Double-stranded DNA activity of factor TFIIH and the mechanism of RNA II open complex formation

James Fishburna, Eric Tomkob, Eric Galburtb,1, and Steven Hahna,1

aDivision of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109; and bDepartment of Biochemistry and Molecular Biophysics, Washington University in St. Louis, St. Louis, MO 63110

Edited by Robert G. Roeder, The Rockefeller University, New York, NY, and approved February 24, 2015 (received for review September 12, 2014) Formation of the RNA polymerase II (Pol II) open complex (OC) re- downstream from the site of DNA unwinding in the OC (20–24). quires DNA unwinding mediated by the Therefore, XPB/Ssl2 cannot function as a conventional TFIIH helicase-related subunit XPB/Ssl2. Because XPB/Ssl2 binds to promote OC formation (20). Three models have been pro- DNA downstream from the location of DNA unwinding, it cannot posed to explain the role of XPB/Ssl2 in transcription. First, it function using a conventional helicase mechanism. Here we show was postulated that XPB acts as a molecular wrench, binding to that yeast TFIIH contains an Ssl2-dependent double-stranded DNA its site on downstream DNA and using its ATPase to rotate up- translocase activity. Ssl2 tracks along one DNA strand in the 5′ → stream DNA within the Pol II cleft (20). Because upstream DNA 3′ direction, implying it uses the nontemplate strand to is constrained by TBP, TFIIB, and other factors, DNA rotation reel downstream DNA into the Pol II cleft, creating torsional strain could lead to DNA opening. Second, it was proposed that the and leading to DNA unwinding. Analysis of the Ssl2 and DNA-de- XPB ATPase activity promoted DNA opening via a conforma- pendent ATPase activity of TFIIH suggests that Ssl2 has a proces- tional change in PIC components, leading to a structural rear- sivity of approximately one DNA turn, consistent with the length rangement of both and DNA, analogous to the ATP- of DNA unwound during transcription initiation. Our results can dependent mechanism of OC formation in the bacterial σ54 system explain why maintaining the OC requires continuous ATP hydrolysis (25). Third, comparing structure models of the PIC and OC sug- and the function of TFIIH in promoter escape. Our results also sug- gested that ∼15 bp of downstream promoter DNA inserts into the gest that XPB/Ssl2 uses this translocase mechanism during DNA re- Pol II cleft upon OC formation (22). Based on this and other data, pair rather than physically wedging open damaged DNA. it was proposed that XPB/Ssl2 functions as a double-stranded DNA (dsDNA) translocase (22, 23, 26). According to this model, transcription initiation | DNA unwinding | DNA helicase XPB/Ssl2 attempts to track away from the PIC, but, because it is bound to the PIC via TFIIH, it instead feeds and rotates dsDNA or all multisubunit RNA (Pols), a universal step into the Pol II cleft, leading to DNA opening. However, a key Fin the transcription initiation pathway is formation of the feature of this latter model, whether XPB/Ssl2 has dsDNA trans- open complex (OC) (1, 2). The OC forms when Pol and its as- locase function, had not been tested. sociated transcription machinery bind to promoter DNA, gen- Here we show that yeast TFIIH has Ssl2-dependent dsDNA erating a series of conformational changes in both DNA and translocase activity and that it primarily tracks along one DNA protein, including the unwinding of ∼11 bp of DNA upstream strand in the 5′ → 3′ direction. The kinetic properties of the from the transcription start site (TSS). This open state is stabi- suggest a rate-limiting step between DNA binding and lized by interactions of Pol with the unwound strands of pro- translocation and a processivity on the order of a turn of DNA, moter DNA and by the binding of downstream double-stranded – promoter DNA to the Pol Cleft/Jaw domains (3 5). All tested Significance multisubunit Pols spontaneously form OCs, except for RNA Pol II, where ATP and the general transcription factor TFIIH are required for DNA unwinding (6–8). For Pol II, this unwound How ATP hydrolysis is coupled to promoter DNA unwinding state is unstable, decaying with a half-life of 30–60 s (8, 9). and open complex formation at RNA polymerase II (Poll II) The general transcription factor TFIIH contains two subunits promoters is a longstanding question. Of the multisubunit RNA with DNA-dependent ATPase activity, XPD/Rad3 and XPB/Ssl2 polymerases, only Pol II requires ATP for DNA unwinding. Here (human/yeast ), which are members of the SF2 helicase- we show that the general transcription factor TFIIH subunit BIOCHEMISTRY translocase family (10). The XPB/Ssl2 ATPase is required for Ssl2 is a double-stranded DNA translocase. These and other DNA unwinding in