©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Lethality in Yeast of Trichothiodystrophy (TTD) Mutations in the Human Xeroderma Pigmentosum Group D Gene
IMPLICATIONS FOR TRANSCRIPTIONAL DEFECT IN TTD (*)

(Received for publication, April 28, 1995; and in revised form, June 6, 1995)

Sami N. Guzder Patrick Sung Satya Prakash Louise Prakash (§)

From the 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

Mutations in the human XPD gene result in a defect in nucleotide excision repair of ultraviolet damaged DNA and cause the cancer-prone syndrome xeroderma pigmentosum (XP). Besides XP, mutations in XPD can cause another seemingly unrelated syndrome, trichothiodystrophy (TTD), characterized by sulfur-deficient brittle hair, ichthyosis, and physical and mental retardation. To ascertain the underlying defect responsible for TTD, we have expressed the TTD mutant proteins in the yeast Saccharomyces cerevisiae and determined if these mutations can rescue the inviability of a rad3 null mutation. RAD3, the S. cerevisiae counterpart of XPD, is required for nucleotide excision repair and also has an essential role in RNA polymerase II transcription. Expression of the wild type XPD protein or the XPD Arg-48 protein carrying a mutation in the DNA helicase domain restores viability to the rad3 null mutation. Interestingly, the XPD variants containing TTD mutations fail to complement the lethality of the rad3 null mutation, strongly suggesting that TTD mutations impair the ability of XPD protein to function normally in RNA polymerase II transcription. From our studies, we conclude that XPD DNA helicase activity is not essential for transcription and infer that TTD mutations in XPD result in a defect in transcription.


INTRODUCTION

Nucleotide excision repair (NER)()of ultraviolet damaged DNA in eukaryotes is a complex process requiring many genes(1) . In humans, a defect in NER results in xeroderma pigmentosum (XP). XP individuals are highly sensitive to sunlight, and they suffer from a high incidence of skin cancers. Genetic studies with the NER genes in the yeast Saccharomyces cerevisiae have revealed that in addition to their role in DNA repair, several of these genes function in other processes as well. The yeast NER genes RAD3 and RAD25 are essential for viability because of their role in RNA polymerase II transcription(1, 2, 3, 4) . Both Rad3 and Rad25 proteins contain single-stranded DNA-dependent ATPase and DNA helicase activities(3, 5, 6) , and they are components of Pol II transcription factor TFIIH(7) .

In humans, the XPD and XPB genes encode the counterparts of yeast RAD3 and RAD25 genes, respectively(1) . Like Rad3 and Rad25, XPD and XPB are DNA helicases (8, 9) , and they constitute two of the subunits of human TFIIH(10, 11) . Expression of XPD in S. cerevisiae rescues the inviability of the rad3 mutation, indicating that XPD protein can replace Rad3 in Pol II transcription(8) .

In addition to causing XP, mutations in XPD can result in a seemingly unrelated disorder, trichothiodystrophy (TTD), which is characterized by sulfur-deficient brittle hair resulting from a reduction in the levels of cysteine/cystine in hair proteins. Other TTD symptoms include ichthyotic skin, physical and mental retardation, protruding ears, microcephaly, and nail dysplasia(12, 13) . Mutations in XPD that cause TTD have been recently identified in four patients(14) . To examine if TTD mutations might affect Pol II transcription in an adverse manner, we have constructed mutations in XPD that correspond to two of these TTD mutations, TTD1BEL and TTD1B1, and examined if these mutant XPD proteins restore viability to a yeast rad3 mutant strain. We also constructed a mutation in the nucleotide binding domain of XPD to determine the role of XPD DNA helicase in the viability function.


MATERIALS AND METHODS

Generation of XPD Mutations

Mutations were constructed by site-directed mutagenesis (15, 16) using dutung Escherichia coli and M13 derivative phages containing appropriate restriction fragments for mutagenesis. Restriction fragments containing the desired mutations were used to replace the corresponding fragment of the wild type XPD gene, driven by the ADC1 promoter in the TRP1 2µ vector pSCW231(5) , and all mutations were verified by double-stranded sequencing in the plasmids prior to introduction into appropriate yeast strains. The specific mutations constructed in this study are given in Fig. 1.


Figure 1: Mutations in XPD. A, DNA helicase mutation. Mutation of Lys-48 to Arg-48 in the nucleotide binding consensus sequence I of the human XPD protein. B, TTD1BEL mutation. Mutation of XPD Arg-722 to Trp-722 found in the TTD1BEL cell line(14) . C, TTD1B1 mutation. Deletion of a single G residue in the Arg-730 codon produces a truncated protein of 743 amino acids compared with the wild type protein of 760 amino acids. Amino acid sequences of S. cerevisiae Rad3, S. pombe rhp3+, and human XPD proteins are indicated for the regions flanking the mutant site. The sequences were aligned using the GAP program of the Genetics Computer Group, Inc. (22) . Verticallines mark identical residues; colons mark highly conserved residues; dots mark weakly conserved residues.



