. cerevisiae DNA repair gene. RAD14 encodes a protein of 371 amino acids with a predicted size of 43 kDa, and RAD14 immunoprecipitated from RAD+ cells exhibits a size of 48 kDa in SDS-PAGE. The translation-initiating ATG codon in RAD14 is at position −456 in the previously reported sequence (19Bankmann M. Prakash L. Prakash S. Nature. 1992; 355: 555-558Crossref PubMed Scopus (97) Google Scholar), and an 84-base pair intron occurs between positions −429 and −346 (19Bankmann M. Prakash L. Prakash S. Nature. 1992; 355: 555-558Crossref PubMed Scopus (97) Google Scholar). The presence of the intron was confirmed by sequence analysis of reverse transcriptase-polymerase chain reaction product of poly(A)+ mRNA isolated from wild type and rna2-1 strains held at 25°C or at 37°C for 1 h. 2We have observed that the RAD14 gene contains an intron, representing the first example of an intron in an S. cerevisiae DNA repair gene. RAD14 encodes a protein of 371 amino acids with a predicted size of 43 kDa, and RAD14 immunoprecipitated from RAD+ cells exhibits a size of 48 kDa in SDS-PAGE. The translation-initiating ATG codon in RAD14 is at position −456 in the previously reported sequence (19Bankmann M. Prakash L. Prakash S. Nature. 1992; 355: 555-558Crossref PubMed Scopus (97) Google Scholar), and an 84-base pair intron occurs between positions −429 and −346 (19Bankmann M. Prakash L. Prakash S. Nature. 1992; 355: 555-558Crossref PubMed Scopus (97) Google Scholar). The presence of the intron was confirmed by sequence analysis of reverse transcriptase-polymerase chain reaction product of poly(A)+ mRNA isolated from wild type and rna2-1 strains held at 25°C or at 37°C for 1 h. that also functions in damage recognition. Here, we show that TFIIH is complexed with RAD14 via the RAD23 protein. We discuss the possible role of this complex in NER. Antibodies to RAD3 (6Sung P. Prakash L. Weber S. Prakash S. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6045-6049Crossref PubMed Scopus (90) Google Scholar), RAD14 (10Guzder S.N. Sung P. Prakash L. Prakash S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5433-5437Crossref PubMed Scopus (88) Google Scholar), RAD23 (12Watkins J.F. Sung P. Prakash L. Prakash S. Mol. Cell. Biol. 1993; 13: 7757-7765Crossref PubMed Scopus (215) Google Scholar), and RAD25 (3Guzder S. Sung P. Bailly V. Prakash L. Prakash S. Nature. 1994; 369: 578-581Crossref PubMed Scopus (159) Google Scholar) were raised in rabbits as described. Antibodies to TFB1 were raised against a GST-TFB1 hybrid protein (13Gileadi O. Feaver W.J. Kornberg R.D. Science. 1992; 257: 1389-1392Crossref PubMed Scopus (53) Google Scholar) that was expressed in Escherichia coli and purified from inclusion bodies by preparative denaturing polyacrylamide gel electrophoresis. All the antibodies were purified by affinity chromatography as described (14Bailly V. Sommers C.H. Sung P. Prakash L. Prakash S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8273-8277Crossref PubMed Scopus (56) Google Scholar). Yeast extracts were prepared in buffer B (50 m M Tris-HCl, pH 7.5, 50 m M NaCl, 0.2% Triton X-100, and protease inhibitors) using a French press as described previously (14Bailly V. Sommers C.H. Sung P. Prakash L. Prakash S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8273-8277Crossref PubMed Scopus (56) Google Scholar). Extract from 0.7 g of cells was mixed for 60 min at 25°C with 30 μl of protein A-agarose beads containing 3 mg/ml of covalently conjugated anti-RAD14 antibodies or non-immune IgG. After being washed twice with 600 μl of buffer B, bound proteins were eluted from the immunoprecipitate by a 5-min treatment with 70 μl of 2% SDS at 42°C, and 10 μl of the SDS eluates were analyzed by immunoblotting. RAD23 protein (2 mg) was dialyzed against 1 liter of coupling buffer (0.1 M MOPS, pH 7.5) at 4°C for 12 h. Affi-Gel 15 matrix (Bio-Rad), 0.5 ml, previously washed with cold water, was mixed with purified RAD23 protein in a final volume of 1 ml overnight at 4°C. The unreacted active groups on the matrix were blocked by incubation with 1 M ethanolamine, pH 8.0, for 4 h at 4°C. Bovine serum albumin (4 mg) was coupled to Affi-Gel 15 using the same procedure. 