the OC, whereas the XPD/Rad3 ATPase data suggest that Ssl2 promotes DNA opening by tracking activity does not function in transcription (11–14). In DNA along the nontemplate promoter strand, rotating and inserting strand displacement assays, the two isolated human subunits DNA into the Pol II cleft, leading to DNA unwinding. have DNA helicase activity of opposite polarity with XPD having Our accompanying biochemical studies explain why the open 5′ → 3′ activity and XPB having 3′ → 5′ activity. XPD is at least complex is unstable and how TFIIH can promote Pol II escape eightfold more active than XPB in strand displacement (15). In from the promoter. Our findings also have important implica- contrast to the activity of purified XPB, the most purified prepa- tions for the mechanism of TFIIH-mediated DNA repair. rations of native or recombinant human TFIIH have only the XPD ′ → ′ ′ → ′ Author contributions: J.F., E.T., E.G., and S.H. designed research; J.F. and E.T. performed 5 3 helicase activity, with no detectable 3 5 helicase research; J.F., E.T., E.G., and S.H. analyzed data; and J.F., E.T., E.G., and S.H. wrote function (14, 15). In addition to its role in transcription initiation, the paper. TFIIH can assist in Pol II promoter escape in an XPB-dependent The authors declare no conflict of interest. mechanism (16, 17), and both the XPB and XPD subunits of This article is a PNAS Direct Submission. TFIIH play an essential role in general and transcription-coupled 1To whom correspondence may be addressed. Email: [email protected] or nucleotide excision repair (NER) (18, 19). [email protected]. Mapping the location of XPB/Ssl2 in RNA Pol II preinitiation This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. complexes (PICs) revealed that this factor binds promoter DNA 1073/pnas.1417709112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1417709112 PNAS | March 31, 2015 | vol. 112 | no. 13 | 3961–3966 Downloaded by guest on September 29, 2021 consistent with the amount of DNA thought to be unwound in E236Q) that is predicted to be defective in ATP hydrolysis (27) the OC. Our findings have important implications for the mech- is defective in helicase function. In contrast, both WT and Rad3 anism of OC formation, the relative instability of the OC, and the mutant TFIIH preparations are equally active in supporting role of XPB/Ssl2 in NER. transcription with purified factors and Pol II from the yeast HIS4 promoter (Fig. S1). Results As an initial test for dsDNA translocase function, we assayed TFIIH Has Ssl2-Dependent dsDNA Translocase Activity. We first mea- whether TFIIH can displace a 22 nucleotide DNA triplex, a well- sured DNA helicase activity of purified yeast TFIIH using a established assay for dsDNA translocase function (28–30). The substrate consisting of a 100-base oligonucleotide with a labeled DNA triplex was formed by annealing a polypyrimidine triplex- 25-base oligonucleotide annealed at either the 3′ or 5′ end forming oligonucleotide (TFO) to a 142-bp dsDNA containing (Fig. 1A). Consistent with the function of human TFIIH, we found a complementary polypurine sequence at one end (Fig. 1A). The that yeast TFIIH contains 5′ → 3′ helicase but not 3′ → 5′ helicase triplex is disrupted by heating, but is stable at 26 °C with or without the addition of ATP (Fig. 1C, lanes 1–3). Incubation of function (Fig. 1B). The 5′ → 3′ helicase activity requires both WT TFIIH, ATP, and the triplex substrate results in near com- hydrolysable ATP and the Rad3 ATPase, because a TFIIH de- plete dissociation of the TFO (Fig. 1C, lanes 4–6). The triplex rivative containing a mutation in the Rad3 Walker B motif (Rad3 displacement activity of TFIIH is unaffected by the Rad3 E236Q ATPase mutation but is abolished by the equivalent Walker B ATPase mutation in Ssl2 (E489Q; Fig. 1C, lanes 7–11). To assay whether TFIIH can simply bind and displace the triplex without translocation, we used a 22 nucleotide triplex substrate that did not contain additional dsDNA (Fig. 2A). The short triplex substrate is less stable than the longer triplex, likely due to the lack of stabilizing dsDNA. Nevertheless, as predicted for a dsDNA translocase, TFIIH cannot displace the TFO from this shorter substrate lacking dsDNA (Fig. 2B). The TFIIH triplex displace- ment activity was not dependent on a free DNA end, because TFIIH can also displace the TFO annealed to a 3.2-kb double- stranded plasmid, although with much slower kinetics (Fig. 2C). Both ATP and dATP function to promote OC formation (7) and, as expected, both nucleotides can promote triplex displacement (compare Fig. 1C and Fig. 2C, Right). Our combined results are consistent with the prediction that TFIIH contains an Ssl2- dependent dsDNA translocase activity.

