ABSTRACT
Several proteins, including Rad3 and Rad25(Ssl2), are essential for nucleotide
excision repair (NER) and function in the RNA polymerase II transcription
initiation complex TFIIH. Mutations in genes encoding two other subunits of
TFIIH,
TFB1
and
SSL1
, result in UV sensitivity and have been shown to take part in NER in an
in vitro
system. However, a deficiency in global NER does not exclude the possibility
that such repair-deficient mutants can perform transcription-coupled repair (TCR), as shown for xeroderma pigmentosum group C. To
date, temperature-sensitive C-terminal truncations of Tfb1 are the only TFIIH mutations that
result in intermediate UV sensitivity, which might indicate a deficiency in
either the global NER or TCR pathways. We have directly analyzed both TCR and
global NER in these mutants. We found that
ssl1
,
rad3
and
tfb1
mutants, like
rad25(ssl2-xp)
mutants, are deficient in both the global NER and TCR pathways. Our results
support the view that the mutations in any one of the genes encoding subunits
of TFIIH result in deficiencies in both global and TCR pathways of NER. We
suggest that when subunits of TFIIH are in limiting amounts, TCR may preclude
global NER.
DNA damage sensitive mutants of yeast have been assigned to three epistasis
groups by complementation analyses which were originally based upon survival
studies of cells responding to UV light or ionizing radiation (
1
,
2
). The classification of mutants into epistasis groups has been largely
supported by biochemical evidence from investigations into the repair of one or
more DNA lesions. These three epistasis groups contain mutants with defects in
three generally non-overlapping biochemical pathways: nucleotide excision repair (
RAD3
group), spontaneous and DNA-damage induced mutagenesis (
RAD6
group), and recombinogenic mechanisms for damage tolerance (
RAD52
group).
Nucleotide excision repair (NER) is a multistep process initiated by the
recognition of DNA damage. Dual single-strand incisions 3' and 5' to the damage in the strand containing the damage are then
introduced and the damage is excised as part of an oligonucleotide. DNA
polymerases, DNA ligase, and additional proteins fill in the resulting gap to
regenerate intact duplex DNA. NER has been reconstituted
in vitro
using
Escherichia coli
, human, and yeast cell-free systems. NER occurs via similar biochemical mechanisms in all three
systems (
3
,
4
).
There are at least two classes of NER: a global repair pathway that deals with
lesions throughout the genome and a repair pathway specific for the transcribed
strands of active genes termed transcription-coupled repair (TCR). The consequence of TCR
in vivo
is a preferential repair of the transcribed strands of active genes over the
non-transcribed strands and the genome overall.
Escherichia coli mfd
-
mutants (
5
), yeast
rad26
[Delta] mutants (
6
) and Cockayne's syndrome cells (
7
), exhibit a deficiency of TCR that results in a reduction of the rate of repair
of the transcribed strands of active genes to the same rate as that of the non-transcribed strands and the overall genome. The converse situation exists
in xeroderma pigmentosum group C cells and yeast
rad7
[Delta] and
rad16
[Delta] mutants. UV-induced DNA damage in these cells is not repaired in the genome
overall but the transcribed strands of active genes are repaired very rapidly (
8
,
9
). These two classes of NER have been demonstrated in
E
.
coli
,
Saccharomyces cerevisiae
and mammalian cells (
10
).
Several yeast proteins required for NER have been shown to be components of the
transcription initiation factor TFIIH (
11
,
12
). As has been demonstrated for their mammalian homologs (
13
-
15
), these repair proteins are either integral parts of the TFIIH protein complex
or are accessory factors which co-purify with TFIIH through multiple chromatographic steps. Currently, there
are five proteins which associate to make up the heart of the TFIIH complex,
which is often referred to as core TFIIH. Core TFIIH consists of the Rad3 (89
kDa), Ssl1 (62 kDa), Tfb1 (73 kDa) and two additional proteins (p55 and p38).
