(Received for publication, January 3, 1996; and in revised form, March 10, 1996)
From the
Yeast TFIIH is composed of six subunits: Rad3, Rad25, TFB1, SSL1, p55, and p38. In addition to TFIIH, we have purified a subassembly of the factor that lacks Rad3 and Rad25 and which we refer to as TFIIHi. In the in vitro nucleotide excision repair (NER) system that consists entirely of purified proteins, we show that neither TFIIHi nor a mixture of purified Rad3 and Rad25 proteins is active in NER but that the combination of TFIIHi with Rad3 and Rad25 promotes the incision of UV-damaged DNA. These results provide the first evidence for a direct requirement of Rad3, Rad25, and of one or more of the TFIIHi subunits in the incision step of NER. The NER efficacy of TFIIH is greatly diminished or abolished upon substitution of Rad3 with the rad3 Arg-48 mutant protein or Rad25 with the rad25 Arg-392 mutant protein, respectively, thus indicating a role of the Rad3 and Rad25 DNA helicase functions in the incision of damaged DNA. Our results further indicate that the carboxyl-terminal domain kinase (CTD) TFIIK is dispensable for the incision of damaged DNA in vitro. These studies reveal the differential requirement of Rad3 DNA helicase and CTD kinase activities in damage-specific incision versus RNA polymerase II transcription.
Extensive genetic studies in Saccharomyces cerevisiae have indicated the requirement of RAD1, RAD2, RAD3, RAD4,
RAD10, RAD14, and RAD25 genes in nucleotide excision
repair (NER). ()Mutations in these genes cause a high degree
of sensitivity to UV light and a defect in the incision of UV-damaged
DNA(1) . Besides their function in NER, the RAD3 and RAD25 genes are essential for cell viability because of their
involvement in RNA polymerase II (Pol II)
transcription(2, 3, 4) . The RAD3-
and RAD25-encoded products both possess an ATP-dependent DNA
helicase activity (4, 5) and are constituents of
TFIIH, which also contains four additional polypeptides, TFB1, SSL1,
p55, and p38(6, 7) . TFIIK, consisting of the KIN28
kinase and a cyclin component, associates with TFIIH and confers to
TFIIH the ability to phosphorylate the carboxyl-terminal domain (CTD)
of the RPB1 subunit of Pol II(8) . In a reconstituted
transcription system consisting of purified factors, only the
combination of TFIIH and TFIIK promotes transcription(9) .
Recently, we have reconstituted the incision step of NER using purified
Rad14, the Rad4-Rad23 complex, the Rad1-Rad10 nuclease, Rad2 nuclease,
replication protein A (RPA), and the six-subunit core TFIIH. The
combination of these protein factors promotes ATP-dependent dual
incision of UV-damaged DNA(7) .
In NER, the direct
requirement for Rad3 and Rad25 in an early step of the repair process
is indicated by the extreme sensitivity of various rad3 and rad25 mutants to UV light and to other DNA-damaging agents by
the existence of mutants that are only inactivated in the repair
function and by the fact that these mutations confer a total defect in
the incision of damaged
DNA(1, 3, 4, 10) . On the other
hand, whether the other subunits of TFIIH, TFB1, SSL1, p55, and p38,
are also directly required for the incision of UV-damaged DNA has not
yet been established. Although UV-sensitive mutations of SSL1 and of TFB1 have been identified, these ssl1 and tfb1 mutations cause conditional lethality, indicating a
defect in Pol II transcription, but confer only a low level of UV
sensitivity, making it possible that the moderate UV sensitivity
represents a side effect engendered by the primary defect in
transcription(11) . ()No UV-sensitive mutations of
p55 and p38 have yet been reported. Thus, it is unclear whether only
the Rad3 and Rad25 proteins participate in the incision of damaged DNA
or whether the entire TFIIH is in fact required in this process.
