HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Feaver, W. J. Articles by Friedberg, E. C. Search for Related Content PubMed PubMed Citation Articles by Feaver, W. J. Articles by Friedberg, E. C. Social Bookmarking                      What's this?

J Biol Chem, Vol. 274, Issue 41, 29564-29567, October 8, 1999

The TFB4 Subunit of Yeast TFIIH Is Required for Both Nucleotide Excision Repair and RNA Polymerase II Transcription^*

William J. Feaver, Wenya Huang^§, and Errol C. Friedberg^¶

From the Laboratory of Molecular Pathology, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9072

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The N-degron strategy has been used to generate a yeast strain harboring a temperature-sensitive allele of TFB4 (tfb4^td), the gene that encodes the 37-kDa subunit of the transcription/repair factor TFIIH. The tfb4^td strain was sensitive to UV radiation and is defective in nucleotide excision repair in vitro. The mutant strain was also found to be an inositol auxotroph due at least in part to an inability to properly induce expression of the INO1 gene. These results indicate that like other subunits of TFIIH, Tfb4 is required for both RNA polymerase II transcription and DNA repair.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The holo transcription factor IIH (TFIIH)¹ is comprised of nine subunits and has been shown to be required for both transcription by RNA polymerase II (RNAP II) and nucleotide excision repair (NER) (1, 2). The recent identification of the TFB2, TFB3, and TFB4 genes encoding the 55-, 38-, and 37-kDa subunits, respectively, completed the molecular definition of this yeast transcription factor (2). All yeast IIH subunits are encoded by essential genes, consistent with an essential role in transcription. In addition each yeast subunit has a highly conserved counterpart in human TFIIH (2).

The NER pathway is required for the repair by excision of a myriad of helix-distorting lesions, although it is perhaps best characterized with respect to its ability to remove 6-4 photoproducts and cyclobutane pyrimidine dimers, resulting from exposure to UV radiation (3). Following base damage recognition, DNA surrounding lesions is locally unwound and incised at double-strand/single-strand junctions by two junction-specific endonucleases with opposite strand polarity. Damaged bases are then excised as short, single-stranded oligonucleotides. Repair synthesis and ligation complete the process of NER. In yeast, localized unwinding during NER is catalyzed by the Rad3 and Ssl2 DNA helicases, both subunits of TFIIH (1). Similarly, promoter melting during initiation by RNAP II is thought to require the activity of Ssl2 (4). It seems likely that the common requirement for localized unwinding is the functional basis for TFIIH involvement in both NER and RNAP II transcription.

To date viable and conditional mutations in the yeast IIH subunits Ssl2, Rad3, Tfb1, Tfb2, Ssl1, and TFB3 have been utilized to demonstrate a direct requirement for these subunits in NER (2, 5, 6).² Holo TFIIH can be further divided into core TFIIH, comprising the seven subunits described above, and the subcomplex TFIIK (8). TFIIK, comprised of the Ccl1 and Kin28 proteins, has protein kinase activity directed toward the C-terminal domain of Rpb1, the largest subunit of RNAP II (9-11). No role for TFIIK in NER has been demonstrated.

In this study we have used the N-degron approach of Varshavsky and colleagues (12) to generate a strain carrying a temperature-sensitive allele of TFB4. Characterization of this strain revealed UV radiation sensitivity, defective NER in vitro, and impaired induction of INO1, leading to inositol auxotrophy. Based on these results we conclude that Tfb4 is required for both RNAP II transcription and NER.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Construction of the tfb4^td Strain-- The TFB4 open reading frame was amplified by high fidelity polymerase chain reaction (PCR) from yeast genomic DNA with primers 5'-ATATAAGCTTGGATCCGAATGGATGCAATATCTGATCC -3' and 5'-ATATGGATCCTCATGGTTTCGTCACCTTCT-3', introducing HindIII and BamH1 restriction sites (underlined) on the 5' end of the amplified fragment. The PCR product was digested with HindIII and BsaAI, and the fragment containing the 5' portion of the open reading frame was cloned into pPW66R (12) digested with HindIII and XhoI (blunt) to give pPW66R/TFB4. pPW66R/TFB4 was digested with NheI and used to transform strains SX46a (MATa ade2 his3-532 trp1-289 ura3-52) and SX46A-UBR1::HIS3 to yield strains tfb4^td and UBR1::HIS3-tfb4^td. SX46a-UBR1::HIS3 was made by transforming SX46a with EcoRI-digested pJDubr14-B (12). Integration at the TFB4 locus was confirmed by PCR using a DHFR primer and the downstream TFB4 primer described above.