Phenotype of XPD Mutations

Plasmids containing various XPD mutant alleles were introduced into the diploid yeast strain YR3-20 (MATa/MAT leu2-3, leu2-112/leu2-3, leu2-112 trp1/trp1 ura3-52/ura3 rad3::LEU2/rad3-2). The viability and genotype of spores obtained following sporulation of these diploids were determined.

Expression of Wild Type and Mutant XPD Proteins in Yeast

Yeast cells of strain YR3-20 harboring various plasmids were grown at 30 °C in synthetic complete medium lacking tryptophan. Lysis of cells was carried out using the French press in 50 mM Tris, pH 7.5, 10 mM EDTA, 10% sucrose, 0.6 M KCl containing 10 mM -mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 2 mM benzamidine hydrochloride, and 10 µg/ml each of the protease inhibitors leupeptin, pepstatin A, chymostatin, and aprotinin. Lysates were clarified by centrifugation at 100,000 g for 90 min, and equal amounts of proteins were electrophoresed in a 7.5% denaturing polyacrylamide gel. After electrotransfer of the proteins to a nitrocellulose membrane, the membrane was probed with affinity-purified anti-XPD antibodies as described previously(8) .


RESULTS

Rationale of Studies

Because of the dual involvement of XPD in NER and Pol II transcription, in principle, mutations in XPD can cause defects in NER only, in transcription only, or in both processes simultaneously. Consistent with this idea, mutations in RAD3 that affect either the NER function or the transcription function, or both of these functions simultaneously, have been identified in S. cerevisiae. In humans, mutations in XPD that differentially inactivate the NER function do exist; however, mutations that affect the transcriptional function have not yet been identified. Interestingly, the TTD mutations in XPD manifest a distinct clinical picture from those causing XP, and they could potentially represent a subtle defect in Pol II transcription. But, in the absence of isogenic wild type and mutant TTD cell lines, it may not be possible to ascertain whether such TTD cell lines have transcriptional abnormalities, because any effect detected could simply reflect differences in the genetic background of different cell lines. To examine the effect of TTD mutations on transcription, we have sought a model system where the wild type XPD and its TTD mutant derivatives could be compared in an isogenic background and where the biological differences between the mutant and wild type alleles could be phenotypically amplified, allowing the detection of any possible transcriptional abnormalities that the TTD mutations might engender. Previously, we showed that expression of XPD complements the lethality of the rad3 mutation of S. cerevisiae, indicating that XPD can form a functional complex with the Pol II transcriptional machinery in yeast(8) . However, rad3 cells carrying the XPD gene grow at a slower rate than do wild type yeast cells(8) , suggesting that XPD protein interacts less efficiently with the yeast transcriptional machinery than does Rad3. Because XPD does not rescue the NER defect of rad3 mutant alleles, domains controlling interactions between XPD and yeast NER proteins have apparently diverged to a greater extent than have the domains that mediate interaction between XPD and the yeast transcriptional machinery(8) .

The reduced efficiency of XPD interaction with the yeast transcriptional system suggested to us that introduction of TTD mutations into the XPD gene could reduce the capacity of XPD protein to interact with the yeast transcriptional complex even further, and this might produce an easily detectable phenotype.

Mutation in the XPD ATP Binding Domain Does Not Affect Transcription

Residues 35-51 in XPD comprise the highly conserved motif I found in proteins containing DNA-dependent ATPase and helicase activities. This region is conserved to a remarkable degree among the homologous S. cerevisiae Rad3, Schizosaccharomyces pombe rhp3+, and human XPD proteins (Fig. 1). Previously, we showed that the Rad3 Arg-48 mutant protein, in which the lysine residue at position 48 has been changed to arginine, lacks the ATPase and DNA helicase activities but retains the ability to bind ATP and DNA(17, 18) . The rad3 Arg-48 mutant is defective in NER, and its UV sensitivity is the same as that of the totally incision-defective rad3 mutants. However, the rad3 Arg-48 mutation has no effect on viability, indicating that Rad3 DNA helicase activity is dispensable for Pol II transcription(17) . A mutation of lysine 48 to arginine in the S. pombe rhp3+ gene also engenders a defect in NER but has no adverse effect on viability or growth rate(19) . To determine if the XPD DNA helicase is essential for Pol II transcription, we introduced plasmid pXPD.1, which expresses the mutant XPD Arg-48 protein in yeast (Fig. 2, lane3), into the diploid yeast strain YR3-20 (rad3::LEU2/ rad3-2). The diploid strain was sporulated and the genotype of spores in tetrads determined. We found that Leurad3 spores carrying plasmid pXPD.1 are viable, and as shown in Fig. 3, rad3 strain carrying the XPD Arg-48 mutant gene grows as well as the rad3 strain carrying the wild type XPD gene. In multiple experiments, we found the growth rates of the rad3 strain carrying the wild type XPD or the XPD Arg-48 mutant allele to be the same. This result indicates that the XPD Arg-48 protein is as efficient as the wild type XPD protein in forming a functional complex with yeast TFIIH and that like the Rad3 and rhp3+ DNA helicases, XPD DNA helicase activity is dispensable for Pol II transcription.