In this work, we purify a subassembly of TFIIH that contains the TFB1, SSL1, p55, and p38 subunits but lacks the Rad3 and Rad25 proteins, which we refer to as TFIIH-incomplete or TFIIHi. The availability of a defined in vitro NER system and of purified Rad3 protein (12) , Rad25 protein(4) , and TFIIHi (this work) has now enabled us to address whether Rad3 and Rad25 together are sufficient for damage-specific incision to occur or whether the incision reaction in fact also requires the other TFIIH subunits. Here, we show that in addition to Rad3 and Rad25, the incision of UV-damaged DNA is absolutely dependent upon the presence of TFIIHi. These reconstitution studies provide the first direct evidence for the requirement of Rad3, Rad25, and of one or more of the TFIIHi subunits in the incision of damaged DNA.
In addition, we determine whether the DNA helicase activities of Rad3 and Rad25 are essential for the incision step of NER and examine the role of TFIIK in this process. Interestingly, our studies indicate that in contrast to Pol II transcription, where only the Rad25 DNA helicase activity is required(4) , the DNA helicase activities of both Rad3 and Rad25 are essential for the incision of damaged DNA. TFIIK is dispensable for the incision of UV-damaged DNA in vitro, suggesting that association of TFIIK with TFIIH has functional relevance only for Pol II transcription.
Figure 1:
Different forms of TFIIH. A, fractions from the Mono Q column containing TFIIHi-TFIIK (lane
1), TFIIH-TFIIK (lane 2), and TFIIH (lane 3)
were concentrated separately, and 1.2 pmol of the concentrated factors
were run in an 8% denaturing polyacrylamide gel and silver-stained. The
TFIIH subunits (Rad3, Rad25, TFB1, SSL1, p55, and p38) and the TFIIK
subunits (p47, p45, and KIN28 in italics) are indicated.
Because the Rad25 and KIN28 proteins do not stain well with silver
under the conditions used, their actual amount was higher than the
staining intensity would suggest. B, TFIIH (lane 1)
and TFIIHi-TFIIK (lane 2), 0.6 pmol each, were analyzed for
their content of Rad3, Rad25, TFB1, and SSL1 by immunoblotting. C, purified RNA polymerase II, 4 µg, was run in an 11% denaturing
polyacrylamide gel and stained with Coomassie Blue. The RNA polymerase
II subunits RPB1-RPB8 are indicated. D, purified RNA
polymerase II, 200 ng, was incubated alone (lane 1) and with
TFIIHi-TFIIK (lane 2), TFIIH (lane 4), and
TFIIH-TFIIK (lane 6) in the presence of
[-
P]ATP as described under ``Materials
and Methods.'' TFIIHi-TFIIK (lane 3), TFIIH (lane
5), and TFIIH-TFIIK (lane 7) were also incubated with
[
-
P]ATP without RNA polymerase II. The
level of RPB1 phosphorylation (
P-RPB1) was
revealed by autoradiography of the dried gel. IIH, TFIIH; IIH-IIK, TFIIH-TFIIK; IIHi-IIK, TFIIHi-TFIIK; Pol
II, RNA polymerase II.
Interestingly, in Mono Q fractions 7-15, polypeptides with sizes identical to the TFB1, SSL1, p55, and p38 subunits of TFIIH were present, but no polypeptide with the size of Rad3 or Rad25 was evident in these fractions (Fig. 1A, lane 1). By immunoblotting, we established that TFB1 and SSL1 were indeed present in these fractions but that there was neither Rad3 nor Rad25 (Fig. 1B, lane 2). We refer to this TFIIH subassembly as TFIIHi. It should be emphasized that TFIIHi was not an anomaly of this particular protein preparation, as we obtained the same results in three other independent preparations (data not shown). Interestingly, TFIIK also associates with TFIIHi (Fig. 1A, lane 1), and TFIIHi-TFIIK is as active in RPB1 phosphorylation as TFIIH-TFIIK (Fig. 1D, compare lanes 2 and 6).
