Reconstitution of TFIIH and requirement of its DNA helicase subunits, Rad3 and Rad25, in the incision step of nucleotide excision repair.

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). 1 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). 2 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 dispensa-* This work was supported by National Cancer Institute Grant CA35035 and Department of Energy Grant DEFG03-93 ER61706. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  ble for the incision of UV-damaged DNA in vitro, suggesting that association of TFIIK with TFIIH has functional relevance only for Pol II transcription.
Purification of Different Forms of TFIIH-Extract was prepared from 1.3 kg of yeast strain YPH/TFB1-6HIS with the use of a French press in the presence of protease inhibitors (12) and was clarified by centrifugation (100,000 ϫ g for 90 min). The supernatant obtained (fraction I, 900 ml) was dialyzed overnight against 10 liters of A, diluted with A to give ionic strength equivalent to 100 mM KOAc, and applied onto Bio-Rex 70 (5 ϫ 15 cm; 300 ml total) equilibrated with A ϩ 200. The column was washed with 500 ml of A ϩ 200 and treated with A ϩ 600 mM KOAc to elute bound proteins (fraction II, 150 ml). After being dialyzed against 2 liters of B ϩ 100 overnight, the Bio-Rex 70 protein pool was applied onto DEAE-Sephacel (2.6 ϫ 14 cm; 75 ml total) equilibrated in B ϩ 100. After washing with 150 ml of B ϩ 200, proteins were eluted with B ϩ 500 to give fraction III (40 ml). The DEAE pool was dialyzed against 1 liter of B ϩ 70 for 12 h and applied onto Bio-Rad HTP hydroxyapatite (1.6 ϫ 6 cm; 12 ml total) equilibrated in C without EDTA and developed with a 160-ml gradient from C to D, collecting 3.2-ml fractions. The HTP column fractions that contained TFIIH were identified by immunoblotting with affinity-purified antibodies specific for the Rad3, Rad25, TFB1, and SSL1 subunits of TFIIH, pooled (fraction IV, 22.4 ml), and dialyzed against 1 liter of E overnight. The dialysate was mixed with 1.2 ml of nickel-nitriloacetate-agarose (Qiagen) on a rocking platform for 3 h and then centrifuged to collect the matrix. The nickel matrix was resuspended in 6 ml of E ϩ 10 mM imidazole and transferred into a glass chromatography column with internal diameter of 0.6 cm. The nickel matrix was washed sequentially with 5 ml each of E ϩ 20 mM imidazole, E ϩ 30 mM imidazole, E ϩ 40 mM imidazole, and E ϩ 100 mM imidazole. The majority of the TFIIH (Ϸ75%) was present in the E ϩ 100 mM imidazole wash (fraction V), which was dialyzed against F for 2 h to lower the ionic strength to 100 mM KOAc and chromatographed on Mono S (HR5/5) with a 20-ml gradient from F ϩ 100 to F ϩ 550, collecting 0.5-ml fractions. The Mono S fractions that contained the various TFIIH subunits, eluting at Ϸ300 mM KOAc, were identified by immunoblotting and pooled (fraction VI, 2.5 ml). The Mono S pool was fractionated on Mono Q (HR5/5) with a 12-ml gradient of 300-1500 mM KOAc in F, collecting 0.3-ml fractions; TFIIHi eluted at Ϸ600 mM KOAc, while TFIIH eluted at Ϸ1000 mM KOAc.
RNA Polymerase II Purification-For the purification of RNA polymerase II, hydroxyapatite fractions 9 -12 from TFIIH purification (see above) containing core RNA polymerase II were applied directly onto a Mono Q column (HR5/5), which was developed with a 40-ml gradient of 100-1500 mM KOAc in buffer F. Fractions containing RNA polymerase II, which elutes at Ϸ1,100 mM KOAc, were pooled and concentrated to 2 mg/ml. Rad3, and 20 ng of Rad25 were used in various combinations, as indicated. All the protein components were purified to near homogeneity as described previously (Ref. 7 and references therein). The protein components were preincubated for 10 min at 25°C before the DNA substrate was added. After incubation at 30°C for 12 min, reaction mixtures were deproteinized by treatment with 0.4% SDS and proteinase K at 250 g/ml for 10 min at 37°C and then subjected to electrophoresis in 0.8% agarose gels in TAE buffer (40 mM Tris acetate, pH 7.4, 0.5 mM EDTA). Gels were treated with ethidium bromide (1 g/ml) for 60 min to stain DNA, soaked in a large volume of H 2 O for 4 h to reduce background staining, and then photographed through a red filter using Polaroid type 55 films. For detecting the excision fragments, reaction mixtures were assembled exactly the same way as in the incision assay, except that they had a final volume of 50 l, contained 1 g of DNA substrate and five times the amounts of NER factors used in the incision assay, and were incubated at 30°C for 30 min. The reaction mixtures were extracted once with 50 l of buffered phenol, and the DNA was precipitated at Ϫ20°C for Ͼ2 h after adding 150 l of ethanol. The DNA precipitate was dissolved in 10 l of TE (10 mM Tris-HCl, pH 7.5, 0.2 mM EDTA), and 3 l of the DNA solution was treated with 5 Ci of [␣-32 P]dideoxy-ATP (5000 Ci/mmol, Amersham Corp.) and 3 units of calf thymus terminal transferase at 37°C for 30 min in a final volume of 20 l of the buffer supplied by the vendor (Boehringer Mannheim). After labeling, the DNA was bound to 5 l of glass matrix in the Mermaid kit according to the instructions supplied by the vendor (Bio-101). The DNA was eluted from the matrix into 10 l of H 2 O, and 6 l of the final DNA solution was dried down in vacuum, dissolved in 3 l of loading buffer, and run in a 15% sequencing gel with size markers. The excision fragments were visualized by autoradiography using Kodak BioMax films.