In addition, Ssl2(Rad25) (85 kDa) co-purifies with TFIIH (
11
). Association of additional proteins with core TFIIH/Ssl2 results in formation
of holo-TFIIH. Holo-TFIIH possesses protein kinase activity and is the transcriptionally
active form of TFIIH (
36
). Yeast strains harboring mutations in either of two genes,
rad3
and
rad25
(
ssl2
), exhibit a complete lack of nucleotide excision repair throughout the genome
as well as within expressed genes under control of class II promoters (
16
-
20
, this report). Furthermore, temperature sensitive
rad3
and
rad25
mutants are completely deficient in RNA polymerase II transcription at the non-permissive temperature (
21
,
22
). The similarities in the repair and transcription deficiencies in the
rad3
and
rad25
mutants suggested that their protein products are required both in repair and
in transcription. Protein-protein interactions between Rad3 and Rad25, as part of transcription
initiation factor TFIIH, have been demonstrated
in vivo
using the two-hybrid system (
23
) and
in vitro
(
11
,
12
).
It appears that core TFIIH/Ssl2 functions as an intact complex in repair and in
transcription. Therefore, it is possible that mutations in genes encoding each
of the remaining subunits of TFIIH might be deficient in either TCR or global
NER (or both), as in
rad3
and
rad25
mutants. Ssl1 and Tfb1 proteins have been shown to be integral components of
TFIIH. Strains with mutations in the genes encoding Ssl1 and Tfb1 are very UV
sensitive and the proteins they encode are required in an
in vitro
repair system (
24
-
27
). While cell-free systems have been developed for both transcription and NER
in vitro
, there is as yet no eukaryotic
in vitro
system capable of TCR. Thus, investigation of the role of TFIIH and its
subunits in TCR requires determination of DNA repair deficiencies
in vivo
. We therefore examined the fine structure of DNA repair in yeast
ssl1
,
tfb1
and
rad3
mutants. We found that mutations in
ssl1
and
rad3
, like the
ssl2-xp
mutation (
20
), result in severe deficiency in NER, both in global and transcription-coupled pathways.
Various
tfb1
mutants have been used to study the role of Tfb1 in transcription initiation (
26
). The mutants are temperature-sensitive for growth due to their inability to transcribe at the non-permissive temperature. At the permissive temperature, these
temperature-sensitive mutants also exhibit a UV sensitivity intermediate between that
of repair-proficient strains and that of
rad3
and
ssl2-xp
mutants (
26
). The
tfb1
mutants produce different truncated versions of the protein, and the extent of
the truncations correlated well with the degree of the UV sensitivity, i.e.,
the greater the truncation the more UV sensitive the strain. Tfb1 was recently
demonstrated to be essential for nucleotide excision repair in an
in vitro
system (
27
). We found that these truncations of Tfb1 also correlate well with the DNA
repair deficiencies of the resultant mutant strains at the permissive
temperature.
The temperature-sensitive
tfb1
strains used in this study (mentioned above) all contain a disrupted chromsomal
TFB1
gene. Each strain, including the parent strain, carries a single allele of
TFB1
on a centromeric
ARS
plasmid. The level of protein expression in these temperature-sensitive strains relative to expression from the chromosomal locus is not
yet known. Expression of
TFB1
on the plasmid may be higher or lower than the expression of
TFB1
at its chromosomal locus. We found that TCR and NER are deficient in these
temperature-sensitive mutants at the non-permissive temperature. Furthermore, the `parent' strain containing
a plasmid-borne
TFB1
+
gene displayed little repair of the non-transcribed strand but retained good repair of the transcribed strand of
RPB2 at the non-permissive temperature.
YPD medium is 1% yeast extract/2% Bacto-peptone (Difco)/2% glucose (
28
). Synthetic glucose medium (SD) is 2% glucose/0.67% bacto-yeast nitrogen base without vitamins (Difco) supplemented with the
appropriate amino acids and bases (
28
). Agar (1.5%) was added to media for plates. Yeast strains used in this study
are listed in Table
1
. Plasmid pKS212 is a Bluescript vector (Stratagene) which contains the internal
1.0 kb
Eco
RI-
Xho
I from
RPB2
(
29
). Strand-specific RNA probes for
RPB2
were synthesized by cleaving pKS212 with
Xho
I or
Eco
RI and incubating the linearized plasmid with rNTPs and T7 RNA polymerase or T3
RNA polymerase, respectively, under conditions recommended by the manufacturer.