Ssl2 Tracks in the 5′ → 3′ Direction Along the DNA Duplex. dsDNA are thought to track along one strand of the DNA backbone (31). If true for Ssl2, discerning the polarity of trans- location will reveal which promoter strand is used by Ssl2 during DNA unwinding. As an initial test of polarity, we assayed triplex displacement using substrates where the TFO was annealed to either the top or bottom strand of the 142-bp duplex DNA (Fig. 3A). As shown above, TFIIH can readily displace the TFO from the bottom strand triplex substrate (Fig. 3B, lanes 4–6). In contrast, the top strand triplex substrate is almost completely resistant to TFO displacement (Fig. 3B, lanes 1–3). One model consistent with this result is that Ssl2 tracks along one strand of duplex DNA in the 5′ → 3′ direction and that this tracking is blocked by the annealed TFO. As a further test of this model, we introduced biotin as a tracking barrier on the triplex substrates (Fig. 3A). These substrates contain a single-strand DNA nick with biotin attached to the 5′ end of one strand of duplex DNA. We found that the top-strand biotin was a much stronger block to TFO displacement, with only one third of the TFO displaced after 6 h (Fig. 3 C and D). Although the bottom-strand biotin modestly inhibited TFO displacement, the TFO was >80% dis- placed after 6 h, similar to the nonbiotinylated template. As a further test of whether the integrity of the top strand is the most critical for TFO displacement, we generated triplex substrates with 5-bp single-stranded gaps on either the top or bottom du- Fig. 1. ATP-dependent helicase and translocase activities of TFIIH. (A) Heli- plex strand, directly adjacent to the triplex (Fig. S2). Consistent case and triple helix substrates. Helicase substrates are designed to with the above results, we found that TFO displacement was most distinguish 5′ → 3′ and 3′ → 5′ enzyme polarity. The short displaced oligo- inhibited by the gap on the top strand. Under conditions of the nucleotide is fluorescently labeled for visualization. The triplex substrate is assay, 80–90% of the TFO was displaced from the nongapped formed via Hoogsteen base pairs between the 22 nucleotide (nt) 32P-labeled template, 45% displaced from the template with the gap on the triplex forming oligo (TFO) and the purine rich strand of the PCR generated – duplex. (B) Helicase assay testing ATP-dependence and polarity of WT TFIIH bottom strand, and only 20 25% displaced from the template or TFIIH containing the ATPase mutant Rad3-E236Q. (C) Triplex disruption as- with the gap on the top strand. Our combined results strongly say testing ATPase requirements for triplex displacement with either WT TFIIH suggest that Ssl2 primarily tracks along one strand of duplex DNA or TFIIH containing the ATPase defective mutants Rad3-E236Q or Ssl2-E489Q. in the 5′ → 3′ direction.

3962 | www.pnas.org/cgi/doi/10.1073/pnas.1417709112 Fishburn et al. Downloaded by guest on September 29, 2021 we created HIS4 promoter derivatives containing a 12 nucleotide single-strand bubble beginning 21 bp downstream from TATA (Fig. 4C). Pol II initiating from this bubble in the absence of any general factors transcribed past the Cy3 block with 47–77% ef- ficiency compared with a bubble template lacking Cy3 (Fig. 4D, lanes 4–7). Taken together, our results show that the integrity of the nontemplate strand is important for transcription initiation and is consistent with the dsDNA translocase model for open complex formation.

