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. Author manuscript; available in PMC: 2016 Sep 17.
Published in final edited form as: Mol Cell. 2015 Sep 17;59(6):1025–1034. doi: 10.1016/j.molcel.2015.08.012

Tripartite DNA Lesion Recognition and Verification by XPC, TFIIH, and XPA in Nucleotide Excision Repair

Chia-Lung Li 1,4,5, Filip M Golebiowski 1,4, Yuki Onishi 2,3, Nadine L Samara 1, Kaoru Sugasawa 2,3, Wei Yang 1,*
PMCID: PMC4617536  NIHMSID: NIHMS717692  PMID: 26384665

SUMMARY

Transcription factor IIH (TFIIH) is essential for both transcription and nucleotide excision repair (NER). DNA lesions are initially detected by NER factors XPC and XPE or stalled RNA polymerases, but only bulky lesions are preferentially repaired by NER. To elucidate substrate specificity in NER, we have prepared homogeneous human ten-subunit TFIIH and its seven-subunit core (Core7) without the CAK module and show that bulky lesions in DNA inhibit the ATPase and helicase activities of both XPB and XPD in Core7 to promote NER, whereas non-genuine NER substrates have no such effect. Moreover, the NER factor XPA activates unwinding of normal DNA by Core7, but inhibits the Core7 helicase activity in the presence of bulky lesions. Finally, the CAK module inhibits DNA binding by TFIIH and thereby enhances XPC-dependent specific recruitment of TFIIH. Our results support a tripartite lesion verification mechanism involving XPC, TFIIH, and XPA for efficient NER.

Graphical Abstract

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INTRODUCTION

The multi-subunit transcription factor II H (TFIIH) is essential for transcription and nucleotide excision repair (NER) of DNA lesions induced by ultraviolet (UV) light or environmental toxins in all eukaryotes (Compe and Egly, 2012). TFIIH consists of a seven-subunit core (Core7), which in mammals is composed of XPB, XPD, p62, p52, p44, p34, and p8 (TTDA), and a three-subunit CDK-activating kinase (CAK) module composed of CDK7, MAT1, and cyclin H (Egly and Coin, 2011). The CAK module phosphorylates RNA polymerase II to regulate transcription but is dissociated from Core7 during NER (Coin et al., 2008). Core7 contains two ATP-dependent DNA helicases: XPB with 3′ to 5′ polarity and XPD with 5′ to 3′ polarity (Compe and Egly, 2012; Egly and Coin, 2011; Fuss and Tainer, 2011). During transcription, the helicase activity of XPB is essential for melting promoter DNA and facilitating promoter escape (Dvir et al., 2003; Lin et al., 2005); XPD appears to play a structural role and assists the CAK module in phosphorylating the C-terminal region of RNA polymerase II (Seroz et al., 2000; Tirode et al., 1999). In contrast, the ATPase activities of both XPB and XPD are required for NER, and mutation of the Walker A motif in either enzyme impairs the NER activity (Coin et al., 2007). Mutations in human XPB, XPD, and TTDA have been associated with rare genetic diseases, e.g., xeroderma pigmentosum (XP), Cockayne syndrome (CS), and trichothiodystrophy (TTD), which manifest as predisposition to cancer among XP patients and UV sensitivity, pre-mature aging, growth impairment, and developmental and neurological abnormalities in general (Compe and Egly, 2012; Hoeijmakers, 2009). Interestingly, mutations in XPD alone are implicated in XP, CS, and TTD (Fan et al., 2008; Schäfer et al., 2013).

NER removes a wide range of DNA lesions including chemically induced bulky base adducts and ubiquitous UV-induced cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimi-done (6-4) photoproducts. There are two sub-pathways of NER: transcription-coupled NER (TC-NER) and global genome NER (GG-NER), which differ in how DNA lesions are initially recognized (Lagerwerf et al., 2011; Sugasawa, 2010). In TCNER, a damaged site is initially recognized by a stalled RNA polymerase on the transcribed DNA strand (Hanawalt et al., 2000; Lagerwerf et al., 2011). In GG-NER, a lesion site and associated DNA helical distortion are detected by either the heterotrimeric XPC protein complex composed of XPC, RAD23B, and Centrin-2 (Araki et al., 2001; Masutani et al., 1994; Sugasawa, 2010) or the UV-damaged DNA-binding protein (UV-DDB, also known as XPE) (Fitch et al., 2003; Moser et al., 2005; Scrima et al., 2008; Sugasawa et al., 2005), which binds UV lesions specifically and facilitates XPC loading. After initial lesion recognition, both TC-NER and GG-NER rely on the same repair factors to remove the lesion (Mocquet et al., 2008; Naegeli and Sugasawa, 2011; Wood et al., 2000). Briefly, TFIIH complex is recruited to unwind DNA around a lesion and form a DNA bubble (Lainé and Egly, 2006). XPA and RPA bind the lesion-containing and undamaged single-stranded DNA (ssDNA), respectively (Wakasugi and Sancar, 1999), and further stabilize the pre-incision complex. Lesions are excised first on the 5′ side by XPF-ERCC1 and then the 3′ side by XPG (Staresincic et al., 2009). Removal of the ~27-nucleotide (nt) lesion-containing DNA fragment is facilitated by the coupled DNA repair synthesis and ligation (Kemp et al., 2014), which completes NER.