All strains were grown and irradiated as described previously (
20
). Briefly, cells were grown to log phase in YPD or minimal SD media
supplemented with the appropriate amino acids. Cells were collected by
centrifugation and resuspended in ice-cold phosphate-buffered saline (PBS) at 1 * 10
7
cells/ml. The cell suspensions were transferred to Pyrex dishes (25 * 15 * 4 cm
3
) such that the depth of the suspension was ~0.2 cm to ensure a uniform UV dose to all cells. Shaking cell suspensions
were irradiated with 30 J/m
2
or 40 J/m
2
of predominantly 254nm UV light at 0.33 J/m
2
/s using a Westinghouse L782-30 germicidal lamp. Initial dimer frequencies were 0.73 +- 0.08 or 0.97 +- 0.17 per fragment following exposure to 30 or 40 J/m
2
, respectively. The cells were collected by centrifugation after irradiation and
either lysed immediately or resuspended in their original growth media. Cells
were incubated for various times to allow DNA repair and then lysed. All
manipulations were performed under yellow light to preclude photoreactivation.
Table 1
Cells were digested with Zymolyase 100T as described (
29
). After digestion, spheroplasts were collected by centrifugation and
resuspended in 0.2 ml of Zymolyase buffer lacking Zymolyase. Spheroplasts were
then diluted with 2.8 ml of 0.05 M Tris-HCl (pH 8.5)/0.05 M EDTA and lysed by the addition of 0.2 ml of 20%
Sarkosyl (
30
). The mixture was incubated at 70oC for 10 min and then chilled on ice. Cellular debris and Sarkosyl were
precipitated by the addition of 0.64 ml of 5 M potassium acetate. Mixtures were
incubated at 4oC overnight and centrifuged at 6000 r.p.m. in a Sorvall HS-4 rotor at 4oC for 20 min. Supernatants containing chromosomal DNA were
transferred to fresh tubes and precipitated by the addition of 2 vol ice-cold ethanol (
31
). Pellets were washed once with ice-cold 70% ethanol. If necessary, samples were incubated in the presence of
RNaseA (final concentration 50 [mu]g/ml) to digest RNA and the DNA was precipitated with ethanol and washed as
before. Purified DNA was resuspended in 10 mM Tris-HCl (pH 7.5)/1 mM EDTA (TE) and stored at 4oC.
The incidence of CPDs in a particular restriction fragment was determined by
methods developed in this laboratory (
32
,
33
). Briefly, 1 [mu]g of purified and restricted DNA in 10 mM Tris-HCl (pH 7.5)/0.1 M NaCl/10 mM EDTA/1 mg/ml BSA was digested with T4
endonuclease V, a CPD-specific DNA glycosylase/AP lyase, in 40 [mu]l for 30 min at 37oC. The specific activity of the T4 endonuclease V, prepared by B.
Donahue from an overproducing
E
.
coli
strain, was ~9 * 10
14
U/[mu]g of protein on irradiated DNA and ~1.4 * 10
11
U/[mu]g of protein on unirradiated DNA (A. Ganesan and P. Hanawalt, unpublished
observation). One unit (U) is defined as 1 nick/min on 250 ng of pSV2gpt DNA
irradiated with 32 J/m
2
(~1 CPD/plasmid). T4 endonuclease V digestion of yeast DNA samples was
stopped by the addition of 10 [mu]l 12.5% Ficoll/5 mM EDTA/ 0.125% bromophenol blue/0.25 M NaOH. Samples were
immediately loaded into 1.0% alkaline agarose gels under 30 mM NaOH/1 mM EDTA
and electrophoresed at 1.7 V/cm overnight with recirculating buffer. DNA was
transferred to Hybond N
+
membrane (Amersham). Membranes were prehybridized for at least 2 h, then
hybridized with strand-specific RNA probes made from pKS212. Autoradiographic signal intensities
were quantified using a Helena Quick Scan R & D Densitometer.
Monoclonal antibodies against CPDs were used to determine the frequency of CPDs
in genomic DNA in a modification of the technique of Stapleton
et al
. (
34
). Antibodies were generated by a mouse immunized with UV-irradiated DNA (
35
). Denatured genomic DNA (~0.5 [mu]g) in 10* SSPE was applied to Hybond N+ membranes using a slot blot
apparatus after which the wells were rinsed with 20* SSPE. DNA was fixed to the Hybond N+ membranes with 0.4 N NaOH as per
the manufacturer's instructions and the membranes were rinsed for 1 min in 5* SSPE. Non-specific binding of antibodies to the membranes was prevented by
incubation of the membranes in 5% dried non-fat milk in PBS-Tween20 (PBS-T) for at least 1 h at 24oC after which the membranes were washed several times in
PBS-T and incubated with the primary mouse monoclonal antibody to CPDs [1:2000
dilution of TDM-2 in PBS (
35
)] for 4 h at 24oC. Membranes were washed several times in PBS-T and incubated with the secondary goat anti-mouse monoclonal antibody (1:3000 dilution in PBS)
radioactively labelled with
35
S (Amersham) for 4 h at 24oC, then washed several times in PBS-T and wrapped in Saran wrap. Radioactivity was detected by exposure
of X-ray film or phophor screen to the membranes. The amount of DNA bound to
the membranes was then determined by hybridization with radioactive RNA or DNA
probes specific for the
RPB2
gene. Antibody binding was then corrected for the amount of DNA bound to the
membrane.