Figure 2:
Damage-specific incision mediated by the
combination of Rad3, Rad25, and TFIIHi. A, undamaged DNA
(-UV) and UV-damaged DNA (+UV) were
incubated alone without any NER factor (lanes 1 and 4) and with various combinations of NER factors for 12 min at
30 °C. The reaction mixtures in lanes 2, 3, and 5-10 all contained Rad1-Rad10 complex, Rad2, Rad4-Rad23
complex, Rad14, and RPA. Either TFIIH (lanes 2, 5,
and 6), Rad3-Rad25 (lane 7), TFIIHi (lane
8), or the combination of TFIIHi and Rad3-Rad25 (lanes 3, 9, and 10) was also added to the reaction mixtures,
as indicated above the gel. ATP was omitted from the reaction mixtures
in lanes 6 and 10, as indicated. OC, open
circular; SC, supercoiled. B, UV-irradiated DNA
(+UV) and undamaged DNA (-UV) were
incubated without any NER factor (lane 2) and with various
combinations of NER factors for 30 min at 30 °C, followed by
purification of the excision DNA fragments and their labeling with
[-
P]dideoxy-ATP and terminal transferase as
described under ``Materials and Methods.'' The reaction
mixtures in lanes 1 and 3-8 contained
Rad1-Rad10 complex, Rad2, Rad4-Rad23 complex, Rad14, and RPA. Either
TFIIH (lanes 1, 3, and 4), TFIIHi (lane
5), Rad3-Rad25 (lane 6), or the combination of TFIIHi and
Rad3-Rad25 (lanes 7 and 8) was also added to the
reaction mixtures, as indicated above the autoradiogram. ATP was
omitted from the reaction mixtures in lanes 3 and 8,
as indicated.
The rad3 Arg-48 mutant allele exhibits
negative dominance over the wild type RAD3 gene, as its
overexpression renders wild type yeast cells sensitive to UV light (Fig. 3A). Since the rad3 Arg-48 mutation does
not affect viability, we could purify the rad3 Arg-48 protein from a rad3 yeast strain(14) . To facilitate the
purification of rad25 Arg-392 mutant protein, we attached a 6-histidine
tag to the amino terminus of the rad25 Arg-392-encoded
protein, whose expression in yeast is driven by the PGK promoter in plasmid pR25.21. When we attempted to introduce
plasmid pR25.21 into wild type Rad
yeast cells, we
failed to recover any transformant that overexpresses the rad25 Arg-392
mutant protein. However, transformants that overexpress the rad25
Arg-392 mutant protein were obtained at the expected frequency when
plasmid pR25.21 was introduced into yeast cells harboring plasmid
pR25.22 that overexpresses the rad25
-encoded
protein from the ADCI promoter. The rad25
mutant protein that lacks the 45 carboxyl-terminal amino acids of
Rad25 protein is defective in NER (1, 10) but is
competent in RNA polymerase II transcription(4) . The failure
to overexpress the 6-histidine-tagged rad25 Arg-392 mutant protein in
Rad
cells strongly suggests that the rad25 Arg-392 mutant allele also exerts negative dominance over the RAD25 gene. The rad25 Arg-392 mutant protein was purified using a
five-step procedure that includes affinity chromatography on
nickel-agarose.
Figure 3:
Rad3 and Rad25 helicase functions are
required for incision. A, UV sensitivity of Rad (
), rad3 Arg-48 mutant (
), and Rad
strain harboring the ADC1::rad3 Arg-48 plasmid pPS5 that
overproduces rad3 Arg-48 protein (
). All the strains are
derivatives of CMY135. UV survival of the rad3 Arg-48 mutant
was not affected by plasmid pPS5. B, undamaged DNA
(-UV) and UV-irradiated DNA (+UV) were
incubated without any NER factor (lanes 1 and 5) and
with various combinations of NER factors at 30 °C for 12 min. The
reaction mixtures in lanes 2-4 and in lanes
6-9 all contained Rad1-Rad10 complex, Rad2, Rad4-Rad23
complex, Rad14, and RPA. TFIIHi was added to the reaction mixtures in lanes 2-4 and in lanes 6-9, as indicated
above the gel. The combinations of Rad3-Rad25 (lanes 2 and 7), Rad3-rad25 Arg-392 (lanes 4 and 9), and
rad3 Arg-48-Rad25 (lanes 3 and 8) were used in
various reaction mixtures, as indicated. C, UV-irradiated DNA
(+UV) was incubated at 30 °C for 30 min with or
without NER factors. The reaction mixtures in lanes 2-4 all contained Rad1-Rad10 complex, Rad2, Rad4-Rad23 complex, Rad14,
RPA, and TFIIHi and either the combination of Rad3 and Rad25 (lane
2), rad3 Arg-48 and Rad25 (lane 3), or Rad3 and rad25
Arg-392 (lane 4), as indicated. TFIIHi is abbreviated as IIHi in C, rad3 Arg-48 as rad3, and rad25 Arg-392 as
rad25 in B and C.