Ssl2 Translocates with Low Processivity. To further characterize the Ssl2 motor within the TFIIH complex, we next investigated stim- ulation of the ATPase by nucleic acid. Measurements of how the steady-state rate of ATP hydrolysis depends on DNA template length and concentration can be used to assay for translocation and to evaluate different models of translocation (33–35). For

Fig. 2. Template requirements for triplex disruption by TFIIH. (A) Triple helix substrates used to monitor TFIIH translocation. TFO22 was generated by annealing the 22-nt TFO to the 22-bp triplex target sequence. This smaller triplex was somewhat less stable than the 142-bp triplex DNA, likely due to no additional dsDNA. To make the 142-bp and circular triplex templates, the TFO was annealed to a PCR-generated 142-bp duplex or a 3.2-kb plasmid containing the triplex target sequence. (B) Triplex disruption assay using the TFO22 triple helix template and the TFIIH derivative Rad3 E236Q. (C) Time course of triplex disruption comparing the circular 3.2-kb plasmid triplex and the linear 142-bp triplex templates. Intact triplex was measured at each time point and quantified by comparison with a standard curve. The percent disruption of each triplex is indicated. ATP and dATP both function in the TFO displacement assay (compare with Fig. 1C) and in OC formation (7).

Blocking Ssl2 Translocation Inhibits Transcription Initiation. The above results predict that blocking Ssl2 translocation should in- hibit transcription initiation. Mapping the location of Ssl2/XPB- DNA binding in PICs has suggested Ssl2 binds 30–36 (yeast) and 40–50 bp (human) downstream from TATA (20–23). To test the role of Ssl2 translocase function in transcription initiation, we created three promoter derivatives with Cy3 dye inserted in the phosphodiester backbone of the nontemplate DNA strand 37, 41, or 46 bp downstream from the HIS4 TATA (Fig. 4A). Cy3 posi- tioned in the DNA backbone is a strong inhibitor of Ssl2 trans-

location in the TFO displacement assay (Fig. S3). BIOCHEMISTRY The modified and unmodified DNA templates were tested for in vitro transcription activity using the reconstituted system (32), and transcription from these templates was completely TFIIH dependent (Fig. 4B, compare lanes 4–7and8–11). We found that Cy3 positioned on the nontemplate strand 41 and 46 bp from TATA inhibited transcription approximately fivefold (Fig. 4B, Fig. 3. Polarity of Ssl2 catalyzed TFIIH translocation. (A) Triple helix sub- lanes 4, 6, and 7), consistent with the dsDNA translocase model. strates designed to test the polarity of Ssl2 translocation. Top and bottom Cy3 positioned 37 bp downstream from TATA inhibited tran- strand triplexes are annealed as shown using PCR-generated duplex DNA scription approximately twofold (Fig. 4B,lanes4–5). We speculate and the TFO. PAGE-purified oligonucleotides were annealed with the TFO to that transcription escaping this more upstream Cy3 insertion may generate the biotin-containing triplexes where a 5′-biotin labeled oligonu- be due to inherent flexibility in the Ssl2–DNA interaction, allow- cleotide is positioned immediately upstream of the triplex on the top or B ing Ssl2 to escape the Cy3 block by binding DNA just downstream bottom duplex strand. ( ) Triplex disruption assay comparing templates with the TFO annealed to either the top or bottom strand of the duplex. Either of Cy3 37 in a position nearly equivalent to the observed XPB- WT or Rad3 E236Q TFIIH was used as indicated. (C) Time course of triplex human promoter interaction (23). disruption from biotin templates by TFIIH (Rad3-E236Q). Reactions were To confirm that the observed transcription inhibition by Cy3 incubated for the indicated times with 1 mM dATP at 26 °C and quantified could not be explained by an inhibition of transcription elongation, for intact triplex remaining. (D) Quantitation of results in C.