Purification of rad3 Arg-48 and rad25 Arg-392 Mutant Proteins-The rad3 Arg-48 mutant protein was purified as described previously (14). To purify rad25 Arg-392 protein, extract was prepared from 210 g of strain EPY52.01 harboring plasmids pR25.21 and pR25.22, as described (12). After centrifugation (100,000 ϫ g for 90 min), the extract (fraction I) was treated with ammonium sulfate (0.21 g/ml). The protein precipitate was collected by centrifugation (20,000 ϫ g, 30 min), dissolved in 100 ml of buffer K (20 mM KH 2 PO 4 , pH 7.5, 10% glycerol, 0.5 mM EDTA, and 1 mM DTT), and dialyzed against 2 liters of buffer K ϩ 50 mM KCl for 12 h on ice. The dialysate (fraction II) was fractionated in a column of Q-Sepharose (1.6 ϫ 10 cm; 20-ml matrix) using a 200-ml gradient of 100 -500 mM KCl in buffer K. Fractions containing the peak of 6-histidine-tagged rad25 Arg-392 protein, eluting at Ϸ300 mM KCl, were identified by immunoblotting and pooled to give fraction III (30 ml total), which was treated with ammonium sulfate (0.3 g/ml). The protein precipitate was collected by centrifugation, dissolved in 10 ml of buffer K, dialyzed against buffer K for 3 h to lower the ionic strength to 40 mM KCl, and then fractionated in SP-Sepharose (1 ϫ 3.8 cm; 3-ml matrix) with a 42-ml gradient of 50 -400 mM KCl in buffer. The rad25 mutant protein eluted between 100 and 220 mM KCl from SP-Sepharose, and the peak fractions were pooled to yield fraction IV (15 ml total), applied directly onto Mono Q (HR5/5), which was developed with a 25-ml gradient of 100 -500 mM KCl in buffer K that contained 0.01% (w/v) Nonidet P-40 and 1 mM 2-mercaptoethanol instead of DTT. The pool of rad25 protein (fraction V; 5 ml total), eluting at Ϸ320 mM KCl, was mixed with 0.3 ml of nickel-nitrilotriacetate agarose (Qiagen) at 4°C for 2 h. The nickel matrix was washed in an Eppendorf tube with 0.6 ml each of 10, 20, 30, 40, and 100 mM imidazole in buffer H containing 300 mM KCl. The 40 and 100 mM imidazole eluates were combined (fraction VI; 1.2 ml total), concentrated to 0.2 ml, and diluted with 3 ml of buffer K that contained 0.01% Nonidet P-40 before being fractionated in Mono S (HR5/5) using a 9-ml, 30 -600 mM KCl gradient. The pool of rad25 mutant protein, eluting at Ϸ200 mM KCl, was concentrated and used in the NER studies.

RESULTS
Purification of TFIIH and TFIIHi-In the last step of purification in a Mono Q column, TFIIH containing the six core subunits was found in fractions 21-26. Fractions 21-23 also contained three additional polypeptides with molecular sizes of 47, 45, and 33 kDa (Fig. 1A, lane 2); the level of these three proteins in fractions 24 -26 was much lower (Fig. 1A, lane 3). These three additional protein species are constituents of the RNA polymerase II CTD kinase, TFIIK, because: (i) they had the same molecular sizes described for the three subunits of TFIIK (8), (ii) they eluted in the same position in the Mono Q gradient relative to TFIIH as reported for TFIIK (8), and (iii) the ability to specifically phosphorylate the RPB1 subunit of purified RNA polymerase II (Fig. 1C) coincided with the abundance of the three polypeptides (compare Fig. 1A, lanes 2 and 3 with Fig. 1D, lanes 6 and 4, respectively). For use in NER studies, fractions 21-23 containing the combination of TFIIH and TFIIK (Fig. 1A, lane 2) were pooled and concentrated, and fractions 24 -26, which contained TFIIH and a much smaller amount of TFIIK (Fig. 1A, lane 3), were pooled and concentrated separately.
