©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Yeast DNA Repair Protein RAD23 Promotes Complex Formation between Transcription Factor TFIIH and DNA Damage Recognition Factor RAD14 (*)

Sami N. Guzder (§) , Véronique Bailly (§) , Patrick Sung , Louise Prakash , Satya Prakash (¶)

From the (1) Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555-1061

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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).() Among these genes, RAD3 and RAD25 are of particular interest because in addition to their role in NER, they are essential for cell viability (1) . Studies with temperature-sensitive conditional lethal mutations have indicated a direct and essential role of RAD3 and RAD25 in RNA polymerase II transcription (2, 3, 4) . Both RAD3 and RAD25 proteins contain single-stranded DNA-dependent ATPase and DNA helicase activities (3, 5, 6) . The DNA helicase activity of RAD3 is required for NER but is dispensable for polymerase II transcription (7) . In contrast, the DNA helicase activity of RAD25 is essential for both transcription and DNA repair (3, 4) . 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 (8) . 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 (9) .

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 (10) . Interestingly, RAD3 also binds preferentially to UV-damaged DNA in a manner dependent upon ATP and negative superhelicity (11) . 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 (11) .

Because RAD3 is a damage recognition protein, it is important to determine how TFIIH is assembled with RAD14() 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.


MATERIALS AND METHODS

Antibodies

Antibodies to RAD3 (6) , RAD14 (10) , RAD23 (12) , and RAD25 (3) were raised in rabbits as described. Antibodies to TFB1 were raised against a GST-TFB1 hybrid protein (13) 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 (14) .

Immunoprecipitation

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 (14) . 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.

Preparation of RAD23 Affinity Matrix

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.

Affinity Binding of S-Labeled Proteins to RAD23 Affi-Gel

S-Labeled RAD3, RAD14, RAD25, and TFB1 proteins were obtained by coupled in vitro transcription and translation in 50-µl reactions containing 40 µCi of [S]methionine with the use of the TNT T7 reticulocyte lysate system (Promega). The S-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 MgCl, 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.

Affinity Binding of TFIIH to RAD23 Column

Extract was prepared from 80 g of the rad23 yeast strain JWY36 with the use of a French press and passed through a Bio-Rex 70 column (1.6 8 cm) as described (15) . 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 (15) . 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.

In Vitro Transcription Reactions

Extracts for transcription were prepared as described previously (16) . The template for transcription, pSL187, contains the promoter of the yeast CYC1 gene and yields transcripts of 375 and 350 nucleotides (17) . Reaction mixtures were assembled and processed as described (2, 3) .


RESULTS

A Complex of Nucleotide Excision Repair Proteins and TFIIH

To investigate whether RAD14 protein forms a complex with TFIIH, affinity-purified antibodies to RAD14 (10) were covalently conjugated to protein A-agarose, and the resulting immunobeads were mixed with wild type RADyeast 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 (1, 12) , 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.


Figure 1: A higher order complex of RAD14, RAD23, and TFIIH. Extracts from the wild type yeast ( WT) strain LP3041-6D ( lanes 3 and 4) and from its derivative rad23 (23) strain ( lanes 1 and 2) were incubated with protein A-agarose beads containing either rabbit immunoglobulins ( lanes 1 and 3) or antibodies to RAD14 protein ( lanes 2 and 4). The SDS eluates from the immunoprecipitates were examined by immunoblotting for the presence of the various proteins.



Mutations in the RAD23 gene greatly compromise the efficiency of NER (18) , 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 (1, 12) . 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 rad23 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.

Purification of 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 combination of ammonium sulfate precipitation and chromatographic fractionation in columns of Q-Sepharose, hydroxylapatite, and Mono Q. The purified RAD23 protein was analyzed by SDS-PAGE and staining with Coomassie Blue, which revealed that the protein preparation was nearly homogeneous (Fig. 2 A). We obtained 5 mg of RAD23 protein from 200 g of starting yeast paste. With the use of nitrocellulose filter DNA binding assay and DNA mobility shift assay in agarose gels, using a wide pH range, we found no interaction of RAD23 protein with DNA. We also found no ATPase or nuclease activity in RAD23.