Fishburn et al. PNAS | March 31, 2015 | vol. 112 | no. 13 | 3963 Downloaded by guest on September 29, 2021 a Michaelis–Menten curve, we were able to determine both a Michaelis constant (KM) and a maximal velocity (Vmax)foreach length of DNA (Fig. 5B). Although KM does not show a length dependence (Fig. 5C), Vmax is clearly sensitive to DNA length (Fig. 5D). The dependence of Vmax on template length is a clear indication that Ssl2 is a dsDNA translocase as nontranslocating do not show this behavior (33–35) (Fig. S4A). Models of translocation can be used to fit the dependence of Vmax on DNA length to extract quantitative values for parame- ters such as occluded site size, kinetic step size, and motor pro- cessivity, i.e., the probability that the motor takes a step forward instead of dissociating (Materials and Methods)(33–35). In the simplest model for translocation, the motor binds DNA at any site and steps forward coupled to ATP hydrolysis. At each site, the motor may either hydrolyze ATP and take a step forward or dis- sociate (Fig. S4B). At the end of the template, the motor either dissociates or translocates off the end of the DNA. This model predicts that KM will vary with the length of the DNA template, whereas Vmax will not (Fig. S4B). This behavior is inconsistent with our observed results (Fig. 5 C and D). However, in contrast to the simple translocation model above, models that incorporate a slow kinetic step between unbound and the active DNA-bound motor states result in a length- dependent Vmax and a length-independent KM (33–35). Two lim- iting cases of this behavior are (i) a slow step after binding and before translocation or (ii) a slow step before dissociation from the end of the DNA (Fig. S4C). These two cases predict different

Fig. 4. In vitro transcription from promoters with DNA backbone blocks on the nontemplate DNA strand. (A) HIS4 promoter derivatives with Cy3 DNA backbone insertions. were constructed from synthetic oligonucleotides and contained Cy3 positioned 37, 41, or 46 bp downstream from HIS4 TATA. Transcription was assayed by primer extension using the lacI oligonucleotide as shown. (B) In vitro transcription using the reconstituted yeast Pol II system and the promoters in A. Lanes 1–4 contain the indicated amounts of a transcription reaction used to generate a standard for quantitation of tran- scription signals relative to the unmodified template. Lanes 5–7 are tran- scription reactions using the indicated Cy3 templates. Percent transcription relative to the unblocked template is indicated. No transcription is observed when TFIIH is omitted (lanes 8–11). (C) HIS4 promoter derivatives identical to those in A except that they contain the 12 nucleotide single-stranded DNA bubble as shown. This bubble allows transcription initiation by Pol II in the absence of other general factors (33). (D) In vitro transcription using purified yeast Pol II on the bubble templates and assayed by primer extension. Lanes 1–4 contain the indicated amounts of a transcription reaction using the non- Cy3 bubble template. Lanes 5–7 are transcription reactions using the Cy3- modified bubble templates. Lanes 8–9 are mock transcription reactions lacking Pol II and assayed by primer extension. The products marked by * are due to primer extension of the DNA template, which is blocked by Cy3. These blocked products are not visible in B, lanes 9–11, as they are significantly longer than the RNA products initiated at the HIS4 TSS.

these measurements, we used the TFIIH preparation contain- ing Rad3 E236Q so that Ssl2 was the only functional ATPase. TFIIH and DNA were preincubated for 40 min, and then ATP was added, and phosphate release was quantitated at different – Fig. 5. Dependence of the ATP hydrolysis rate on DNA length. In all reac- times (1 20 min). The steady-state rate of ATP hydrolysis was tions, TFIIH (Rad3 E236Q) was preincubated with DNA for 40 min before ATP