35S-Labeled RAD3, RAD14, RAD25, and TFB1 proteins were obtained by coupled in vitro transcription and translation in 50-μl reactions containing 40 μCi of [35S]methionine with the use of the TNT T7 reticulocyte lysate system (Promega). The 35S-labeled translation products were partially purified by precipitation with 50% ammonium sulfate (0.31 g/ml). The protein pellet was dissolved in 50 μl buffer A (20 m M HEPES-KOH, pH 7.5, 70 m M KCl, 5 m M sodium bisulfite, 4 m M MgCl2, 0.5 m M EDTA, 1 m M dithiothreitol, 0.1% Tween 20, and 2.5% glycerol). A 10-μl aliquot of the protein solution was mixed with 90 μl of buffer A and 10 μl of BSA-Affi-Gel or RAD23-Affi-Gel beads for 30 min at 25°C. After washing with 500 μl of ice-cold buffer A, bound proteins were eluted from the beads with the use of 2% SDS and fractionated in 9% denaturing polyacrylamide gels. The gels were dried onto Whatman 3MM paper and subjected to fluorography. Extract was prepared from 80 g of the rad23Δ yeast strain JWY36 with the use of a French press and passed through a Bio-Rex 70 column (1.6 × 8 cm) as described (15Sayer M.H. Tschochner H. Kornberg R.D. J. Biol. Chem. 1992; 267: 23376-23382Abstract Full Text PDF PubMed Google Scholar). After washing the Bio-Rex 70 column with buffer A containing 300 m M KOAc, TFIIH and other proteins were eluted with 600 m M KOAc (15Sayer M.H. Tschochner H. Kornberg R.D. J. Biol. Chem. 1992; 267: 23376-23382Abstract Full Text PDF PubMed Google Scholar). Fractions corresponding to the protein peak were pooled (5 ml), concentrated to 2 ml in a Centricon-30 (Amicon), and dialyzed overnight against 1 liter of buffer C (50 m M Tris-HCl, pH 7.5, 0.1 m M EDTA, 5% glycerol, 1 m M dithiothreitol, and protease inhibitors containing 50 m M KOAc). RAD23-Affi-Gel and BSA-Affi-Gel, 0.15 ml each, were packed in a 1-ml pipette tip plugged with glass wool and 1 ml of the dialyzed TFIIH pool was passed through the RAD23 affinity column or the BSA control column twice at 25°C. The columns were eluted with 0.45 ml of 0.2 M, 0.5 M, 1 M, and 2 M KOAc in buffer C, collecting two 0.225-ml fractions during each elution step. Five μl of the starting Bio-Rex 70 TFIIH pool of the flow-through fraction from the RAD23-Affi-Gel and BSA-Affi-Gel columns and of the various salt eluates were examined for their content of the RAD25, RAD3, and the TFB1 proteins. Extracts for transcription were prepared as described previously (16Woontner M. Wade P.A. Bonner J. Jaehning J.A. Mol. Cell. Biol. 1991; 11: 4555-4560Crossref PubMed Scopus (79) Google Scholar). The template for transcription, pSL187, contains the promoter of the yeast CYC1 gene and yields transcripts of 375 and 350 nucleotides (17Koleske A. Buratowski S. Nonet M. Young R.A. Cell. 1992; 69: 883-894Abstract Full Text PDF PubMed Scopus (139) Google Scholar). Reaction mixtures were assembled and processed as described (2Guzder S.N. Qiu H. Sommers C.H. Sung P. Prakash L. Prakash S. Nature. 1994; 367: 91-94Crossref PubMed Scopus (121) Google Scholar, 3Guzder S. Sung P. Bailly V. Prakash L. Prakash S. Nature. 