We determined whether rad3 Arg-48 and rad25 Arg-392
mutant proteins can function in the NER reaction. As shown in Fig. 3B, when Rad3, Rad25, and TFIIHi were mixed with
the remainder of the NER factors, >85% of the supercoiled UV-damaged
plasmid DNA was converted to the open circular form (lane 7).
By contrast, when purified rad25 Arg-392 mutant protein was used
instead of wild type Rad25, no incision of the UV-damaged DNA occurred (lane 9). Consistent with the results from the agarose gel
assay, there was no excision DNA fragment formed by the rad25 Arg-392
protein as assayed by the P-labeling protocol (Fig. 3C, lane 4). We have also tagged the
wild type Rad25 protein with the same 6-histidine sequence and
overproduced the tagged protein by use of the PGK promoter.
The 6-histidine-tagged Rad25 protein, unlike the 6-histidine-tagged
rad25 Arg-392 mutant protein, could be overproduced in Rad
yeast cells. The purified 6-histidine-tagged Rad25 protein has a
level of activity comparable with the untagged form in the in vitro NER system (data not shown).
When we substituted wild type Rad3
protein with the rad3 Arg-48 mutant protein in the incision assay,
reproducibly a low level of incision activity was observed, as
evidenced by the conversion of 3% of the supercoiled plasmid to the
open circular form (Fig. 3B, lane 8). This
incision activity is specific for UV damage, because the undamaged
plasmid DNA was not acted on by the rad3 Arg-48 mutant protein (Fig. 3B, lane 3). However, we could not detect any
excision DNA fragment using the
P-labeling protocol (Fig. 3C, lane 3) when rad3 Arg-48 protein was used.
Since the excision fragments are formed by a dual incision event (15) , the failure to detect any excision DNA fragment with
rad3 Arg-48 protein raises the possibility that the small amount of
open circular form (Fig. 3B, lane 8) arose by
the introduction of one of the two incision nicks that are normally
made.
Figure 4:
TFIIK has no effect on NER in vitro.A, undamaged DNA (-UV) was incubated alone (lane 1), with TFIIH-TFIIK and the remainder of NER factors (lane 2), and with TFIIH and the remainder of NER factors (lane 3) at 30 °C for 12 min. UV-irradiated DNA
(+UV) was incubated alone (lane 4), with
TFIIH-TFIIK and the remainder of NER factors (lanes
5-8), and with TFIIH and the remainder of NER factors (lanes 9-12) at 30 °C for the indicated times. OC, open circular; SC, supercoiled. B, the
gel in A was subjected to image analysis to obtain data points
for a graphical representation of the results. , TFIIH-TFIIK;
, TFIIH. C, UV-irradiated DNA was incubated alone (lane 1), with TFIIH-TFIIK and the remainder of the NER
factors (lane 2), and with TFIIH and the remainder of the NER
factors (lane 3) at 30 °C for 30 min. IIH, TFIIH; IIH-IIK, TFIIH-TFIIK.