UV Radiation Sensitivity-- Cells were grown to late log phase in media supplemented with 0.5 mM CuSO₄. Following dilution and plating on the same media containing 2% (w/v) agar, cells were exposed to the indicated amount of UV radiation at a dose of 1 J/s/m² and incubated at 23 °C until colonies were large enough to be counted. To construct pRS314/TFB4, the TFB4 locus was amplified by high fidelity PCR from yeast genomic DNA with primers 5'-ATATGGATCCTTTATGCGGCTCCAGTGAAG-3' and 5'-ATATCTCGAGTCTTAATCGATATGGCGTTG-3' introducing BamHI and XhoI restriction sites (underlined) on the 5' and 3' ends of the amplified fragment, respectively. The PCR product was digested with BamHI and XhoI and cloned into the same sites of pRS314 (13).

Inositol Auxotrophy-- To construct the INO1 reporter plasmid pJH359/TRP the URA3 marker of pJH359 (14) was replaced with TRP1 as follows. The NaeI/AatII (blunt) fragment from pRS303 (13) was cloned into StuI + SalI (blunt)-digested pJH359 to give pJH359/TRP. Inositol starvation medium was prepared as described (15). Small whole cell extracts were prepared and assayed for -galactosidase activity as described (16).

Other Methods-- Growth of cells, preparation of whole cell extracts, and measurement of in vitro NER activity were as reported previously (17). Yeast were transformed using a standard LiOAc protocol. YPD medium contained 1% (w/v) yeast extract, 2% (w/v) bacto peptone, and 2% (w/v) dextrose.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Generation of a Temperature-sensitive Allele of TFB4-- As had been done with other yeast TFIIH subunits described above, we wished to investigate the requirement of TFB4 for NER and/or RNAP II transcription. To this end a conditional, temperature-sensitive (ts) allele of TFB4 was required. To avoid a labor-intensive and potentially unsuccessful conventional screen for ts mutants, we chose to use the relatively rapid "N-degron" strategy described by Varshavsky and colleagues (12) for generating temperature-degradable (td) yeast mutants. In short, with this method, the chromosomal copy of the gene encoding the protein of interest is replaced by a fusion consisting of ubiquitin, the N terminus of DHFR, and finally the gene of interest, all under control of the CUP1 promoter. Post-translational proteolytic removal of the ubiquitin moiety results in a nonmethionine N terminus of the DHFR fusion protein. This N terminus, together with a temperature-destabalizing point mutation in the DHFR domain, results in degradation of the entire fusion protein by the polyubiqutination/proteasome pathway at elevated temperatures. In addition, expression of the fusion protein can be down-regulated by removing Cu⁺² from the growth medium. We have previously successfully used this method to generate temperature-degradable alleles of the yeast DNA replication proteins PCNA and RPA1 (pcna^td and rfa1^td; Ref. 17). A complete description of the construction of the tfb4^td strain is provided under "Experimental Procedures." Successful integration at the TFB4 locus, i.e. the generation of a viable strain, indicates that fusion to the N-degron domain did not impair the essential function of Tfb4 protein.

As anticipated, the tfb4^td mutant strain exhibited growth at 23 °C but not at 37 °C (Fig. 1A). The parental wild-type strain grew equally well at both temperatures (Fig. 1A). The tfb4^td strain grew slightly more slowly than the wild type at 23 °C.³ This growth difference is due in part to a prolonged lag phase for the mutant, as well as a slightly reduced doubling time (174 min as opposed to 144 min for wild type at 23 °C). Temperature sensitivity of tfb4^td could be suppressed by disruption of the UBR1 gene (Fig. 1A). The UBR1 gene product is required for the recognition of destabilized N termini (12). Disruption of UBR1 alone did not result in temperature sensitivity (Fig. 1A). The temperature sensitivity of the tfb4^td mutant could also be suppressed by transformation with a low copy plasmid expressing wild-type Tfb4 protein (Fig. 1B). Transformation with empty vector had no effect (Fig. 1B). We conclude from these experiments that the temperature sensitivity of tfb4^td is due to temperature-induced instability of Tfb4 protein, resulting in proteosome-mediated degradation.