In each DNA-excision repair process, whether base excision, nucleotide excision, or mismatch repair, lesion recognition often requires a verification step to increase repair specificity (Reardon and Sancar, 2004; Yang, 2008). Although RNA polymerases can be stalled by a variety of DNA lesions and XPC binds the undamaged strand opposite either a genuine NER-specific lesion or other DNA damage including mismatched bases and abasic sites (Min and Pavletich, 2007), DNA dual incisions in NER are only efficient in the presence of a bulky DNA base lesion. A mechanism for NER lesion verification appears inevitable after initial binding of XPC or stalling of RNA polymerases. It has been shown that the human NER machinery searches for lesions using a 5′ to 3′ DNA helicase activity (Sugasawa et al., 2009), which suggests an important role of XPD in lesion recognition. Earlier studies of the yeast homolog of XPD (Rad3) suggest that its helicase activity is suppressed by DNA lesions (Naegeli et al., 1993). However, recent studies of archaeal XPD homologs produce conflicting results, suggesting that DNA lesions on the 5′ to 3′ translocated strand either reduce the XPD helicase activity or have no effect (Buechner et al., 2014; Mathieu et al., 2010; Rudolf et al., 2010).

Prior to this report, human TFIIH had not been purified to homogeneity for in vitro studies of its DNA binding, ATP hydrolysis, and DNA unwinding activities. Building on previous knowledge (Sugasawa et al., 2009; Tirode et al., 1999), here we have purified both the ten-subunit holo-enzyme and seven-subunit Core7 of human TFIIH and prepared XPB and XPD ATPase-deficient Core7 proteins. We show that (1) bulky lesions inhibit both the XPB and XPD helicase activities of Core7, (2) XPA modulates the TFIIH helicase activity and further inhibits the Core7 helicase activity in the presence of a bulky lesion, and (3) the CAK module of TFIIH inhibits DNA binding by TFIIH and thus enhance lesion-dependent recruitment of TFIIH by XPC.

RESULTS

Purification of TFIIH Complexes

The human ten-subunit TFIIH and seven-subunit Core7 proteins (Figure 1A) were expressed in insect cells (Experimental Procedures) (Sugasawa et al., 2009). The FLAG tag originally placed at the highly conserved N terminus of XPD (Bedez et al., 2013) was moved to the C terminus to avoid unwanted functional disturbance. The recombinant human TFIIH and Core7 proteins were purified to homogeneity by modification of a previously described procedure (Sugasawa et al., 2009) (Figure 1B). Because XPD helicase contains an iron-sulfur cluster sensitive to oxidation (Pugh et al., 2008; Rudolf et al., 2006), we used a helium-sparging method to remove oxygen from all buffers. This improved the TFIIH stability, purity, and yield in each purification step (Figures 1C and S1). After five chromatographic steps, TFIIH and Core7 were purified to better than 95% homogeneity with yields of 1 mg and 0.6 mg of protein per liter of insect cell culture, respectively (Figure 1B). The integrity of the purified TFIIH complex was confirmed by negatively stained electron microscopic images (Figure S1D), which resembled the cryo-EM structure of yeast and human TFIIH (Gibbons et al., 2012; He et al., 2013; Schultz et al., 2000), and by its activity in an in vitro reconstituted NER assay (Figure S2). X-ray absorption spectroscopy further confirmed the presence of iron in the purified TFIIH (data not shown).

Figure 1. Purification of TFIIH and Core7.

Figure 1

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(A) An illustration of TFIIH complex (Zhovmer et al., 2010) with Core7 colored yellow and CAK blue.

(B) Purified TFIIH and Core7 (5 μg each) were analyzed on a 4%–12% Bis-Tris NuPAGE SDS gel and stained with Coomassie blue.

(C) Samples of Core7 purification steps (5 μg each), crude extract (1), and eluent from Heparin (2), M2 beads (3), HisTrap (4), Superose 6 (5), are shown with the molecular weight markers (M) on a 4%–12% SDS-PAGE gel.

Stimulation of TFIIH ATPase Activity by ssDNA

Purified TFIIH and Core7 complexes exhibited clear ATPase activities (Figure 2A). To examine the effect of DNA on the ATPase activities, ssDNA or double-stranded (ds) DNA (Table S1) was added at 50-fold molar excess to the proteins. The ATPase activity of Core7 responded to DNA stimulation more strongly than that of TFIIH (Figure 2A), suggesting that the CAK module negatively regulates the enzymatic activity of TFIIH. In accordance, the CAK sub-complex has previously been shown to downregulate the helicase activity of XPD (Abdulrahman et al., 2013) and dissociate from TFIIH during NER (Coin et al., 2008).

Figure 2. ATPase Activity of TFIIH and Core7.

Figure 2

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(A) ATPase activities of TFIIH and Core7 were assayed alone or in the presence of 50-fold molar excess of ssDNA (20 nt) or dsDNA (25 bp) (Table S1) and shown in the bar graph.

(B) ATPase activities of Core7 in the presence of ssDNA, dsDNA, or bubble-structured DNA of variant lengths.

(C) ATPase activities of WT, XPBKR, XPDKR, and double KR (2KR) mutant Core7 as protein alone or with different ssDNAs and different bulky lesions.

(D) ssDNA, with or without an AP lesion, stimulated the Core7 ATPase equally. Standard deviations were estimated based on at least triplicates.