1994; 369: 578-581Crossref PubMed Scopus (159) Google Scholar). To investigate whether RAD14 protein forms a complex with TFIIH, affinity-purified antibodies to RAD14 (10Guzder S.N. Sung P. Prakash L. Prakash S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5433-5437Crossref PubMed Scopus (88) Google Scholar) were covalently conjugated to protein A-agarose, and the resulting immunobeads were mixed with wild type RAD+ yeast extract at 25°C for 60 min to bind RAD14 and proteins that are associated with RAD14. As control, the extract was also incubated with protein A-agarose beads containing non-immune IgG. After washing the immunoprecipitates with a large volume of buffer, bound proteins were eluted with SDS and analyzed by immunoblotting with the appropriate antibodies for RAD14 and the TFIIH components RAD3, RAD25, and TFB1. We found that the amount of the TFIIH components associated with the anti-RAD14 immunobeads was ≈6-fold higher than the background level of these components in the control (Fig. 1, compare lanes 3 and 4). Interestingly, another NER protein, RAD23 (1Prakash S. Sung P. Prakash L. Annu. Rev. Genet. 1993; 27: 33-70Crossref PubMed Scopus (256) Google Scholar, 12Watkins J.F. Sung P. Prakash L. Prakash S. Mol. Cell. Biol. 1993; 13: 7757-7765Crossref PubMed Scopus (215) Google Scholar), also co-precipitated specifically with RAD14, as the level of RAD23 in the anti-RAD14 immunoprecipitate was ≈6-fold higher than the control (Fig. 1, compare lanes 3 and 4). These results suggest the existence of a higher order protein complex in wild type yeast cells consisting of TFIIH, RAD14, and RAD23. Mutations in the RAD23 gene greatly compromise the efficiency of NER (18Miller R.D. Prakash L. Prakash S. Mol. & Gen. Genet. 1982; 188: 235-239Crossref PubMed Scopus (49) Google Scholar), and we have previously suggested that RAD23 protein is a non-catalytic NER component that could act in the assembly of a functional nucleotide excision repair complex (1Prakash S. Sung P. Prakash L. Annu. Rev. Genet. 1993; 27: 33-70Crossref PubMed Scopus (256) Google Scholar, 12Watkins J.F. Sung P. Prakash L. Prakash S. Mol. Cell. Biol. 1993; 13: 7757-7765Crossref PubMed Scopus (215) Google Scholar). To directly test this possibility, we carried out anti-RAD14 immunoprecipitation using extracts prepared from a yeast strain lacking the genomic RAD23 gene. In the absence of RAD23 protein, as in the case of rad23Δ extract, the amount of TFB1, RAD3, and RAD25 proteins bound in the anti-RAD14 immunoprecipitate was only slightly higher (20-40%) than the background level of these proteins that were associated nonspecifically with beads containing the non-immune IgG (Fig. 1, compare lanes 1 and 2). Thus, co-precipitation of TFIIH with RAD14 protein is strongly dependent on the RAD23 protein, lending support to the notion that efficient assembly of the complex of NER proteins requires the RAD23 protein. To further establish the role of RAD23 in complex formation, we purified this protein from yeast cells. To facilitate the purification, the RAD23 gene was joined to the highly expressed yeast ADC1 promoter to yield the multicopy plasmid pJW112 (2μ,ADC1-RAD23). Purification of RAD23 from the protease-deficient yeast strain LP2749-9B harboring pJW112 was achieved by a of ammonium sulfate precipitation and in columns of and The purified RAD23 protein was analyzed by and with that the protein was (Fig. We obtained 5 of RAD23 protein from g of starting yeast the use of DNA binding and DNA in using a pH we found of RAD23 protein with We also found ATPase or activity in RAD23. immunoprecipitation studies indicated that RAD23 is of a higher order complex of excision repair proteins and TFIIH and that it in the assembly of this