determined by a linear fit of these data (Fig. 5A). ATP hydrolysis addition. (A) The phosphate released (Pi) as a function of time for a range of in the absence of nucleic acid was undetectable, and dsDNA DNA lengths shows a linear dependence that can be fit to give a steady-state consistently stimulated ATPase activity four- to fivefold higher rate of ATP hydrolysis. (B) The steady-state rate of ATPase hydrolysis as than that of single-stranded DNA (Table 1), consistent with the a function of DNA length and DNA concentration. Concentration is plotted enzyme being a dsDNA translocase. as micromolar-base pairs (μMbp), and the data are fit to Michaelis–Menten K V V The steady-state rate of ATP hydrolysis was measured as a curves, allowing for the extraction of M and max parameters. The max of the plasmid data and its associated error are shown by two dashed hori- function of DNA concentration and DNA template length zontal lines. (C) K as a function of DNA length. (D) V as a function of DNA over a range of DNA lengths from 30 to 80 bp and on a circular M max length. Fit shown using analytical expression for the dependence of Vmax on plasmid representing an infinitely long template (Fig. 5 A and DNA length for the model described in the text with a step size of 1 bp and B).Asthedependenceofrateon DNA concentration follows a processivity of 10 bp (SI Materials and Methods).

3964 | www.pnas.org/cgi/doi/10.1073/pnas.1417709112 Fishburn et al. Downloaded by guest on September 29, 2021 Table 1. Vmax of the Ssl2 ATPase with single- or double- the contacts between Pol II and unwound DNA are not strong stranded DNA enough to stabilize the fully unwound state in the absence of con-