RAD23 Interacts with TFIIH Subunits and RAD14

Our immunoprecipitation studies indicated that RAD23 is part of a higher order complex of excision repair proteins and TFIIH and that it in fact promotes the assembly of this protein complex. To determine whether RAD23 contacts TFIIH and RAD14 directly or does so via some other protein component(s), we covalently conjugated purified RAD23 protein to Affi-Gel-15 and used the resulting RAD23 matrix as affinity beads for binding S-labeled TFB1, RAD3, RAD25, and RAD14 proteins. To obtain radiolabeled proteins for this work, the protein coding frames of the TFB1, RAD3, RAD25, and RAD14 genes were placed under the bacteriophage T7 promoter, and the resulting constructs were transcribed in vitro to obtain mRNAs that code for these proteins, followed by translation of the mRNAs in rabbit reticulocyte lysate in the presence of [S]methionine. The radiolabeled proteins thus obtained were partially purified by ammonium sulfate precipitation, dissolved in reaction buffer, and mixed with the RAD23 Affi-Gel-15 beads. Affinity binding to the RAD23 matrix was allowed to proceed at 25 °C for 30 min. After washing with binding buffer, the bound S-labeled proteins were eluted from the RAD23 Affi-Gel beads with the use of SDS and revealed by fluorography after denaturing polyacrylamide gel electrophoresis. As a control in these experiments, we also mixed the S-labeled proteins with Affi-Gel-15 beads containing per unit volume of matrix an amount of BSA twice that of RAD23 used. The BSA beads were treated with SDS, and the eluates were run in polyacrylamide gels alongside the SDS eluates from the RAD23 beads. As shown in Fig. 2 B, the level of the S-labeled TFB1 and RAD14 proteins in the SDS eluates from the RAD23 affinity beads was 10- and 7-fold, respectively, of that in the BSA control, indicating a specific and direct interaction of TFB1 and RAD14 with RAD23. The amount of S-labeled RAD25 protein bound to the RAD23 beads was about 3-fold higher than to the BSA beads, suggesting an interaction between RAD25 and RAD23 proteins as well (Fig. 2 B). Reproducibly, in three separate experiments (Fig. 2 B and data not shown), the level of RAD3 bound to the RAD23 beads was the same as that found in the control, indicating that these two proteins do not interact directly.


Figure 2: Interaction of RAD23 protein with TFB1, RAD25, and RAD14 proteins. A, SDS-PAGE of purified RAD23 protein. A 9% denaturing polyacrylamide gel containing molecular size markers ( lane 1) and RAD23 protein, 2 µg ( lane 2), was stained with Coomassie Blue. B, in vitro translated S-labeled RAD25 ( lanes 1 and 2), TFB1 ( lanes 3 and 4), RAD3 ( lanes 5 and 6), and RAD14 ( lanes 7 and 8) proteins were incubated with Affi-Gel-15 beads containing covalently conjugated RAD23 protein ( lanes 1, 3, 5, and 7) or with Affi-Gel-15 containing BSA ( lanes 2, 4, 6, and 8). Bound S-labeled proteins were eluted from the Affi-Gel beads by SDS, separated on a 9% denaturing polyacrylamide gel, and visualized by fluorography.



Binding of TFIIH to RAD23 Affinity Matrix

To obtain further evidence that RAD23 and TFIIH interact physically, a Bio-Rex 70 column fraction derived from rad23 extract that was enriched in TFIIH was passed through a column of RAD23 Affi-Gel-15, and BSA Affi-Gel-15 was used as a control. The RAD23 column and control BSA column were washed with buffer and then eluted with 0.2 M, 0.5 M, 1 M, and 2 M potassium acetate, and the content of the TFIIH components RAD3, RAD25, and TFB1 in the various salt washes was examined by immunoblotting. The results from this analysis indicate that a sizable proportion (>70%) of TFB1, RAD3, and RAD25 proteins were retained on the RAD23-Affi-Gel column and that these proteins are eluted from the RAD23 column from 0.2-2 M acetate (Fig. 3 A). Only a trace amount of these proteins bound nonselectively to the control BSA column, and all of the retained proteins were readily eluted by 0.2 M acetate (Fig. 3 A). We have previously described conditional lethal mutations of RAD3 and RAD25 which result in defective RNA polymerase II transcription at the restrictive temperature both in vivo and in vitro (2, 3, 4) . As shown in Fig. 3 B, the transcriptional defect in the rad3-tsand the rad25-tsextracts can be complemented specifically by the eluate from the RAD23 affinity column. These results are again consistent with interaction of TFIIH with the RAD23 protein.


Figure 3: Interaction of RAD23 protein with TFIIH. A, TFIIH is retained on an RAD23 affinity column. A Bio-Rex 70 fraction enriched in TFIIH ( lane 1) was passed through the RAD23-Affi-Gel column or the BSA-Affi-Gel column. The flow-through fraction ( lane 2) and the 0.2 M ( lanes 3 and 4), 0.5 M ( lanes 5 and 6), 1 M ( lanes 7 and 8), and 2 M ( lanes 9 and 10) KOAc eluates from the columns were subjected to immunoblotting to examine their content of the TFB1, RAD3, and RAD25 proteins. B, eluate from the RAD23 column complements the rad3-tsand rad25-tstranscriptional defects. The 0.2, 0.5, and 1 M eluates from the RAD23-Affi-Gel or BSA-Affi-Gel columns in A were combined and concentrated to 50 µl. One µl of the concentrated pooled eluate from the BSA-Affi-Gel ( lane 2) and from the RAD23-Affi-Gel ( lane 3) columns were subjected to immunoblotting to examine their content of RAD3 ( upper panel of I) and RAD25 ( upper panel of II) proteins along with 10 ng of purified RAD3 and RAD25 ( lane 1, upper panels). In the transcription reaction, in lower panel of I, wild type extract ( lane 1), rad3-tsextract ( lane 2), rad3-tsextract together with 1 µl of the concentrated eluate from the BSA column ( lane 3), and rad3-tsextract with 1 µl of the concentrated eluate from the RAD23 column ( lane 4) were treated at 39 °C for 5 min before use. In the lower panel of II, wild type extract ( lane 1), rad25-tsextract ( lane 2), rad25-tsextract with 1 µl of the concentrated eluate from the BSA column ( lane 3), and rad25-tsextract with 1 µl of the concentrated eluate from the RAD23 column ( lane 4) were treated at 37 °C for 5 min and used for the transcription reaction. Transcription reactions were incubated for 10 min at 25 °C in both I and II.