We measured removal of CPDs from the
RPB2
gene in yeast strains which possess the wild type
SSL1
gene (JJ565) or suppressor alleles
ssl1-1
(JJ636) and
ssl1-3
(JJ638). Exponentially growing cultures at 30oC were irradiated with 30 J/m
2
UV. Cells were either harvested and lysed immediately or returned to their
original medium and allowed to repair their DNA before lysis. DNA was isolated
as described in Materials and Methods and digested with the appropriate
restriction endonucleases. DNA samples were divided into halves, one of which
was digested with the CPD-specific enzyme T4 endonuclease V while the other was mock-treated. The DNA was then denatured and electrophoresed through
alkaline agarose gels, transferred to nylon membranes and hybridized with
radioactive RNA probes specific for either the transcribed or non-transcribed strand of the
RPB2
gene. Membranes that were hybridized with probes for the transcribed strand of
RPB2
in
SSL1
+
and
ssl1-1
are shown in Figure
1
. Restoration of hybridization signal at the size corresponding to the full-length restriction fragment in the T4 endonuclease-treated lanes is indicative of repair. It is apparent (Fig.
1
) that repair of the transcribed strand of
RPB2
was very rapid in the repair-proficient parent strain
SSL1
+
. By 5 min after UV irradiation ~30% of the CPDs had been removed from the transcribed strand and within 30
min, 70% of CPDs had been removed. In contrast, repair of the transcribed
strand of
RPB2
was completely absent in the
ssl1-1
strain. There was no restoration of full length restriction fragments which
hybridize to the RNA probe for the transcribed strand (Fig.
1
).
The Rad3 protein, as part of TFIIH, is essential for both transcription
initiation (
22
) and nucleotide excision repair (
16
-
18
). The
rad3-2
allele contains a missense mutation leading to incorporation of arginine
instead of glycine at position 461. Glycine at postion 461 is conserved among
the Rad3 homologues of fission yeast and humans. A repair deficient
rad3-2
mutant is completely deficient in global NER. However, the capacity of
rad3-2
mutants for TCR had not previously been determined.
We measured removal of CPDs from the
RPB2
gene in yeast
rad3-2
mutants. These mutants displayed almost no repair of either the transcribed
strand or the non-transcribed strand (Fig.
2
). The small amount of repair observed in these experiments is not significantly different from the
ssl1
mutants. The repair deficiency observed for the
rad3-2
mutant is similar to that of
ssl1
and
ssl2-xp
mutants (Fig.
2
;
20
).
TFB1 is another protein essential for the transcription initiation function by
TFIIH. Although originally isolated as a factor associated with a kinase
activity which phosphorylated the C-terminal domain (CTD) of RNA polymerase II, it has since been demonstrated
that Tfb1 itself does not possess this kinase activity (
36
).
Mutations which result in truncations of Tfb1 protein at the C-terminus yield strains that display both UV sensitivity and temperature-sensitivity for growth. The extent of the truncations correlates
well with the UV sensitivity of the mutant strains at the permissive
temperature. At the permissive temperature, the UV sensitivity observed in
these temperature-sensitive mutants is intermediate between that of repair-proficient strains and that of
rad3
and
ssl2-xp
mutants (
26
). We found that these truncations of Tfb1 also correlate well with the DNA
repair deficiencies of the resultant mutant strains at the permissive
temperature.
We examined the removal of CPDs from the
RPB2
gene in these yeast strains and observed decreased repair of the transcribed
strand at the permissive temperature (24oC). The least UV sensitive mutant,
tfb1-6
, exhibited a substantial decrease in the rate of removal of CPDs from the
transcribed strand when compared with repair observed for the
TFB1
+
parent strain (Fig.