To avoid the negative impact of CAK on TFIIH and complications of the ATP-dependent kinase activity of CAK, Core7 alone was tested for ATPase activity in the presence of different lengths of ssDNA, dsDNA, and DNA bubbles (Figure 2B and Table S1). With ssDNA, at least 15 nt in length was required to stimulate the Core7 ATPase activity, and with dsDNA, 20 base pairs (bp) was the minimal length to have a stimulatory effect. For the same length, ssDNA was more effective than dsDNA in activating the Core7 ATPase activity. The dominant effect of ssDNA was confirmed with DNA bubbles. When the overall DNA length was fixed at 50 nt, the level of ATPase stimulation increased with the size of the bubble (Figure 2B).

DNA with a Cisplatin or Cy5 Adduct Inhibits the Core7 ATPase Activity

To examine whether NER substrates (bulky DNA lesions) have any effect on the Core7 ATPase activity, we prepared a 44-nt ssDNA containing an intrastrand cisplatin [1,2] dGpdG cross-linked lesion (Pt-44, CisPt) and a 45-nt ssDNA with two internal adjacent guanines replaced by the fluorescent probe Cy5 (Cy5-45) mimicking a bulky DNA lesion (Table S2). For comparison, ss44 and ss45 with no lesion or with an abasic site (AP) were also prepared. The Core7 ATPase activity was activated to different levels by ss44 and ss45 (Figure 2C), likely due to their different GC contents (Table S2). Compared to DNAs with the same sequences, the presence of Cy5 or CisPt reduced the Core7 ATPase activity to 45% and 33%, respectively, (Figure 2C). Interestingly, an AP lesion, which is not a preferred NER substrate, did not have measurable effect on the ATPase activity of Core7 (Figure 2D).

Differential Activation of XPB and XPD by ssDNA

To determine how XPB and XPD each contribute to the Core7 ATPase activity, the Lys in the Walker A motif was replaced by Arg in XPB (K346R) and XPD (K48R) to eliminate ATP hydrolysis, and three Core7 variants containing either XPBKR, XPDKR, or both (2KR) were expressed and purified (Figure S3). In the absence of DNA, neither 2KR nor XPBKR mutant Core7 had detectable ATPase activity, and the ATPase activity of the XPDKR mutant Core7 appeared equal to that of wild-type (WT) (Figure 2C). However, in the presence of DNA, the ATPase activities of XPBKR and XPDKR mutant Core7 were similar (Figure 2C). The addition of normal ssDNA (ss44 or ss45) increased the ATPase activity of WT Core7 by 15- to 20-fold and the XPB ATPase (XPDKR) by only 4- to 5-fold. In contrast, the XPD ATPase activity (XPBKR) entirely depended on DNA. When DNA lesions Cy5 (Cy5-45) or CisPt (Pt-44) were introduced, the ATPase activities of WT and the XPBKR mutant Core7 (XPD) were reduced by 50%–60% compared to undamaged ssDNA. Surprisingly, the XPB ATPase activity (XPDKR) was also reduced by 25%– 45% by the presence of Cy5 or CisPt, although previously only XPD homologs have been shown to respond to DNA lesions (Buechner et al., 2014; Mathieu et al., 2010; Naegeli et al., 1993). In the context of Core7, the levels of inhibition of XPB and XPD by bulky lesions varied within 2-fold. We conclude that the presence of a bulky lesion impedes the ATPase activities of both XPB and XPD.

Core7 Prefers to Bind ssDNA With or Without Lesion

Stimulation of the Core7 ATPase activity by ssDNA could result from simple DNA binding or active translocation along the DNA, and the inhibitory effect of DNA lesions may be due to weakened binding of Core7 to damaged DNA or hindered DNA translocation. We first examined Core7 binding to DNA by electrophoretic mobility shift assay (EMSA). Core7 preferred to bind ssDNA (ss45) and had a very low affinity for blunt-ended dsDNA (ds45) (Table S1 and Figure 3A). The binding affinity of Core7 for ssDNA increased with DNA length (Figures 3B and 3C), from not detectable at 15 nt to stable Core7-DNA complexes at 25 nt with a dissociation constant (KD) of 7.6 ± 0.9 nM. We note that the length of ssDNA required for the protein-DNA complex formation mirrored the activation of the Core7 ATPase activity (Figure 2B). When the length of ssDNA exceeded 30 nt, a higher-molecular-weight protein-DNA complex (complex 2) was detected, consistent with a second Core7 binding to the DNA substrate (Figures 3A and 3B). With ss45, the KD of complexes 1 and 2 were estimated to be 2.5 ± 0.2 nM and 12.4 ± 1.0 nM, respectively. Interestingly, the presence of DNA lesions did not alter the binding affinity of Core7 for ssDNA (Figures 3D and 3E). Moreover, the addition of ATP, which likely leads to DNA translocation, resulted in little to no change in Core7 binding to ssDNA, with or without lesions (Figures 3D and 3E). This might be because the EMSA assays were conducted under equilibrium conditions with ATP turnover. We conclude that the reduced ATPase activity of Core7 in the presence of bulky lesions is not due to reduced protein-DNA interactions.

Figure 3. EMSA of DNA Binding by Core7.

Figure 3

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(A) Core7 has different affinity for ssDNA (45 nt, ss45) and dsDNA (45 bp, ds45). The 5′-32P-labeled DNAs (0.5 nM) were incubated with Core7 (0, 0.25, 0.5, 1, 2, 4, 8, 16, and 20 nM). Two protein-DNA complexes were detected with ss45 (1 and 2).

(B) Different lengths of 32P-labeled ssDNAs (0.5 nM) were incubated with Core7 (0, 0.5, 4, and 20 nM). The longer the ssDNA used, the more the protein-DNA complexes formed.