View larger version (65K): [in this window] [in a new window]   Fig. 1.   A, temperature sensitivity of the tfb4td strain. Serial dilutions of the indicated strains were spotted on YPD plates supplemented with 0.5 mM CuSO4 and incubated at either 23 or 37 °C for 3-4 days. B, the temperature sensitivity of the tfb4td strain can be rescued by transformation with a low copy plasmid expressing wild-type Tfb4 protein. The tfb4td strain was transformed with either pRS314/TFB4 or empty vector, pRS314. Four individual transformants of each were streaked on minimal selective plates supplemented with 0.5 mM CuSO4 and incubated as described in A.

The tfb4^td Mutant Is Sensitive to UV Radiation and Is Defective for NER in Vitro-- Impaired ability to repair UV radiation-induced DNA damage is a hallmark of defective NER. Hence, tfb4^td and control strains were tested for UV radiation sensitivity. The tfb4^td mutant was shown to be significantly more sensitive to UV radiation than the wild-type control (Fig. 2A). Remarkably, this sensitivity was observed at the permissive temperature of 23 °C. As was the case with temperature sensitivity, UV radiation sensitivity could be suppressed by disruption of UBR1 (Fig. 2A). The ubr1 disruption strain itself did not exhibit increased UV sensitivity (Fig. 2A). UV radiation sensitivity of tfb4^td could also be suppressed by transformation with a low copy plasmid expressing wild-type Tfb4 protein but not with the empty vector (Fig. 2B). We conclude from these experiments that impairment of Tfb4 function results in sensitivity to UV radiation consistent with a role for this protein in NER.

View larger version (11K): [in this window] [in a new window]   Fig. 2.   A, the tfb4td strain exhibits increased sensitivity to UV radiation. Strains were grown in YPD at 23 °C and UV irradiated as described under "Experimental Procedures." , TFB4; , tfb4td; , UBR1::HIS3; , tfb4td-UBR1::HIS3. B, the UV radiation sensitivity of the tfb4td strain can be rescued by transformation with a plasmid expressing wild-type Tfb4. The tfb4td stain was transformed with either pRS314/TFB4 or pRS314. Individual transformants were grown in minimal selective media at 23 °C and exposed to UV irradiation as described under "Experimental Procedures." , tfb4td[pRS314]; , tfb4td[pRS314/TFB4]. For A and B, following irradiation the plates were incubated at 23 °C for 3-4 days, and the colonies were counted. The results shown represent the averages of at least two independent experiments.

To directly test for a role of Tfb4 in NER, we measured the ability of extracts from tfb4^td to support NER in an in vitro repair synthesis assay (18, 19). Whole cell extracts were prepared from tfb4^td grown at the permissive temperature (23 °C) either prior to or subsequent to a shift to 37 °C for 2 or 4 h. Together with radioactively labeled dNTPs, extracts were incubated simultaneously with damaged and undamaged plasmid substrates. Following DNA recovery, gel electrophoresis, and autoradiography, NER activity was determined by measuring incorporation of label specifically into the damaged substrate. NER activity was reduced to a level of ~59% of the control after 4 h at 37 °C (Fig. 3). Thus conditions that result in a decrease in the steady state level of Tfb4 also result in a decrease in NER activity. Extracts from pcna^td and rfa1^td strains prepared in the same manner showed similar decreases in NER activity (17). In the latter two cases defective in vitro NER could be complemented by addition of recombinant PCNA or RFA proteins, respectively. With tfb4^td this was not possible given our inability to generate soluble recombinant Tfb4 (2).³ Taken together the UV radiation sensitivity and in vitro repair synthesis assays support a role for Tfb4 in NER.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   The tfb4td strain is defective for NER in vitro. tfb4td was grown in YPD at 23 °C with 0.5 mM CuSO4 to mid log phase, collected by centrifugation, and resuspended in media lacking CuSO4 prewarmed to 37 °C. Growth was allowed to continue for 4 h at 37 °C. Aliquots of cells were harvested at 0, 2, and 4 h post-transfer, and whole cell extracts were prepared. A, 100 µg of each whole cell extract was assayed for in vitro NER activity.  AAF, undamaged DNA substrate; + AAF, damaged DNA substrate. Top panel, ethidium bromide stained gel; bottom panel, autoradiogram. B, PhosphorImager quantitation of A.

