(Received for publication, April 28, 1995; and in revised form, June 6, 1995)
From the
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. Nucleotide excision repair (NER) 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 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
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.
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.
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
Figure 4:
TTD mutations do not rescue the
inviability of the rad3
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 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 We also examined the effect of the XPD Arg-48 mutation on viability of the rad3 Our observation that TTD1BEL and TTD1B1 mutant
proteins are unable to restore viability to the rad3 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.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)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) .
mutation, indicating that XPD protein can replace
Rad3 in Pol II transcription(8) .
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.
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.
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) .
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) .
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 Leu
rad3
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.
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
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
Strain
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.
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.
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.
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.
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.
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.
We thank Ed Miller for excellent technical assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.