protein To determine whether RAD23 TFIIH and RAD14 directly or via some other protein we covalently conjugated purified RAD23 protein to and used the resulting RAD23 matrix as affinity beads for binding 35S-labeled TFB1, RAD3, RAD25, and RAD14 proteins. To proteins for this the protein of the TFB1, RAD3, RAD25, and RAD14 genes were the T7 and the resulting were in vitro to that for these by translation of the in reticulocyte lysate in the presence of The proteins obtained were partially purified by ammonium sulfate dissolved in reaction buffer, and mixed with the RAD23 binding to the RAD23 matrix was to at 25°C for 30 After washing with binding buffer, the bound 35S-labeled proteins were eluted from the RAD23 Affi-Gel beads with the use of SDS and by denaturing polyacrylamide gel electrophoresis. As a control in these we also mixed the 35S-labeled proteins with beads containing volume of matrix an amount of BSA twice that of RAD23 The BSA beads were with and the eluates were in polyacrylamide gels the SDS eluates from the RAD23 As shown in 2 B, the level of the 35S-labeled TFB1 and RAD14 proteins in the SDS eluates from the RAD23 affinity beads was and of that in the BSA control, indicating a and direct of TFB1 and RAD14 with RAD23. The amount of 35S-labeled RAD25 protein bound to the RAD23 beads was higher than to the BSA an between RAD25 and RAD23 proteins as (Fig. in (Fig. and not the level of RAD3 bound to the RAD23 beads was the same as that found in the control, indicating that these two proteins not To further that RAD23 and TFIIH a Bio-Rex 70 column fraction from rad23Δ extract that was in TFIIH was passed through a column of RAD23 and BSA was used as a The RAD23 column and control BSA column were washed with buffer and eluted with 0.2 M, 0.5 M, 1 M, and 2 M and the content of the TFIIH components RAD3, RAD25, and TFB1 in the various salt was examined by immunoblotting. The results from this analysis that a of TFB1, RAD3, and RAD25 proteins were on the RAD23-Affi-Gel column and that these proteins are eluted from the RAD23 column from M (Fig. a amount of these proteins bound to the control BSA and of the proteins were eluted by 0.2 M (Fig. We have previously described conditional lethal mutations of RAD3 and RAD25 in defective RNA polymerase II transcription at the both in and in vitro (2Guzder S.N. Qiu H. Sommers C.H. Sung P. Prakash L. Prakash S. Nature. 1994; 367: 91-94Crossref PubMed Scopus (121) Google Scholar, 3Guzder S. Sung P. Bailly V. Prakash L. Prakash S. Nature. 1994; 369: 578-581Crossref PubMed Scopus (159) Google H. Park E. Prakash L. Prakash S. Genes & Dev. 1993; 7: 2161-2171Crossref PubMed Scopus (73) Google Scholar). As shown in the defect in the and the extracts be specifically by the from the RAD23 affinity These results are with of TFIIH with the RAD23 protein. The of the RAD14 and RAD23 genes to determine whether they are essential for cell viability their known role in nucleotide excision Yeast strains genomic of these genes show at (12Watkins J.F. Sung P. Prakash L. Prakash S. Mol. Cell. Biol. 1993; 13: 7757-7765Crossref PubMed Scopus (215) Google Scholar, M. Prakash L. Prakash S. Nature. 1992; 355: 555-558Crossref PubMed Scopus (97) Google Scholar) or at V. P. L. and S. indicating that they are not required for RNA polymerase II In with the genetic we found that extracts prepared from the and the rad23Δ strains are as in RNA polymerase II transcription as the wild type extract, of whether the mutant extracts were subjected to treatment at 37°C for 5 min to the transcription reaction (Fig. 4). The requirement for RAD14 and RAD23 proteins in NER, but not in RNA polymerase II transcription, in with RAD3 and RAD25 we have shown to be indispensable for (2Guzder S.N. Qiu H. Sommers C.H. Sung P. Prakash L. Prakash S. Nature. 