Figure 2: Expression of wild type XPD and mutant XPD proteins in yeast. The nitrocellulose blot of a 7.5% denaturing polyacrylamide gel was probed with affinity-purified antibodies specific for XPD(8) . Each lane contains an equal amount of whole-cell extract from strain YR3-20 harboring plasmid pPM1 (2µ, ADC1), which contains the ADC1 promoter but lacks the XPD gene (lane1); plasmid pPM7 (2µ, ADC1-XPD) carrying the wild type XPD gene (lane2); pXPD.1 (2µ, ADC1-XPD Arg-48) carrying the XPD-Arg48 mutant gene (lane3); plasmid pXPD.3 (2µ, ADC1-TTD1B1) carrying the TTD1Bl mutant gene (lane4); plasmid pXPD.4 (2µ, ADC1 TTD1BEL) carrying the TTD1BEL mutant allele (lane5).




Figure 3: Growth of rad3 strain, carrying either the wild type XPD gene or the XPD Arg-48 mutant allele, on YPD plate at 30 °C. Top, rad3-2 carrying plasmid pPM7 (2µ, ADC1-XPD). These yeast cells have wild type growth rate and viability. Left, rad3 strain carrying the XPD gene in plasmid pPM7 (2µ, ADC1-XPD); right, rad3 strain carrying the XPD Arg-48 mutant gene in plasmid pXPD.1 (2µ, ADC1-XPD Arg-48).



Inviability of TTD(XPD) Mutations in the rad3 Strain

The TTD1BEL cell line is heterozygous for two mutations in the XPD gene in which one allele has a change of Arg-616 to Pro and the other allele carried a change of Arg-722 to Trp(14) . The TTD1B1 cell line carries an XPD allele that is not expressed, and the other allele contains a -G frameshift in codon 730 that causes the protein to terminate 13 amino acids downstream of the mutation(14) . To examine the effect of TTD mutations in rad3 yeast strains, we introduced the TTD1BEL mutation (Arg-722 to Trp) and the -G frameshift TTD1B1 mutation (Fig. 1) carried on plasmids pXPD.4 and pXPD.3, respectively, into the rad3/rad3-2 diploid strain YR3-20. As expected, the TTD1B1 mutation produces a shorter protein than the wild type XPD protein (Fig. 2, lane4), and the level of mutant protein is somewhat lower than that of the XPD protein. The TTD1BEL mutation yields a normal size protein, and this mutant protein is expressed in yeast at the same level as the wild type XPD protein (Fig. 2, lane5). The YR3-20 diploid carrying the TTD1BEL or TTD1B1 mutation was sporulated and genotypes of spores in tetrads determined. A total of 40 tetrads was analyzed in each case. In both cases, we found no more than two viable spores (Fig. 4), and none of the viable spores was Leu, which marks the rad3 mutation. The Leu spores all carried the rad3-2 mutant allele. These results indicate that the two mutant XPD proteins harboring the TTD mutations are expressed in yeast, but they are unable to rescue the inviability of the rad3 mutation.


Figure 4: TTD mutations do not rescue the inviability of the rad3 mutation. Diploid strain YR3-20 (rad3::LEU2/rad3-2) harboring either plasmid pXPD.3 carrying the TTD1B1 mutation (top) or plasmid pXPD.4 harboring the TTD1BEL mutation (bottom) was sporulated and subjected to tetrad analysis. None of the 40 tetrads examined yielded more than two viable spores. In each case, no Leu spores were recovered, indicating inviability of rad3 spores harboring either TTD mutant gene. All the spores that grew were Leu and UV sensitive, and they carried the rad3-2 mutant gene.