3
). The
tfb1-6
strain removed ~50% of the CPDs within the first 15 min after UV irradiation while the
parent strain removed 80-90% of the CPDs in this period. The rapid repair of the transcribed
strand observed for the parent strain, relative to repair observed for repair-proficient JJ565 grown in supplemented minimal medium, may be due to the
rapid growth (and transcription) that occurs when strains are grown in rich
medium. Within 30 min after UV irradiation,
tfb1-6
removed ~65% of the CPDs while
TFB1
+
removed 80-90% of the CPDs in this period. Yeast strains
tfb1-1
and
tfb1-101
, containing greater truncations of the Tfb1 protein, were correspondingly more
deficient in repair of the transcribed strand of
RPB2
(Fig.
3
). In summary, we found that at the permissive temperature the deficiencies in
NER for the temperature-sensitive truncation mutants reflected the degree of UV sensitivity.
We analyzed the rates and extent of removal of CPDs from genomic DNA in
tfb1
,
ssl1
and
rad3
mutants using an assay that utilizes monoclonal antibodies against CPDs (
35
). We observed repair of CPDs in genomic DNA of the repair-proficient strains
TFB1
+
and
SSL1
+
(Fig.
5
). This genomic DNA repair was similar to the repair observed for the non-transcribed strand of
RPB2
in these same mutants (Figs
2
and
3
). Genomic repair was also proficient in the least UV sensitive
tfb1
mutant,
tfb1-6
(Fig.
5
). Strains containing larger deletions of TFB1 (
tfb1-1
, Fig.
5
, and
tfb1-101
, data not shown) exhibited little or no repair of genomic DNA. Strains
containing
ssl1-1
(Fig.
5
) and
ssl1-3
(data not shown) alleles also exhibited little or no repair of genomic DNA.
Previously, we examined the DNA repair deficiency of a yeast strain harboring a
mutation in the
RAD25(SSL2)
gene, which encodes for a subunit of the transcription initiation factor TFIIH
(
11
,
20
). This mutation resulted in a total lack of repair throughout the genome
regardless of the transcriptional status of the DNA. We concluded that TFIIH,
including Ssl2, must not function solely in the recognition of DNA damage in
the transcribed strand, i.e., transcription-coupled repair. Rather, we proposed TFIIH forms an integral part of the
DNA incision complex responsible for DNA cleavage at the site of DNA damage (
20
,
37
).
In the present study, we examined DNA repair in yeast strains with mutations in
RAD3
,
SSL1
and
TFB1
, genes encoding other subunits of core TFIIH. Our expectation was that the
repair deficiencies in
rad3
and
ssl1
mutants would be similar to those in
ssl2-xp
mutants. We found that the mutations in the
RAD3
and
SSL1
genes resulted in strains with a complete deficiency in the removal of CPDs
from the entire genome, not just from transcriptionally active regions of the
genome. Our results are consistent with the lack of global repair reported for
yeast
rad3
mutants (
16
,
17
) and a yeast cell-free repair system made from
rad3
or
ssl1
mutants (
36
). We extend those results by demonstrating that
rad3
and
ssl1
mutants are defective for preferential repair of the transcribed strand of
RPB2
.
To date, the C-terminal truncations of Tfb1 are the only mutations in a subunit of TFIIH
which result in UV survival that is intermediate between that observed for
repair-proficient strains and
rad3
or
ssl2-xp
mutants. We examined DNA repair in
tfb1
mutants to test whether they might be deficient in global NER, but not in TCR.
We found that increasingly greater truncations of the C-terminus of the Tfb1 protein resulted in strains with diminishing repair
capabilities in both TCR and global NER at the permissive temperature. These
results strengthen our previous conclusion that TFIIH functions in overall NER
as an integral component of the DNA incision complex.
The truncation of Tfb1 might destabilize the protein-protein interactions within TFIIH. It has already been demonstrated that
the C-terminus of Tfb1 is required for interactions between Tfb1 and Ssl1 in the
TFIIH complex (
26
). Weakening of the interactions holding TFIIH together would likely result in
diminution or total loss of function of TFIIH in NER and/or transcription
initiation. When C-terminal truncated Tfb1 protein was used in a reconstituted
in vitro
transcription system, transcription was diminished (
27
), consistent with the idea of a destabilized TFIIH complex. The repair
deficiencies we observed for
tfb1
truncation mutants at the permissive temperature support this idea.