For both RAD3 and RAD25, mutations
conferring extreme UV sensitivity and a total defect in incision as
well as mutations that affect only the DNA repair function or the
transcription function have been
identified(1, 3, 4, 10) . While
these and other observations (16) have suggested a direct
involvement of Rad3 and Rad25 in incision, it has remained unclear
whether the other TFIIH subunits also play a direct role in this
process. All the existing tfb1 and ssl1 mutants
exhibit a much lower level of UV sensitivity than rad3 and rad25 mutants, and all the UV-sensitive tfb1 and ssl1 mutants also exhibit temperature-sensitive
lethality(11) , indicative of a transcriptional
defect. A reduction in repair synthesis is seen in nuclear extracts
prepared from tfb1 and ssl1 mutants, and this could
be augmented by the addition of a chromatographic fraction that
contained partially purified TFIIH (11) . Since the repair
synthesis method does not directly measure the incision of damaged DNA
but rather measures the level of DNA synthesis presumably tied to
incision, it is not clear whether reduced repair synthesis in the tfb1 and ssl1 mutant extracts was due to reduced
incision activity or reduced DNA synthesis activity. In addition, since
the chromatographic fraction used for complementing the repair
synthesis deficiency in the mutant extracts contained, besides TFIIH,
other proteins(11) , it is possible that the repair
synthesis-stimulating activity observed was due to factors other than
TFIIH present in the partially purified fraction. It is also important
to consider that because nuclear extracts rather than a purified system
were used(11) , there might conceivably be inhibitory factors
present in the extracts that limit the extent of incision and repair
synthesis, and stimulation of repair synthesis by an added protein
fraction could be due to the removal of the inhibitory factors and may
not necessarily indicate a direct involvement of the added protein
fraction in incision or repair synthesis. Finally, it remains possible
that the transcriptional defect caused by tfb1 and ssl1 mutations results in a lowering of the levels of protein factors
involved in some stage of NER and of protein factors that function in
other cellular processes. In fact, it has been reported that ssl1 mutants exhibit various ribosomal abnormalities including a
reduction in polysomes and an in vitro deficiency in
translation(17) . Thus, previous results concerning the NER
deficiency in tfb1 and ssl1 mutants could reflect
either a side effect engendered by the primary defect in transcription
or a direct role of TFB1 and SSL1 protein in incision or repair
synthesis or an indirect role of TFB1 and SSL1 in helping overcome the
effect of inhibitors that might be present in the nuclear extracts
used, but for reasons outlined above, they do not distinguish among
these possibilities.
Here, using a purified NER system, we show that the combination of Rad3, Rad25, and TFIIHi promotes the dual incision of UV-damaged DNA just as efficiently as does core TFIIH. Importantly, omission of any of Rad3, Rad25, and TFIIHi resulted in abolition of damage-specific incision, providing direct biochemical evidence that NER requires all three purified entities, probably as a reflection that TFIIH in its entirety functions in the incision of UV-damaged DNA. While our results clearly indicate a direct involvement of Rad3, Rad25, and TFIIHi in the incision reaction, it remains possible that they also function in postincision reactions such as in the turnover of the incision protein complex and in repair synthesis.
Previous work has indicated a requirement of Rad25 helicase activity in RNA polymerase II transcription initiation(4) , but unexpectedly, Rad3 helicase activity is dispensable for transcription(14) . By contrast, here we show that both the Rad3 and Rad25 helicase activities are required in the incision phase of NER. Our results are consistent with a model in which DNA unwinding by the combined action of the Rad3 and Rad25 helicase activities creates an unwound ``bubble'' DNA structure appropriate for dual incision to occur. The observed specificities of the Rad1-Rad10 endonuclease (18) and of the Rad2 endonuclease (19) on model DNA substrates are congruent with nicking of the damaged DNA strand by these nucleases at the 5`- and 3`-side of the damage, respectively. Our data, however, do not exclude the possibility that Rad3 and Rad25 helicase functions are also required in postincision reactions.
While stoichiometric amounts of TFIIK are essential for Pol II transcription(9) , our results indicate that TFIIK does not affect the rate of incision of UV-damaged DNA in vitro. From results based on complementation of a rad3 mutant extract using the repair synthesis assay, it has been suggested that TFIIK is dispensable for repair synthesis(20) . It remains to be determined whether TFIIK, as a transcription factor, influences the in vivo efficiency of NER by affecting the levels of DNA repair factors.