The tfb4^td Mutant Is an Inositol Auxotroph-- Mutations in many proteins known to be involved in transcription by RNAP II have been found to cause inositol auxotrophy because of an inability to properly derepress the INO1 gene. Such proteins include the TATA-binding protein, SRB2, and several subunits of RNAP II itself (20-29). Recently, the mechanism for this phenomenon has been elucidated (7). Transcription of the inositol 1-phosphate synthase gene (INO1) is up-regulated by a complex of the INO2 and INO4 gene products, which bind to the INO upstream activation sequence. Interestingly, the INO2 gene also contains an INO upstream activation sequence, hence providing for auto-regulatory control. This auto-regulatory loop tends to amplify the effect of mutations in the transcription machinery, leading to a severe reduction in INO1 expression. Thus, inositol auxotrophy and the level of INO1 expression can be used to monitor mutants for subtle defcts in RNAP II transcription.

At 23 °C the tfb4^td strain failed to grow on media lacking inositol, whereas the wild-type control grew normally (Fig. 4A). Inositol auxotrophy could be suppressed by disruption of UBR1 (Fig. 4A). Disruption of UBR1 alone did not result in inositol auxotrophy (Fig. 4A). To test directly for an inability to activate (derepress) INO1, both wild-type and tfb4^td strains were transformed with a CYC1/-galactosidase fusion reporter plasmid under control of the INO1 regulatory region, including the INO1 upstream activation sequence. -Galactosidase activity was measured before and 4 h after transfer to inositol starvation media. As might be expected given the growth phenotype, tfb4^td cells had an ~3-fold reduction in the ability to derepress expression of INO1 compared with wild-type controls (6.7-fold for wild type versus 2.3-fold for tfb4^td) (Fig. 4B). We conclude from these experiments that impairment of Tfb4 function results in improper derepression of INO1 because of a defect in RNAP II transcription.

View larger version (30K): [in this window] [in a new window]   Fig. 4.   The tfb4td strain is auxotrophic for inositol. A, serial dilutions of the indicated strains grown in YPD supplemented with 0.5 mM CuSO4 at 23 °C were spotted on plates either lacking or supplemented with 200 ng/ml inositol. Plates were incubated at 23 °C for 3 days. B, the tfb4td strain fails to properly derepress expression of INO1 upon inositol starvation. Strains were transformed with pJH359/TRP, a CYC1/-galactocidase fusion reporter plasmid under control of the INO1 regulatory region. Cells were grown in minimal selective media and subsequently transferred to media lacking inositol. Prior to and 4 h post-transfer, aliquots of cells were harvested and assayed for -galactosidase activity as described under "Experimental Procedures." Open columns, wild type; filled columns, tfb4td. The results shown represent the averages of two independent experiments. -Galactosidase activity is shown in Miller units (wild type: 0 h, 22.5; 4 h, 151.1) (tfb4td: 0 h, 18.0; 4 h, 40.5).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In these studies we have used a temperature-degradable mutant of Tfb4 (tfb4^td), the 37-kDa subunit of yeast transcription/repair factor TFIIH, to demonstrate a requirement for this polypeptide in both NER and RNAP II transcription. We previously used the same procedure to demonstrate a role for the DNA replication proteins PCNA and Rpa1 in NER (18). In short, the N-degron method of Varshavsky and co-workers (12) provides an effective, straightforward method for generating conditional yeast mutants, valuable tools for the study of protein function both in vivo and in vitro.