The effect of the TTD mutations was also examined by the plasmid shuffle method, in which the wild type RAD3 gene carried on a low copy plasmid in a rad3 haploid strain was replaced by the various mutant alleles of XPD. Strain YR3-7 (MATaleu2-3, -112 trp1 ura3-52 rad3::LEU2) harboring plasmid pSP43 carrying the wild type RAD3 gene on a low copy plasmid (CEN, RAD3, URA3) was transformed to Trp+ with the wild type XPD or the mutant XPD genes fused to the ADC1 promoter and carried on a multicopy 2µ plasmid pPM7 (2µ, ADC1, XPD), pXPD.1 (2µ, ADC1, XPD Arg-48), pXPD.3 (2µ, ADC1, TTD1B1), or pXPD.4 (2µ, ADC1, TTD1BEL). As controls, we also introduced the vector pSCW231 (2µ, ADC1) or the RAD3-containing plasmid pSCW367 (2µ, ADC1, RAD3) into strain YR3-7 harboring plasmid pSP43. Loss of the CEN URA3 RAD3-containing plasmid pSP43 was selected for by growing the transformants on medium containing 5-fluoroorotic acid (5-FOA), as only cells that have become Ura due to the loss of plasmid pSP43 would grow on 5-FOA (20) . We recovered Ura colonies when cells carried either the wild type RAD3 gene (plasmid pSCW367), the wild type XPD gene (plasmid pPM7), or the XPD Arg-48 mutant gene (plasmid pXPD.1). Introduction of the ADC1 vector alone (pSCW231), or of the TTD1B1 mutation in plasmid pXPD.3, or the TTD1BEL mutation in plasmid pXPD.4 did not yield Ura colonies on 5-FOA-containing medium. These observations again indicate that whereas the XPD and XPD Arg-48 proteins can complement the inviability defect of the rad3 mutation, the two TTD mutants are unable to do so.


DISCUSSION

In this work, we have sought to determine whether mutations in XPD that cause trichothiodystrophy produce a defect in Pol II transcription. For this purpose, we determined whether TTD mutations in XPD impair the Pol II transcriptional activity in yeast by examining whether the mutant XPD proteins can rescue the inviability defect of the rad3 mutation. We had shown previously that the requirement of RAD3 in Pol II transcription represents the essential function of this gene(4) . We find that in contrast to the wild type XPD gene, which restores viability to the rad3 strain, the two TTD mutations TTD1BEL and TTD1B1 are unable to do so.

We also examined the effect of the XPD Arg-48 mutation on viability of the rad3 strain. Based upon our previous biochemical and genetic studies with the rad3 Arg-48 mutation(17) , the XPD Arg-48 mutant protein is expected to be defective in DNA-dependent ATPase and DNA helicase activities, and we anticipated that this mutation will have no detrimental effect on Pol II transcription in yeast. As predicted, we find that the XPD Arg-48 mutant protein restores viability to the rad3 strain, and this strain grows as well as that carrying the wild type XPD gene. From these observations, we conclude that XPD ATPase/DNA helicase is dispensable in Pol II transcription.

Our observation that TTD1BEL and TTD1B1 mutant proteins are unable to restore viability to the rad3 strain suggests that these TTD proteins are unable to form a functional complex with yeast TFIIH. This observation is consistent with the premise that the TTD syndrome represents a defect in transcription. However, for reasons mentioned earlier, it would have been difficult to demonstrate such a defect in human TTD cell lines. The use of the heterologous yeast system described herein has circumvented problems associated with studying human cell lines of different genetic background and has enabled us to magnify the underlying transcriptional defect in TTD mutations.

How might TTD mutations in XPD cause a transcriptional defect that would result in specific phenotypes associated with this syndrome? The recent observation that human and yeast TFIIH interact directly with the acidic activation domains of transcriptional activators such as VP16 and p53 suggests a means as to how this might occur(21) . Because of the involvement of many subunits in the formation of TFIIH and because of its large size, we surmise that TFIIH interacts with a variety of transcriptional activators, and this interaction coordinates efficient activation of transcription of many genes. We suggest that TTD mutations in XPD impair the ability of TFIIH to interact with a subset of transcriptional activators that regulate the processes impaired in TTD patients, manifested by sulfur-deficient brittle hair and physical and mental retardation.


FOOTNOTES

*
This work was supported by Department of Energy Grant DE-FG03-93ER61706 and National Cancer Institute Grant CA35035. 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.

§
To whom correspondence should be addressed: Sealy Center for Molecular Science, UTMB, 6.104 Medical Research Bldg., 11th and Mechanic St., Galveston, TX 77555-1061. Tel.: 409-747-8601; Fax: 409-747-8608.

The abbreviations used are: NER, nucleotide excision repair; XP, xeroderma pigmentosum; Pol, polymerase; TTD, trichothiodystrophy; 5-FOA, 5-fluoroorotic acid.


ACKNOWLEDGEMENTS

We thank Ed Miller for excellent technical assistance.


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