DNA Length [bp (ds) or nt (ss)] Vmax (ATP/Ssl2 s) tinual Ssl2 translocation activity. This mechanism predicts multiple cycles of DNA opening and ds 30 5.19 closing during the initial stages of transcription. It was found that ds 40 6.41 the Pol II OC is stabilized by a four-nucleotide RNA (8), so this ds 60 8.82 may be the minimum length of transcript to prevent reversion to ds 80 9.49 the PIC dsDNA state. However, translocase function may be nec- ss 30 1.03 essary to assist in DNA opening until a 7- to 8-base RNA:DNA ss 40 1.20 ss 60 2.46 hybrid is formed. This model can explain the observation that TFIIH ss 80 2.58 stimulates promoter escape from templates with a short stretch of premelted DNA from −9to−1 with respect to the TSS (17). Reactions with single-stranded DNA were carried out as described in Finally, our results have important implications for the action of Materials and Methods μ and contained 12.5 M nt of each template. The XPB during NER. In the general genome repair pathway, TFIIH average Vmax values from two independent experiments are given. For com- V is recruited to DNA lesions bound by factor XPC, where it opens parison, the predicted max from an analogous ds DNA template is shown ∼ (data from Fig. 4D). an asymmetric 27-bp DNA bubble surrounding the lesion (18, 19). Both XPB and XPD are required for this DNA unwinding, although XPB does not seem to promote unwinding via a con- pre–steady-state behavior in the ATPase rate (34). A slow step ventional helicase mechanism. Mutations that abolish the XPB at the end of the DNA predicts a burst of rapid ATP hydrolysis ATPase abolish DNA unwinding activity during NER, but two on ATP addition compared with the steady-state rate. This XPB mutations with reduced 3′ → 5′ helicase function were re- burst phase arises because initial rounds of hydrolysis do not portedly active for NER (13). Based on these results, it was pro- encounter the rate-limiting step that takes place at the DNA end. posed that XPB functions indirectly in DNA opening by using In contrast, a slow step only between DNA binding and trans- its ATPase function to promote a conformational change in the location predicts a lag in ATP hydrolysis rate. This lag phase arises – – because the rate-limiting step occurs before translocation and XPC DNA TFIIH complex, physically wedging open 5 bp of the before ATP hydrolysis occurs. Our data are consistent with the DNA duplex, and positioning the XPD helicase to open the DNA latter case, as there is a lag in ATP hydrolysis after adding ATP surrounding the DNA lesion (10, 18, 19). Given our results, it to TFIIH prebound to DNA (Fig. 5A). The observed lag suggests seems more likely that XPB opens 5 bp of DNA using a dsDNA that Ssl2, in complex with TFIIH, first binds DNA in an inactive translocase mechanism similar to that in OC formation. By gen- conformation and then, after ATP addition, isomerizes to the erating torsional strain in rotationally fixed damaged DNA, the active state before translocation. XPB subunit of TFIIH can lead to initial unwinding, generating To extract quantitative measures of translocase parameters, a substrate for XPD to generate the fully opened 27-bp asym- we analyzed the DNA length dependence of the Ssl2 ATPase metric bubble surrounding DNA lesions. Vmax with an expression derived from the isomerization model (SI Materials and Methods) (34). Assuming a translocation step Materials and Methods size of 1 bp and a contact size on the DNA of 16 bp (Table S1 and DNA Helicase Assay. TFIIH helicase activity was monitored using a fluorescent Materials and Methods), we determined a processivity of Ssl2 in the dye-labeled oligonucleotide (IR700-TTCACCAGTGAGACGGGCAACAGCC) an- context of TFIIH of 0.90 with a fit error of 0.01 corresponding to nealed to PAGE-purified 100-base oligonucleotides, with the resulting templates an average of 10.0 ± 0.9 bp translocated before dissociation from having 5′ or 3′ overhangs of 75 bases (Fig. 1). The assay was performed as the DNA. Because we have no independent measure of step size, previously described (36), with the following modifications: 10-μLreactions we note that a larger step size would lead to a lower processivity. contained 10 mM Hepes (pH 7.6), 100 mM potassium glutamate, 10 mM mag- For example, analyzing the same data with a step size of 2 bp nesium acetate, 3.5% (vol/vol) glycerol, 1 mM DTT, 1 μg BSA, 60 fmol holo-TFIIH, would correspond to 5.3 ± 0.3 bp translocated before dissociation and 30 fmol template DNA. ATP or ATPγS was added to 1 mM, and reactions from the DNA. were incubated 1–2 h at 26 °C. Controls received TE buffer (10 mM Tris, pH 7.5, 1 mM EDTA) in lieu of ATP or were heated to 95 °C for 30 s. Reactions were Discussion quenched by the addition of 10 μL 33% glycerol, 40 mM EDTA, and 0.5% SDS × Our results on the translocase activity of Ssl2 in the context of the and analyzed by PAGE using 10% acrylamide gels in 0.5 TBE buffer (134 mM TFIIH complex are completely consistent with the translocase Tris, 44 mM boric acid, 2.5 mM EDTA, pH 8.8) plus 0.1% SDS. Following elec- model of OC formation and explain how TFIIH might generate trophoresis, gels were visualized using an Odyssey IR scanner (LI-COR). BIOCHEMISTRY torsional stress in promoter DNA, leading to unwinding. Our finding that Ssl2 tracks in the 5′ → 3′ direction implies that Ssl2 uses the nontemplate strand of promoter DNA as it attempts to track away from the PIC but instead reels downstream dsDNA toward the PIC, creating open DNA via torsional or mechanical stress that is then fed into the Pol II cleft (Fig. 6). Interestingly, this is the opposite direction to the characterized helicase activity of the Ssl2 subunit alone, suggesting that the enzyme does not use the same motor mechanism for helicase and dsDNA translocase activities. Our conclusion that the processivity of Ssl2 translocation is similar to the amount of DNA unwound in the OC can explain Fig. 6. dsDNA translocase model for open complex formation. The Ssl2 ′ → ′ why the Pol II OC is unstable. We speculate that DNA opening subunit of TFIIH tracks in the 5 3 direction on the nontemplate promoter DNA strand (red). Because TFIIH movement is constrained due to interaction requires continuous ATP hydrolysis because Ssl2 likely dissociates with other PIC components, translocation results in insertion and rotation of from the DNA after translocating a short distance (while re- promoter DNA into the Pol II cleft, leading to DNA unwinding (right arrows maining in complex with TFIIH and the PIC), resetting the initi- indicate rotation and direction of dsDNA movement). The short persistence ation complex to its closed state. In contrast to the situation with length of Ssl2/TFIIH predicts that the OC state is unstable, in agreement with other prokaryotic and eukaryotic RNA Pols, it seems likely that experimental observations (9, 10).