(C) The same EMSA assays as shown in (A) were performed for ssDNA of variant lengths, and the results were quantified and plotted. Dissociation constants (KD) were calculated by fitting the EMSA data with the non-linear regression model (Prism) and shown in the table next to the plot. Error bars were estimated from triplicate measurements.

(D and E) Effects of Cy5 (D), CisPt (E), and ATP on DNA binding by Core7. Core7 (0.5, 1, 4, and 20 nM) and ssDNA were incubated with or without ATP and MgCl2 and analyzed by EMSA. The percentage of Core7-DNA complexes in each lane was quantified and indicated below the gels.

Bulky DNA Lesions Inhibit the Core7 Helicase Activity

Since bulky DNA lesions do not affect Core7 binding to ssDNA, we next examined whether these lesions altered the Core7 heli-case activity. CisPt or Cy5 was placed on either the long strand with an overhang (labeled as TOP) or the short complementary strand (labeled as BOT) in the 34–35 bp duplex region and examined for strand separation by Core7 (Figure 4 and Table S2). For comparison, DNAs with a mismatch or AP site were also tested (Table S2). Unwinding by WT Core7 was detected on normal DNA duplexes with a 5′ overhang, but not with a 3′ overhang (Figures 4A–4D). The lack of helicase activity on DNA with a 3′ overhang was not due to the absence of Core7 binding (Figure S4). We suggest that a 5′ overhang properly orients the Core7 relative to the DNA duplex so that its helicase activity is productive. The weak unwinding activity observed with a Cy5 placed on a 3′-overhanged substrate near the double- and single-stranded junction is likely a result of the unpaired 5′ end due to the lesion (Figure 4D).

Figure 4. Bulky Lesions on the 5′ Overhang Strand Impede the Core7 Helicase Activity.

Figure 4

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(A) DNA unwinding activities of Core7 on a normal 5′ overhang DNA (N_45/35), with Cy5 on the top strand (Cy5_TOP), a mismatched base pair (M_45/35), or Cy5 on the bottom strand (Cy5_BOT). DNA substrates are diagramed at the bottom of each panel, and asterisks indicate 5′-32P labels. Core7 concentrations were 10, 20, 40, and 80 nM in each assay. Triangles (Δ) indicate the heat-denatured substrates, and the “C” stands for control of DNA substrate alone.

(B) Effects of CisPt on the Core7 helicase activity were compared by placing the lesion CisPt on the top (Pt_TOP) or the bottom strand (Pt_BOT) of the normal DNA duplex N_44/34 or N_54/44, respectively, each with a 5′ overhang (Table S2).

(C) DNA unwinding activities of Core7 with an AP lesion on the top (AP_TOP) or bottom strand (AP_BOT).

(D) Unwinding of DNA with a 3′ overhang by Core7 was undetectable. The Cy5 near the ds-ss junction promoted DNA unwinding, perhaps by melting DNA locally and generating a free 5′ end.

(E) The XPDKR mutant Core7 had very weak helicase activity and was inhibited by CisPt present on the strand scanned by XPD. To detect the weak activity, protein concentrations were increased to 80–320 nM.

(F) The XPBKR mutant Core7 had slightly reduced helicase activity compared with WT.

(G) Relative activities of WT Core7 in unwinding DNAs with a 5′ overhang. Helicase activities (%) were normalized to the corresponding undamaged DNA substrates and plotted in the same colors as DNA diagrams shown in (A)–(C). Standard deviations were estimated based on triplicates.

The presence of Cy5 or CisPt on the strand with a 5′ overhang, on which XPD translocated in the 5′ to 3′ direction, significantly reduced the DNA unwinding activity (Figure 4A left and 4B left). However, the inhibition of helicase activity was not observed when these bulky lesions were placed on the complementary strand (Figure 4A right and 4B right). Inclusion of an AP site in the DNA substrate had no detectable effect on the Core7 heli-case activity (Figure 4C), which is consistent with the ATPase activity measurements (Figure 2D). Inclusion of a mismatched base pair led to a slight increase in strand separation by Core7 (Figure 4A), likely due to the reduced duplex stability. The relative helicase activities of WT Core7 on different DNA substrates were quantified and shown in Figure 4G. The presence of Cy5 and CisPt lesions on the strand translocated (scanned) by XPD reduced the helicase activity of Core7 to 45% and 30%, respectively, which closely matched the reduction of Core7 ATPase activities (Figure 2C). The reduced DNA unwinding by these bulky lesions was verified with XPBKR mutant Core7 (Figure S5A). We conclude that DNA translocation in the 5′ to 3′ direction by the XPD helicase of Core7 was impeded by the presence of NER substrate Cy5 and CisPt.

Interestingly, DNA unwinding was also observed with the XPDKR mutant Core7 on a 5′ overhang substrate, and the very weak helicase activity, presumably due to XPB unwinding DNA in the 3′ to 5′ direction, was inhibited by the CisPt present on the strand scanned by XPD (Figure 4E). The weak helicase activity of XPDKR mutant Core7 was also inhibited by the Cy5 lesion (Figures S5B–S5D). The notion that XPB participated in unwinding of DNA after Core7 loading onto a 5′ overhang was corroborated by the reduced DNA unwinding by the XPBKR mutant Core7 compared with the WT Core7 (Figure 4F). Moreover, lesion-induced inhibition of the XPDKR Core7 helicase activity explains why its DNA-dependent ATPase activity was reduced by the presence of bulky lesions on ssDNA (Figure 2C).