The observation that tfb4^td exhibited both UV radiation sensitivity and inositol auxotrophy at 23 °C indicates that impairment of Tfb4 function results even at the permissive temperature. Suppression of these phenotypes by disruption of UBR1 indicates that most, if not all, of these effects are due to a low level of proteosome-mediated degradation. However, we cannot rule out the possibility that Tfb4 function is also negatively affected by fusion to the N-degron domain itself. The level of UV radiation sensitivity and impairment of INO1 derepression could likely be enhanced by utilizing protocols incorporating elevated temperatures. It is interesting to note that although significantly UV radiation-sensitive, the tfb4^td strain has only a slight growth defect at the permissive temperature. Although the exact function of Tfb4 in NER and transcription remains unknown, this observation suggests different functions in each of these processes. Additionally, we cannot definitively conclude from our data that Tfb4 plays a direct role in transcription and/or NER because we cannot formally exclude the possibility that loss of Tfb4 leads to instability and/or degradation of TFIIH as a whole.

The NER defective phenotype of tfb4^td, one shared by other yeast TFIIH subunits, provides further evidence that Tfb4 is indeed a bona fide component of TFIIH. Tfb4 was originally identified as a 37-kDa polypeptide present in variable amounts in TFIIH preparations (8). This type of chromatographic behavior is similar to that observed for Ssl2 (1). Substochiometirc levels of both proteins are thought to result from selective loss during purification, suggesting that both are relatively loosely associated with other subunits of core TFIIH. Like Tfb4, we have recently observed that a conditional mutation in Tfb3, the 38-kDa subunit of core yeast TFIIH, also renders cells UV radiation-sensitive and defective for NER in vitro.² Thus, all of the subunits of core TFIIH have now been implicated in NER, completing an important chapter in the study of TFIIH.

	ACKNOWLEDGEMENTS

We thank Vicent Bruno and S. Henry for providing pJH359 and A. Varshavsky for providing pPW66R and pJDubr14-B.

	FOOTNOTES

^* This work was supported by Research Grant CA12420 from the United States Public Health Service (to E. C. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Fellow of the Jane Coffin Childs Memorial Fund for Medical Research.

^§ Present address: Dept. of Medical Technology, National Cheng-Kung University, No.1 University Rd., Tainan, Taiwan.

^¶ To whom correspondence should be addressed. Tel.: 214-648-4020; Fax: 214-648-4067; E-mail: friedberg.errol@pathology.swmed.edu.

² W. J. Feaver, W. Huang, O. Gileadi, L. Meyers, C. M. Gustafsson R. D. Kornberg, and E. C. Friedberg, submitted for publication.

³ W. J. Feaver and E. C. Friedberg, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: TFIIH, transcription factor IIH; RNAP II, RNA polymerase II; NER, nucleotide excision repair; PCR, polymerase chain reaction.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				Y. Takagi, H. Komori, W.-H. Chang, A. Hudmon, H. Erdjument-Bromage, P. Tempst, and R. D. Kornberg Revised Subunit Structure of Yeast Transcription Factor IIH (TFIIH) and Reconciliation with Human TFIIH J. Biol. Chem., November 7, 2003; 278(45): 43897 - 43900. [Abstract] [Full Text] [PDF]

				M.-C. Keogh, E.-J. Cho, V. Podolny, and S. Buratowski Kin28 Is Found within TFIIH and a Kin28-Ccl1-Tfb3 Trimer Complex with Differential Sensitivities to T-Loop Phosphorylation Mol. Cell. Biol., March 1, 2002; 22(5): 1288 - 1297. [Abstract] [Full Text] [PDF]

				W. J. Feaver, W. Huang, O. Gileadi, L. Myers, C. M. Gustafsson, R. D. Kornberg, and E. C. Friedberg Subunit Interactions in Yeast Transcription/Repair Factor TFIIH. REQUIREMENT FOR Tfb3 SUBUNIT IN NUCLEOTIDE EXCISION REPAIR J. Biol. Chem., February 25, 2000; 275(8): 5941 - 5946. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Feaver, W. J. Articles by Friedberg, E. C. Search for Related Content PubMed PubMed Citation Articles by Feaver, W. J. Articles by Friedberg, E. C. Social Bookmarking                      What's this?