RAD14 and RAD23 Do Not Affect Transcription

The cloning of the RAD14 and RAD23 genes allowed us to determine whether they are essential for cell viability besides their known role in nucleotide excision repair. Yeast strains bearing genomic deletions of these genes show no notable growth deficiency at 30 °C (12, 19) or at 37 °C,() indicating that they are likely not required for RNA polymerase II transcription. In agreement with the genetic data, we found that extracts prepared from the rad14 and the rad23 strains are as proficient in RNA polymerase II transcription as the wild type extract, regardless of whether the mutant extracts were subjected to high temperature treatment at 37 °C for 5 min prior to the transcription reaction (Fig. 4). The requirement for RAD14 and RAD23 proteins in NER, but not in RNA polymerase II transcription, stands in contrast with RAD3 and RAD25 proteins, which we have shown to be indispensable for either process (2, 3, 4) .


Figure 4:RAD14 and RAD23 genes are not required for transcription by RNA polymerase II. Extracts from the wild type strain ( lanes 1 and 2) and from the isogenic rad14 ( lanes 3 and 4) and the rad23 ( lanes 5 and 6) strains were used in the transcription reaction at 25 °C for the indicated times. Extracts were either held at 37 °C for 5 min ( panel II) or not ( panel I) before use in transcription reactions. The arrows mark the positions of the 375- and 350-nucleotide transcripts.




DISCUSSION

Yeast TFIIH consists of RAD3, RAD25, TFB1, SSL1, and two other as yet uncharacterized proteins with molecular sizes of 38 and 55 kDa (8) . Genetic and biochemical studies have indicated a direct role of RAD3 and RAD25 proteins in both transcription and nucleotide excision repair, and multiple rad3 and rad25 mutant alleles that are differentially inactivated for either their repair or transcriptional function have been isolated (1, 2, 3, 4) . In this study, we demonstrate that the NER proteins RAD14 and RAD23 are associated with TFIIH as indicated by their co-immunoprecipitation from wild type yeast extracts and that formation of this complex is modulated by RAD23. To examine the role of RAD23 in mediating complex formation, we coupled purified RAD23 to Affi-Gel-15 and used it as affinity matrix for binding in vitro translated TFIIH subunits and RAD14. We found that RAD23 interacts directly with the TFIIH subunits TFB1 and RAD25 and with RAD14.

The RAD14 protein functions in damage recognition (10) . More recently, we have shown that RAD3 binds UV-damaged DNA in an ATP-dependent manner (11) . Our study identifies RAD23 protein as an intermediary that promotes association of TFIIH with RAD14. It is possible that the TFIIH-RAD23-RAD14 complex has a higher affinity for UV damaged DNA than can be achieved by the individual components. Both the RAD3 (5) and RAD25 (3) subunits of TFIIH possess a DNA helicase activity that may be utilized for effecting an open conformation of the damaged helix for dual incision by the RAD1-RAD10 and RAD2 endonucleases (20, 21, 22) . In addition, the combined helicase action of RAD3 and RAD25 proteins may be essential for post-incision turnover of the NER complex and the damage containing DNA fragment, as our previous studies have suggested a role of RAD3 helicase in the post-incision step (7) . Although RAD23 has no known catalytic function and does not bind DNA, via its role as an assembly factor, it could facilitate the efficient recognition of the DNA lesion and perhaps influence other phases of NER.


FOOTNOTES

*
This work was supported by Grant CA41261 from the National Cancer Institute and Grant DE-FG03-93ER61706 from the Department of Energy. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
The first two authors contributed equally to this work.

To whom correspondence should be addressed: Sealy Center for Molecular Science, University of Texas Medical Branch, 6.104 Medical Research Bldg., 11th and Mechanic St., Galveston, TX 77555-1061. Tel.: 409-747-8602; Fax: 409-747-8608.

The abbreviations used are: NER, nucleotide excision repair; MOPS, 3-( N-morpholino)propanesulfonic acid; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis.

We 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 RADcells 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 (19), and an 84-base pair intron occurs between positions -429 and -346 (19). 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.

S. N. Guzder, V. Bailly, P. Sung, L. Prakash, and S. Prakash, unpublished observations.


ACKNOWLEDGEMENTS

We thank J. Watkins for the initial work on RAD23 protein; T. Wood, D. Prusak, and C. Kodira for reverse transcriptase-polymerase chain reaction and sequence determination; W. J. Feaver and R. D. Kornberg for the plasmid that expresses the GST-TFB1 hybrid protein; and J. Woolford for the rna2-1 strain.

Addendum-After the preparation of this manuscript, it was reported that several NER proteins associate with TFIIH because they were present in chromatographic column fractions that contained TFIIH (23) .


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