Based on these observations, we conclude that (1) Core7 is loaded and properly oriented on DNA via a 5′ overhang; (2) XPD is the dominant helicase that translocates along ssDNA in the 5′ to 3′ direction; (3) once Core7 is loaded onto DNA via a 5′ overhang, the 3′ to 5′ helicase activity of XPB contributes to DNA unwinding, albeit weakly; (4) a bulky lesion on the strand translocated by XPD stalls both XPB and XPD helicase of Core7; and (5) the “lesion-binding” pocket that stalls XPB trans-location can function without ATP hydrolysis by XPD. Analogous to yeast Rad3 and archaeal XPD (Buechner et al., 2014; Mathieu et al., 2010; Naegeli et al., 1993), we suspect that the presence of a bulky lesion is “sensed” by XPD, and lesion detection by XPD is communicated to XPB via protein-protein interactions, either directly or mediated by other components in Core7, thus resulting in the inhibition of the ATPase and helicase activities of both XPB and XPD (Figures 2C and 4).

XPA Enhances Lesion-Dependent Inhibition of the Core7 Helicase Activity

The inhibition of Core7 helicase activity by lesions on the strand scanned by XPD provides a verification mechanism after initial lesion recognition by XPC or RNA polymerases. However, if the lesion is on the other strand, additional factors may be needed for lesion verification. The NER factor XPA has been shown to play a role in lesion recognition (Reardon and Sancar, 2003; Wakasugi and Sancar, 1999). In addition, during unwinding of DNA around a damaged site by TFIIH, XPA and ssDNA-binding protein RPA further extend the bubble region and protect the undamaged strand (Coin et al., 2008). We thus tested XPA in the Core7 helicase activity assay. The helicase activity of Core7 on normal DNA was significantly enhanced with increasing concentrations of XPA, but little stimulation by XPA was observed in the presence of CisPt on the strand scanned by XPD (Figure 5A). The reduced XPA stimulation was also observed when CisPt was on the opposite strand that was not scanned by XPD (Figure 5B). Moreover, in the presence of XPA, an AP lesion also became distinguishable from normal DNA by its ability of reducing the Core7 helicase activity (Figure 5C). In the presence of a 1:1 molar ratio of XPA to Core7, XPA stimulated the Core7 helicase activity on normal DNA by ~4-fold, but reduced DNA unwinding by Core7 when CisPt was present on either strand (Figures 5D and 5E). Quantitative analysis shows that XPA doubles the inhibition of the Core7 helicase activity by a bulky lesion, regardless of which strand the lesion is on (Figures 5D and 5E).

Figure 5. Inhibition of the Core7 Helicase Activity by Bulky Lesion and XPA.

Figure 5

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(A) XPA significantly enhanced Core7 helicase activities on a normal DNA (N_44/34), but had little stimulatory effect when CisPt was present on the top strand scanned by XPD (Pt_TOP).

(B) XPA had no stimulatory effect when CisPt was present on the bottom strand (Pt_BOT) either.

(C) AP lesions also inhibited the stimulatory effect of XPA on the Core7 helicase. Consistent results were obtained in multiple experiments with 1 or 2 nM DNA, and the latter is shown. Relative heli-case activities of Core7 in the presence of increasing amounts of XPA are plotted beneath each gel in (A)–(C).

(D and E) Comparison of DNA unwinding by Core7 alone or Core7 and XPA together at 1:1 molar ratio with or without DNA lesion. CisPt was on the strand scanned by XPD (D) or the complementary strand (E), and XPA enhances lesion-dependent stalling of Core7 by ~2-fold in both cases. Relative helicase activities are shown in the bar graph beneath. Error bars were estimated from triplicate measurements.

CAK Inhibits DNA Binding by TFIIH

To determine whether the differences observed between mutant and WT Core7 and between Core7 and TFIIH have an impact on NER, the four TFIIH variants were examined in the reconstituted dual-incision assay (Sugasawa et al., 2009) (Figure 6A). As expected, XPBKR and XPDKR mutant Core7 proteins are both inactive, agreeing with previous reports that both ATPase activities are essential for NER. Although the TFIIH ATPase activity is lower than Core7 in the presence of ssDNA (Figure 2A), WT Core7 and TFIIH activities are comparable in the dual-incision assay (Figure 6A).

Figure 6. Effects of CAK on DNA Binding, Unwinding, and Dual Incision.

Figure 6

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(A) TFIIH, WT, and two mutant Core7 were assayed for their ability to activate dual incisions in the reconstituted NER reaction with a 6-4 photoproduct (6-4 PP). The amount of each protein was equalized based on anti-XPB immunoblotting (IB).

(B) EMSA of ssDNA binding by TFIIH and WT Core7 (0, 0.25, 0.5, 1, 2, 4, 8, and 16 nM). The black arrow indicates protein-DNA complexes of Core7 and TFIIH.

(C) DNA helicase activities of Core7 and TFIIH in the presence and absence of XPA.

(D) EMSA examination of Core7 and TFIIH recruitment by XPC/RAD23B/Centrin-2 to a preformed DNA bubble (bubble7, Table S1). DNA complexes with XPC, TFIIH, or Core7 are marked. Supershift indicates the XPC-TFIIH-DNA and XPC-Core7-DNA complexes.