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  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.
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).
Incision of UV-damaged DNA Requires the Combination of TFIIHi, Rad3, and Rad25-We have previously purified Rad3 and Rad25 proteins to near homogeneity from yeast strains genetically tailored to overproduce these proteins (4,12). The availability of TFIIHi has made it possible to define the role of Rad3, Rad25, and TFIIHi in NER. Whereas the incision of DNA damaged by ultraviolet light occurred efficiently when TFIIH was combined with the remainder of the NER factors (viz. Rad1-Rad10, Rad2, Rad4-Rad23, Rad14, and RPA; Fig. 2A, lane 5), neither TFIIHi nor the mixture of Rad3 and Rad25 proteins, when combined with the same set of NER factors, was active in the NER reaction ( Fig. 2A, lanes 7 and 8). Likewise, Rad3 alone, Rad25 alone, the combination of Rad3-TFIIHi, or the combination of Rad25-TFIIHi, when used together with the remainder of the NER factors, also did not promote incision of the UV-damaged DNA (data not shown). Strikingly, when Rad3, Rad25, and TFIIHi were preincubated and then added to the NER reaction, incision of the UV-damaged DNA substrate occurred just as efficiently as when TFIIH was used (Fig. 2A, lane 9). There was a strict dependence of the incision reaction on ATP, regardless of whether TFIIH (Fig. 2A, lanes 5 and 6) or the combination of TFIIHi, Rad3, and Rad25 was used ( Fig. 2A, lanes 9 and 10). We also purified the excision DNA fragments from the NER reactions and then subjected them to labeling with [␣-32 P]dideoxy-ATP and calf thymus terminal transferase. Excision DNA fragments were seen when the UV-damaged DNA was incubated with the combination of TFIIH and the remainder of the NER factors (Fig. 2B, lane 4), and generation of these excision DNA fragments was absolutely dependent on ATP (Fig. 2B, lanes 3  and 4). In agreement with the results obtained using the agarose gel assay ( Fig. 2A), ATP-dependent dual incision of the UVdamaged DNA was observed when TFIIHi was combined with Rad3-Rad25 (Fig. 2B, lanes 7 and 8) but not when TFIIHi or when the mixture of Rad3-Rad25 was used alone (Fig. 2B, lanes  5 and 6). Taken together, our results indicate that in addition to Rad3 and Rad25, TFIIHi is absolutely required during the ATPdependent incision phase of NER.
Requirement for Rad3 and Rad25 Helicase Functions-Both Rad3 and Rad25 proteins possess a DNA helicase activity that is fueled by ATP hydrolysis (4,5). We have previously generated mutations in the highly conserved Walker type A motif required for ATP binding and hydrolysis in Rad3 and Rad25 proteins. Genetic studies on these mutations (rad3 Arg-48 and rad25 Arg-392) have indicated that the Rad3 helicase is re-quired in NER but is dispensable for RNA polymerase II transcription (14), whereas the Rad25 helicase activity is required in both NER and transcription (2,4). However, in these genetic studies, it was not possible to address whether Rad3 and Rad25 helicase activities are required for the incision of damaged DNA or for a later stage of repair, such as for the postincision turnover of the NER protein complex and repair synthesis. Also, because both the Rad3 and Rad25 proteins are essential for Pol II transcription, the possibility existed that the helicase function of these proteins affects the expression of a factor indispensable for incision of UV-damaged DNA or for another phase of the repair process in vivo.
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 ϩ 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 799am -encoded protein from the ADCI promoter. The rad25 799am 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 ϩ 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.
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, Ͼ85% of the supercoiled UVdamaged 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 32 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 ϩ 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 Ϸ3% 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 32 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.
TFIIK Is Dispensable for Incision in Vitro-TFIIH can be purified alone or as a complex with the CTD kinase TFIIK (8) (see also Fig. 1). To investigate whether TFIIK might be important for promoting damage-specific incision, we used the same molar amount of TFIIH that contains either a stoichiometric amount of TFIIK or only a trace of TFIIK (Fig. 1A, lanes  2 and 3) in the NER reaction and assessed the incision rate afforded by the two different forms of TFIIH. As shown in Fig.  4, A and B, there was no detectable difference in the rate of incision of the UV-damaged plasmid with the two different forms of TFIIH. The amount of excision products generated by the TFIIH-TFIIK complex was essentially the same as that obtained with TFIIH containing little TFIIK (Fig. 4C, lanes 2  and 3). DISCUSSION 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), 2 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 5Јand 3Ј-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.