The repair deficiencies observed in
tfb1
mutants and the parent strain (
TFB1
+
) at the non-permissive temperature were very different from the deficiencies observed
at the permissive temperature. All C-terminal truncation mutants,
tfb1-1
,
tfb1-6
and
tfb1-101
, and the parent strain were deficient in repair of the transcribed strand of
RPB2
. All strains completely lacked repair of the non-transcribed strand of
RPB2
at the non-permissive temperature.
The deficiency of repair in
TFB1
+
at 37oC (the non-permissive temperature) is at odds with results previously obtained
with repair-proficient strains (
20
,
29
,
38
; unpublished observations). Repair of both the transcribed and non-transcribed strands of
RPB2
or
GAL7
was faster at 37oC than at 24oC in repair-proficient strains. The repair defect in
TFB1
+
at 37oC resembles the repair deficiency of yeast
rad7
[Delta] and
rad16
[Delta] mutants and the human autosomal recessive disease xeroderma pigmentosum
group C (XP-C). However,
rad7
[Delta],
rad16
[Delta] mutants and XP-C exhibit no significant deficiency in repair of the transcribed
strand of actively transcribing genes compared to repair-proficent cells (
8
,
9
). The possibility exists that the unusual repair deficiency of YSB207(
TFB1
+
) strains is due to abnormal expression of the plasmid-borne
TFB1
gene. If expression of
TFB1
on the plasmid were lower than the expression of
TFB1
at its chromosomal locus, then levels of Tfb1 protein in the cell would be in
limiting amounts. Following exposure to UV radiation, a competition between TCR
and global NER (i. e., two subpathways of NER) is established and TCR is
favored. On the other hand, if expression of
TFB1
on the plasmid were higher than the expression of
TFB1
at its chromosomal locus, then an excess of Tfb1 protein might lead to the
formation of incomplete TFIIH complexes by titrating out the remaining
available constituents of core TFIIH (i. e., Rad3, Ssl2, Rad25 and Ssl1
proteins). To test our proposal, the expression of Tfb1 in the strains we
examined can be determined at both the permissive and non-permissive temperatures.
The authors thank Thomas F. Donahue, Keith D. Gulyas and Stephen Buratowski for
their generosity in providing the yeast strains used in this report; C. A.
Smith, Ann Ganesan and Marie Leithauser for helpful discussions and critical
reading of the manuscript. This work was supported by Postdoctoral Training
Grant T32 ARO7422 (to K.S.S.) from the National Institute of Arthritis and
Musculoskeletal and Skin Diseases and by an Outstanding Investigator Award
CA44349 (to P.C.H.) from the National Cancer Institute.
*To whom correspondence should be addressed at present address: Laboratory for
Cancer Research, Department of Chemical Biology, College of Pharmacy, Rutgers,
The State University of New Jersey, Piscataway, NJ 08855-0789, USA
+
Present address: University of California School of Medicine, San Francisco, CA,
USA
Strain
Genotype
Source
JJ565
MATa, ura3-52, his4-316, ino1-1, SSL2
+
, SSL1
+
T. Donahue
JJ636
MATa, ura3-52, his4-316, ino1-1, SSL2
+
, ssl1-1
T. Donahue
JJ638
MATa, ura3-52, his4-316, ino1-1, SSL2
+
, ssl1-3
T. Donahue
802-7A
MATa, ura3-52, his4-316, ino1-1, ssl2-1, ssl1-1
T. Donahue
LP2649-1A
MAT
[alpha]
, rad3-2, leu2-3,112, ura3-52, can1
Yeast Genetic Stock Center
YSB207
MATa, ura3-52, leu2-3,112, his3
[Delta]
200, tfb1
[Delta]
::LEU2
+
/pRS316-TFB1
+
S. Buratowski
YSB251
MATa, ura3-52, leu2-3,112, his3
[Delta]
200, tfb1
[Delta]
::LEU2
+
/pRS313-tfb1-6
S. Buratowski
YSB151
MATa, ura3-52, leu2-3,112, his3
[Delta]
200, tfb1
[Delta]
::LEU2
+
/pRS313-tfb1-1
S. Buratowski
YSB260
MATa, ura3-52, leu2-3,112, his3
[Delta]
200, tfb1
[Delta]
::LEU2
+
/pRS313-tfb1-101[tfb
[Delta]
Sal]
S. Buratowski
REFERENCES
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