To investigate the role of the CAK module, we compared DNA binding and helicase activities of TFIIH and Core7. At equal molar concentrations (Figure S3), TFIIH has a much lower affinity for ssDNA than Core7 (Figure 6B) and reduced DNA unwinding activity with or without XPA (Figure 6C). CAK appeared to inhibit ssDNA binding by TFIIH and consequently reduced its helicase and DNA-dependent ATPase activities. However, the inhibitory effect of CAK was overcome in the XPC-dependent dual incisions of NER (Figure 6A). Previous studies suggest that TFIIH is associated with DNA independent of lesions in normal cells, and upon UV-induced lesion formation TFIIH relocates to lesion sites and CAK dissociates from TFIIH (Coin et al., 2008). We thus examined dsDNA binding by Core7 and TFIIH and found that they formed different protein-DNA complexes, and TFIIH again had lower affinity than Core7 (Figure S6). Nevertheless, in the presence of a preformed DNA bubble (Table S1), the trimeric XPC significantly increased TFIIH loading onto DNA by forming an XPC-TFIIH-DNA complex, but had limited stimulatory effect on Core7 (Figure 6D).

DISCUSSION

Human TFIIH was partially purified previously by affinity chromatography and shown to have NER and transcription activities (Abdulrahman et al., 2013; Coin et al., 2007; Schultz et al., 2000). Earlier in vitro reconstituted NER assays are indicative that the 5′ to 3′ translocation activity of XPD in TFIIH scans DNA and locates lesions for repair (Sugasawa et al., 2009). By removing soluble oxygen in buffers to keep the iron-sulfur cluster in XPD intact and increasing chromatographic steps to eliminate protein and nucleic acid contaminants, we purified human TFIIH and Core7 to homogeneity, which have significantly improved ATPase, DNA binding, and helicase activities compared to those in previous studies (Abdulrahman et al., 2013).

Our analyses of Core7 show that a 25-nt ssDNA, which matches the size of DNA fragments that are excised in NER, is the preferred binding substrate and most effective in activating its ATPase activity (Figures 2 and 3). As expected, substitution of the Lys in the Walker A motif by Arg in XPB and XPD (2KR) eliminate the ATPase and helicase activities of Core 7 (Figures 2C and S7). The low residual ATPase activity of the 2KR mutant Core7 in the presence of ssDNA was likely due to a trace amount of contaminants. The ATPase activities in the single KR mutant Core7 proteins have to be due to the WT XPB or XPD. Although XPB is the dominant ATPase in the absence of DNA (Figure 2C), the ATPase activities of XPB and XPD are nearly equal in the presence of DNA. Surprisingly, the presence of bulky lesions in ssDNA reduced the ATPase activity of XPB (XPDKR) as well as that of XPD (XPBKR) in Core7.

Growing evidence suggests that the 5′ to 3′ helicase XPD is involved in lesion verification (Araki et al., 2001; Buechner et al., 2014; Buschta-Hedayat et al., 1999; Mathieu et al., 2010; Naegeli et al., 1993; Sugasawa et al., 2009). Several archaeal homologs of XPD have been characterized extensively in vitro. Crystal structures of archaeal homologs of XPD reveal a channel of 8–13Å in diameter between two RecA-like domains and the unique ARCH domain that is sufficient to accommodate ssDNA (Fan et al., 2008; Liu et al., 2008; Wolski et al., 2008). A bulky lesion on the translocated strand may sterically block the channel and thus stall XPD. However, the helicase activity of Sulfolobus tokodaii XPD (stXPD) is unaltered in the presence of lesions (Rudolf et al., 2010), which is likely due to its unstable iron-sulfur domain and an enlarged DNA-binding channel (Liu et al., 2008). In addition to the steric blocking effect of a bulky lesion, it has also been proposed that the iron-sulfur cluster may directly play an important role in damage recognition (Sontz et al., 2012; White, 2009).

In this study we find that XPB also contributes to DNA unwinding and lesion-induced stalling of TFIIH. The apparent 5′ to 3′ direction of the Core7 helicase activity is determined by the requirement of a 5′ overhang for Core7 loading, which leads to the correct orientation not only for the dominant motor XPD but also for the weak motor XPB, so that XPB can translocate in the 3′ to 5′ direction on the complementary strand. The weak 3′ to 5′ helicase activity of XPB is detectable only when Core7 is loaded in the correct orientation (Figures 4E and 4F). The ATPase activity of XPB, however, is essential for lesion-dependent DNA dual incisions in vitro (Figure 6A) and NER in vivo (Coin et al., 2007; Fan et al., 2006; Oksenych et al., 2009). We further show that in the context of Core7, detection of a bulky lesion by XPD, which does not require the XPD ATPase activity, can be communicated to XPB, leading to stalling of both motors (Figures 4A–4E). As a result, the ATPase and helicase activities of both XPB and XPD are strongly inhibited by bulky lesions like CisPt and Cy5 on the XPD-scanned strand, but only weakly or not at all by AP lesions and mismatched base pairs. The very weak inhibitory effect of AP lesions observed with XPA (Figure 5C) is in agreement with the in vivo repair of abasic lesions by TC-NER when the preferred base-excision pathway is impaired (Guo et al., 2013; Kim and Jinks-Robertson, 2010). The level of Core7-helicase inhibition appears to correlate with the lesion excision and NER efficiency.