Fishburn et al. PNAS | March 31, 2015 | vol. 112 | no. 13 | 3965 Downloaded by guest on September 29, 2021 In Vitro Transcription Assay. In vitro transcription using recombinant and indicated times before stopping with 2.5 μL5× GSMB (15% glucose, 3% SDS, purified factors was performed similarly to previously described assays (32). 250 mM Mops, pH 5.5, and 0.04% bromophenol blue). The reactions were See SI Materials and Methods for additional information. analyzed by PAGE using 6% acrylamide gels with buffer: 40 mM Tris-acetate (pH 5.5), 5 mM sodium acetate, and 1 mM magnesium chloride. Gels were TFIIH Purification. WT TFIIH was purified from strain SHY869 (RAD3-(HA)1-TAP visualized using either an Odyssey IR scanner (LI-COR) or dried and visualized tfb6Δ tag, ) as previously described (32) except that, following the ultra- by PhosphorImager (Molecular Dynamics). centrifugation step, potassium acetate was added to the extract to a final concentration of 0.6 M before binding to IgG-Sepharose (GE Healthcare). ATPase Assay. DNA-dependent ATPase activity of TFIIH (Rad3-E236Q) was TFIIH with ATPase-defective Rad3 was purified by the same method from measured using a colorimetric assay kit (Innova; 601-0120). Forty-microliter strain SHY887 (leu2Δ rad3Δ::KanMX, tfb6Δ::HPH) carrying plasmid pJF82 [ars reactions contained 10 mM Hepes (pH 7.6), 100 mM potassium glutamate, cen LEU2 RAD3 (E236Q)-(HA)1-TAP tag]. Because the Ssl2 E489Q mutation is μ lethal, this TFIIH derivative was purified from a WT strain containing the 10 mM magnesium acetate, 3.5% glycerol, 1 mM DTT, 4 g BSA, 40 fmol holo- μ Tap-tagged Ssl2 mutation on a plasmid. Strain SHY861 (leu2Δ SSL2) carrying TFIIH, and 1.25 nM to 1.5 M template DNA. After 40 min at room tem- plasmid pJF62 [ars cen LEU2 ssl2 (E489Q)-(FLAG)1-TAP tag] was grown in perature, purified ATP was added to 0.5 mM, and reactions were incubated glucose complete media lacking leucine, and TFIIH was purified by the 1–20 min at 26 °C. Reactions were stopped by the addition of 10 μL gold mix method described above. and, after 4 min, 4 μL stabilizer 2. After 30 min at room temperature, ab- sorbance was measured at 635 nm and plotted against DNA concentration. Triplex Disruption Assay. Triplex DNA template formation and disruption A standard curve was established for every experiment using the kit-included reactions were performed as previously described (28), with the following phosphate standard and used to determine TFIIH catalyzed ATP hydrolysis. modifications: templates were assembled from duplex DNA containing Templates from 30 to 80 bp were tested at nine concentrations in triplicate, a triplex target sequence (AAGAAAAGAAAGAAGAAAGAAA) and a fluores- spanning a 1,200-fold range of DNA concentration. See SI Materials and 32 cent or P-labeled TFO (TTCTTTTCTTTCTTCTTTCTTT). The DNA and TFO were Methods for information on extracting translocation kinetic parameters from combined at 1 μM final concentration in 25 mM Mes (pH 5.5) and 10 mM the ATPase data. MgCl2 and heated to 57 °C for 15 min and then cooled at 1 °C/min over 35 min to allow annealing. Triplex DNA was stored at -20 °C and diluted in 50 mM ACKNOWLEDGMENTS. We thank members of the S.H. laboratory and Ted Tris·HCl, pH 8.0, 10 mM MgCl , and 1 mM DTT before the assay. Ten- 2 Young for comments and suggestions during the course of this work microliter reactions contained 10 mM Hepes (pH 7.6), 100 mM potassium and S. Grünberg for Fig. 6 and comments on the manuscript. This work μ glutamate, 10 mM magnesium acetate, 3.5% glycerol, 1 mM DTT, 1 g BSA, was supported by National Institute of General Medical Sciences Grant 0.01% Nonidet P-40, 15 fmol holo-TFIIH, and 30 fmol triplex DNA. ATP or 2RO1GM053451 (to S.H.) and National Science Foundation-Molecular and dATP was added to 1 mM, and reactions were incubated at 26 °C for the Cellular Biosciences Grant 1243918 (to E.G.).

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