1994; 367: 91-94Crossref PubMed Scopus (121) Google Scholar, 3Guzder S. Sung P. Bailly V. Prakash L. Prakash S. Nature. 1994; 369: 578-581Crossref PubMed Scopus (159) Google Scholar, 4Qiu H. Park E. Prakash L. Prakash S. Genes & Dev. 1993; 7: 2161-2171Crossref PubMed Scopus (73) Google Scholar). Yeast TFIIH of RAD3, RAD25, TFB1, and two other as yet proteins with of 38 and kDa (8Feaver 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 (281) Google Scholar). and studies have indicated a direct role of RAD3 and RAD25 proteins in both transcription and nucleotide excision and rad3 and rad25 mutant that are for their repair or have been isolated (1Prakash S. Sung P. Prakash L. Annu. Rev. Genet. 1993; 27: 33-70Crossref PubMed Scopus (256) Google Scholar, S.N. Qiu H. Sommers C.H. Sung P. Prakash L. Prakash S. Nature. 1994; 367: 91-94Crossref PubMed Scopus (121) Google Scholar, 3Guzder S. Sung P. Bailly V. Prakash L. Prakash S. Nature. 1994; 369: 578-581Crossref PubMed Scopus (159) Google Scholar, 4Qiu H. Park E. Prakash L. Prakash S. Genes & Dev. 1993; 7: 2161-2171Crossref PubMed Scopus (73) Google Scholar). In this we demonstrate that the NER proteins RAD14 and RAD23 are associated with TFIIH as indicated by their from wild type yeast extracts and that formation of this complex is by RAD23. To the role of RAD23 in complex formation, we coupled purified RAD23 to and used it as affinity matrix for binding in vitro TFIIH subunits and RAD14. We found that RAD23 directly with the TFIIH subunits TFB1 and RAD25 and with RAD14. The RAD14 protein functions in damage recognition (10Guzder S.N. Sung P. Prakash L. Prakash S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5433-5437Crossref PubMed Scopus (88) Google Scholar). we have shown that RAD3 binds UV-damaged DNA in an ATP-dependent manner (11Sung P. Watkins J.F. Prakash L. Prakash S. J. Biol. Chem. 1994; 269: 8303-8308Abstract Full Text PDF PubMed Google Scholar). RAD23 protein as an that of TFIIH with RAD14. It is possible that the complex has a higher affinity for UV damaged DNA than be achieved by the Both the RAD3 P. Prakash L. Matson S.W. Prakash S. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8951-8955Crossref PubMed Scopus (167) Google Scholar) and RAD25 (3Guzder S. Sung P. Bailly V. Prakash L. Prakash S. Nature. 1994; 369: 578-581Crossref PubMed Scopus (159) Google Scholar) subunits of TFIIH a DNA helicase activity that be for an of the damaged for by the and P. P. Prakash L. Prakash S. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar, Sung P. Prakash L. Prakash S. Nature. 1993; PubMed Scopus Google Scholar, Bardwell A.J. Bardwell L. Friedberg E.C. Nature. 1993; PubMed Scopus Google Scholar). In addition, the helicase of RAD3 and RAD25 proteins be essential for of the NER complex and the damage containing DNA as studies have suggested a role of RAD3 helicase in the step (7Sung P. Higgins D. Prakash L. Prakash S. EMBO J. 1988; 7: 3263-3269Crossref PubMed Scopus (221) Google Scholar). RAD23 has known and not bind via its role as an assembly it could facilitate the efficient recognition of the DNA and other of NER. We J. Watkins for the on RAD23 D. and for reverse transcriptase-polymerase chain reaction and sequence J. Feaver and D. Kornberg for the plasmid that the GST-TFB1 hybrid and J. for the rna2-1 the of this it was reported that NER proteins with TFIIH because they were in column fractions that TFIIH J.Q. Z. Feaver W.J. Wu X. Donahue T.F. Friedberg E.C. Kornberg R.D. Cell. Full Text PDF PubMed Scopus Google Scholar).