Using the highly purified Core7 and ten-subunit TFIIH, we also uncovered additional roles of XPA and CAK in NER. First, XPA exhibits lesion-dependent differential effects on the Core7 helicase activity. XPA enhances Core7 translocation along undamaged DNA as it scans for lesions (Figure 5). When a bulky lesion is detected, XPA enhances the stalling of the Core7 helicases whether the lesion is on the XPD-scanned strand or the opposite strand “unseen” by XPD. Recently, RPA is shown to interact directly with the TTDA (p8) subunit of TFIIH (Ziani et al., 2014). The differential stalling of Core7 by XPA may not be sufficient to change the outcome of dual incisions when a lesion is not on the XPD-scanned strand (Sugasawa et al., 2009), but it provides a mechanism for XPA to function in lesion recognition and verification (Reardon and Sancar, 2003; Wakasugi and Sancar, 1999). Second, the CAK module, which has previously been reported to inhibit the XPD helicase activity (Abdulrahman et al., 2013; Coin et al., 2008), effectively prevents DNA binding by TFIIH (Figure 6), indicating that TFIIH has to be recruited for its participation in transcription and NER. The inhibitory effect of CAK is in part overcome by XPC-dependent TFIIH recruitment (Figure 6D) and possibly also by other NER factors (Coin et al., 2008; Kemp et al., 2014), so TFIIH and Core7 are comparable in the dual DNA incision assay (Figure 6A). Inhibition of TFIIH from non-specific DNA binding by CAK shifts the regulation of NER one step before engaging the helicases and thereby enhances the specificity of lesion-dependent strand separation.

To account for a broad substrate range and yet bulky lesionspecific dual incisions in NER, we hypothesize that TFIIH binds chromosomal DNA weakly and non-specifically in the absence of NER lesions (Coin et al., 2008) (Figure S6), but has no unwinding activity owing to a different binding mode and inhibitory effects of CAK (Figure 7A). In the presence of a lesion, TFIIH is either recruited by XPC (Figure 6D) or brought along by an RNA polymerase (Assfalg et al., 2012; Spangler et al., 2001) to the partially unwound lesion site and loaded 5′ to the lesion (Sugasawa et al., 2001, 2009) (Figure 7B). XPD and XPB begin to translocate in parallel on both strands and unwind DNA toward the lesion. Directional DNA unwinding by TFIIH is unaltered by the presence of small base lesions or unpaired DNA loops and accelerated by XPA (Figure 7C). When TFIIH encounters a bulky lesion on the strand scanned by XPD, both motors of XPB and XPD are stalled, and XPA enhances the stalling (Figure 7D). After dissociation of CAK (Coin et al., 2008), Core7 and XPA together demarcate a lesion-containing DNA bubble structure with the help of RPA (Krasikova et al., 2010; Mer et al., 2000), and XPF-ERCC1 and XPG are recruited to make dual incisions. NER is completed by repair-coupled DNA synthesis, which also facilitates incision by XPG and lesion removal.

Figure 7. A Model of Tripartite Lesion Recognition and Verification.

Figure 7

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(A) TFIIH is associated with chromatin weakly in a non-specific manner without DNA lesion.

(B) In the presence of DNA lesions (red hexagon), TFIIH is loaded onto the DNA by XPC or RNA Pol II in a strand- and orientation-specific manner. It scans both DNA strands in parallel with XPD and XPB helicases.

(C) Translocation of TFIIH on normal DNA is accelerated by XPA. Small base lesions and mismatches (small red triangle), which are substrates for base excision and mismatch repair, respectively, behave like normal DNA.

(D) Upon detection of a bulky lesion (red star) on the strand scanned by XPD, both XPB and XPD helicases are stalled by the lesion. XPA enhances the stalling and hence bulky-lesion recognition. After dissociation of CAK, Core7, RPA, and XPA stabilize the bubble structure around the lesion and recruit XPF and XPG to make sequential dual incisions. Repair-coupled DNA synthesis can facilitate incision by XPG and lesion removal.

EXPERIMENTAL PROCEDURES

Protein Expression and Purification

The recombinant human TFIIH complex was expressed in Sf9 cells as described (Sugasawa et al., 2009) with the following modifications. The FLAG tag on XPD was moved from the N to the C terminus in the pFastBac-dual- FLAG-XPD-p8 construct. For TFIIH production, Sf9 cells in suspension were co-infected with five baculoviruses. To produce Core7, the baculovirus encoding CDK7, cyclin H, and MAT1 was omitted. For TFIIH and Core7 purification, all buffers were freshly prepared and degassed with helium sparging. Cell pellets from 4 liters of culture were resuspended in 200 ml extraction buffer (20 mM HEPES-Na [pH 7.0], 250 mM KCl, 1 mM EDTA, 20% glycerol, 0.1% NP-40, 10 mM β-mecaptoethanol [βME], 10 μg/ml Leupeptin, 10 μg/ml Pepstatin A, and 2 mM AEBSF) and lysed by EmulsiFlex-C5 (Avestin). After clearance, cell lysate was loaded onto DEAE (80 ml) and Heparin (50 ml) columns in tandem. After extensive wash, TFIIH and Core7 bound to Heparin were eluted in HP_800 buffer (20 mM HEPES [pH 7.0], 800 mM KCl, 0.1% NP-40, 1 mM EDTA, 20% glycerol, and 10 mM βME). The Heparin eluents were immediately diluted five times with M2 buffer without salt (20 mM HEPES-Na [pH 7.9], 20% glycerol, and 0.01% Triton X-100) and mixed with 6 ml anti-FLAG M2 agarose beads (Sigma) at 4°C. After overnight incubation and thorough wash with 150 mM KCl in M2 buffer, TFIIH and Core7 were eluted by 0.2 mg/ml 1X-FLAG peptide (Sigma). The eluents were further purified with a HisTrap column (1 ml, GE Healthcare Bioscience) followed by a Superose 6 column (GE Healthcare Bioscience) and stored in 20 mM Tris-HCl (pH 7.4), 250 mM NaCl, 1 mM TCEP, and 40% glycerol at 80°C.

