From the
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).
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
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.
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) .
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
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) .
(
)
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
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) .
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-ts
and the
rad25-ts
extracts 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-ts
transcriptional 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-ts
extract ( lane 2),
rad3-ts
extract together with 1 µl of the
concentrated eluate from the BSA column ( lane 3), and
rad3-ts
extract 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-ts
extract ( lane 2),
rad25-ts
extract with 1 µl of the
concentrated eluate from the BSA column ( lane 3), and
rad25-ts
extract 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.
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 (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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.