XPA Preparation

Human XPA was cloned into pET32a vector (Novagen) with a cleavable TRX-6XHis-S tag at the N terminus and expressed in Rosetta2 cells (Invitrogen). Protein expression was induced by the addition of 0.5 mM IPTG and 50 μM ZnCl2 at 15°C for 16 hr. The cells were lysed by EmulsiFlex-C5. XPA was purified using HisTrap columns (GE Healthcare) before and after removing the affinity tag by PreScission Protease. The tag-free XPA was further purified with Mono Q and Superdex 200 columns (GE Healthcare). XPA peak fractions in 20 mM Tris (pH 7.4), 150 mM NaCl, 25 μM ZnCl2, 1mM DTT were pooled and concentrated to 10 mg/ml for storage at −80°C. For activity assays, XPA was thawed and purified over a Superdex 75 column (GE Healthcare) in 25 mM Tris (pH 7.5), 150 mM NaCl, 2 mM DTT, and 5% glycerol.

DNA Substrates

Oligonucleotides were synthesized by the Foundation for Biomedical Research (FBR), gel purified, and 5′-32P-labeled. The Cy5 oligos were synthesized by FBR and gel purified. CisPt oligos were prepared as described (Lee et al., 2014). All oligonucleotides used in this study are listed in Tables S1 and S2. The internally 32P-labeled DNA substrate containing a site-specific 6-4PP for the in vitro dual incision assay was prepared as described (Sugasawa et al., 2001).

ATPase Assay

ATPase activity was measured in a 15 μl reaction buffer containing 20 mM Tris-HCl (pH 7.4) 50 mM KCl, 1 mM DTT, 5 mM MgCl2, 0.1 mg/ml BSA, 5% glycerol, 2 mM [α-32P] ATP (Perkin Elmer), and 20 nM TFIIH or Core7. For DNA-dependent ATPase activity, 1 μM DNA substrate (Table S1) was added. The reactions were incubated at room temperature (normal DNA) or 37°C (lesion containing DNA) for 2 hr before analysis using PEI-Cellulose TLC plate (Grace Discovery Sciences) and quantification by ImageQuant TL. Standard deviations were estimated based on triplicate or more measurements.

EMSA Assay

Core7 or TFIIH was incubated with 0.5 nM 5′-32P-labeled DNA in 20 μl buffer containing 20 mM Tris-HCl (pH 7.4), 50 mM KCl, 1 mM DTT, 0.1 mg/ml BSA, 0.1 mM EDTA, 0.05% NP-40, and 8% glycerol (± 2 mM ATP and MgCl2) for 30 min at 30°C and electrophoresed on a 4% native gel in 1X TGE buffer at 4°C. Standard gel development and analysis were applied. Assays with the trimeric XPC were performed essentially as described (Nishi et al., 2013; Sugasawa et al., 2001). XPC and Core7 or TFIIH (each at 22 nM) and 0.35 nM 5′-32P-labeled bubble7 DNA (Figure 2B and Table S1) were mixed and incubated at 30°C for 1 hr, and the protein-DNA complexes were stabilized by addition of 1 μl of 5% glutaraldehyde and incubation at 30°C for 3 min before EMSA analysis.

Helicase Activity Assay

Helicase activities were measured in 20 μl buffer containing 20 mM Tris-HCl (pH 8.0), 50 mM KCl, 1 mM DTT, 5 mM MgCl2, 5 mM ATP, 0.1 mg/ml BSA, 5% glycerol, 1 nM 32P-labeled DNA (Table S2), and 10–80 nM Core7 for 30 min at 37°C. The XPA effects on Core7 (80 nM) were measured with varying concentrations (0, 20, 40, 80 nM) for 60 min at 37°C. Reaction products were analyzed on a 10% native TBE gel and quantified using ImageQuant TL.

In Vitro NER Dual Incision Assay

In vitro NER dual incision assays were performed as described previously (Nishi et al., 2013).

Supplementary Material

1
2

Highlights.

  • Human ten-subunit TFIIH and its seven-subunit Core7 were purified to homogeneity

  • Bulky lesions in DNA inhibit both the XPB and XPD helicase activities of Core7

  • XPA further inhibits the Core7 helicase activity in the presence of a bulky lesion

  • The CAK module enhances lesion-dependent recruitment of TFIIH by XPC in NER

ACKNOWLEDGMENTS

We thank Dr. Mikalai Lapkouski for help with negative staining electron microscopy; Lindsay Wise for purified XPA protein; and Drs. Bob Craigie, Dan Leahy, and Marty Gellert for critical reading of the manuscript. This work is funded by NIH intramural program (DK075037-06), and Grants-in-Aid for Scientific Research from JSPS and MEXT, Japan.

Footnotes

AUTHOR CONTRIBUTIONS

C.-L.L. and F.M.G. carried out most of the experiments, Y.O. did in vitro dual incision assays, N.L.S. prepared XPA protein, W.Y. and K.S. conceived the project, W.Y. supervised experimental design and interpretation, and all authors were involved in manuscript preparation.

SUPPLEMENTAL INFORMATION

Supplemental Information includes seven figures and two tables and can be found with this article online at http://dx.doi.org/10.1016/j.molcel.2015.08.012.

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Supplementary Materials

1
NIHMS717692-supplement-1.pdf (2.6MB, pdf)
2
NIHMS717692-supplement-2.pdf (3.2MB, pdf)

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