Ordered Conformational Changes in Damaged DNA Induced by Nucleotide Excision Repair Factors^*

Cloning and Vector Constructions

The XPA, ERCC1, XPF, HR23B, and XPC cDNAs were amplified from a human cDNA library and inserted in the pVL1392 vector (Pharmingen). A His₆ tag was added N-terminally to XPA and XPF. The resulting vectors were recombined with baculovirus DNA (BaculoGold DNA, Pharmingen) in Sf9 cells, and viral stocks were prepared by a three-step growth amplification procedure. The human RPA and XPG cDNAs were cloned into expressing vectors (22, 23).

Purification of the Repair Factors

XPF/ERCC1 complex purification was performed as described previously (14). Briefly, Sf9 cells were infected with ERCC1 and XPF-His-tagged baculoviruses. Cells extracts were prepared in buffer A (20 mM Tris-HCl, pH 7.9, 0.15 M NaCl, 20% glycerol, 0.1% Nonidet P-40, 5 mM MgCl₂, 5 mM β-mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride, and 1× protease mixture inhibitor) and centrifuged (15,000 rpm for 40 min at 4 °C). The supernatant was dialyzed for 4 h against buffer B (same as buffer A except for 5% glycerol, 0.02% Nonidet P-40, and 0.5 mM dithiothreitol) and applied to a phosphocellulose column (P11; Whatman), and the flow-through was then applied to a heparin Ultrogel column (Sepracor, Villeneuve la Garenne, France). Following extensive washes, the protein complexes were eluted at 0.6 M NaCl and further incubated for 4 h with 500 μl of the metal affinity resin (Talon; Clontech) in the presence of imidazole (1 mM). After sequential washing with buffer B containing 0.6 M NaCl and 1 mM imidazole, 5 mM imidazole, and, finally, 0.1 M NaCl and 20 mM imidazole, the complex was eluted in buffer B containing 0.1 M NaCl and 0.2 M imidazole. Mutants XPF-D676A and XPF-D720A were purified according to published procedures (15). The XPC/HR23B complex or its individual components (XPC and HR23B) were expressed and purified from insect cells (24). The recombinant heterotrimeric RPA complex was produced in Escherichia coli and purified (22). Recombinant XPG was produced in insect cells and purified as described (23). All of the NER factors were dialyzed against buffer C (50 mM Tris-HCl, pH 7.8, 10% glycerol, 0.1 mM EDTA, 0.5 mM dithiothreitol, and 50 mM KCl).

XPA insect cell extract was applied on a heparin Ultrogel resin (Sepracor) pre-equilibrated in buffer A and washed with buffer A containing 0.3 M NaCl. XPA was then eluted in buffer A containing 0.5 M NaCl, dialyzed for 3 h against buffer D (50 mM Tris-HCl pH 7.8, 10% glycerol, and 0.3 M KCl) and incubated for 2 h at 4 °C with 200 μl of metal affinity resin (Talon; Clontech). After packing the column, the resin was subsequently washed with buffer D containing 20 mM imidazole and 50 mM imidazole. XPA was eluted in buffer D containing 0.1 M imidazole and next with buffer D containing 0.1 M EDTA. The fractions were pooled and dialyzed against buffer C. TFIIH was purified from HeLa cells (25).

Dual Incision Assay

The single cisplatin lesion (Pt-GTG) plasmid, called 105.TS (26), was used for dual incision (7) in a 15-μl reaction mixture containing XPC/HR23B (10 ng), XPA (25 ng), RPA (50 ng), XPG (7.5 ng), XPF/ERCC1 (6.25 ng), and TFIIH (20 ng) in the presence of 2 mM ATP. Following 10 min of pre-incubation at 30 °C, 30 ng of the Pt-GTG plasmid was added, and the reactions were further incubated for 90 min. The excised fragment was detected on 14% urea-PAGE after annealing with the complementary oligonucleotide and the addition of four radiolabeled [α-³² P]dCMP residues by Sequenase V2.1 (USB) (26).

KMnO₄ Footprinting Assays

The damaged strand probe was obtained upon AgeI digestion of the 105.TS plasmid (26) and radiolabeling at the 3′end in a Klenow reaction, the Pt adduct being located at 156 bp from the labeled end. For the non-damaged strand, 105.TS was digested by BssHII, labeled, and then digested by HindIII. The resulting 165-bp fragment in which the lesion is located 92 bp from the labeled 3′ end was purified by the “crush and soak “method after migration in a 5% nondenaturating PAGE (27).

Reactions (75 μl) were carried out in 20 mM Hepes/KOH, pH 7.6, 60 mM KCl, 5 mM MgCl₂, 10% glycerol, 1 mM dithiothreitol, 0.3 mM EGTA, 0.4% polyvinyl alcohol, and 0.4% polyethylene glycol 10000 buffer containing the labeled cisplatinated probe (40 fmol) and, when indicated, 5 mM ATP, XPC/HR23B (40 ng), XPA (80 ng), RPA (60 ng), XPG (10 ng), XPF/ERCC1 (6.25 ng), and TFIIH (40 ng). After incubation at 30 °C for 15 min, 3 μl of 120 mM KMnO₄ was added, and oxidation was allowed to proceed for 3 min at room temperature before reduction by adding 6 μl of 14.6 M β-mercaptoethanol for 5 min in ice. After organic extraction and ethanol precipitation, dried pellets were resuspended in 100 μl of a solution containing 1 M piperidine, 1 mM EDTA, and 1 mM EGTA and incubated at 90 °C for 25 min. Samples were next ethanol precipitated, and final pellets were recovered in 10 μl of loading buffer and analyzed in 8% urea PAGE (damaged strand probe) or in 12% urea PAGE (undamaged strand probe). In parallel, dideoxy sequencing reactions were performed.

Protein-DNA Photo Cross-linking

The synthesis of the photoreactive nucleotide AB-dUTP, complex assembly, and analysis of the photo cross-linked polypeptides were done as described previously (28, 29). For the synthesis of the photoprobes that place two AB-dUMPs on the 3′ side of the cisplatin (photoprobes +12/+15 and +21/+22), we used cisplatinated oligonucleotides of various lengths designed to allow incorporation at the appropriate positions (details are provided the legend to Fig. 6A). For the synthesis of the photoprobes that place two AB-dUMPs on the 5′ side of the cisplatin (photoprobes −23/−20 and −8/−7), we used both a cisplatinated oligonucleotide and an additional 5′ oligonucleotide for directing the incorporation of AB-dUMP. Fill-in with DNA polymerase and ligation were done as described previously (28, 29). After gel purification of the photoprobes, complexes were assembled with either XPC/HR23B alone or XPC/HR23B and TFIIH in the presence of ATP (1 mM).

In Vitro Reconstitution of DNA Opening in the NER Reaction

To investigate the DNA opening step of the NER reaction, we set up a permanganate sensitivity assay that allows the detection of single-stranded DNA around the cisplatinated DNA by generating breaks in phosphate backbone upon piperidine treatment. In such an assay, KMnO₄ oxidized the pyrimidine residues in single-stranded DNA preferentially over those in double-stranded DNA, and the oxidized products could be converted to strand breaks by piperidine treatment (30). In this type of assay, only changes in DNA structure upon protein binding are detected. We thus applied this assay to damaged DNA carrying a single GTG 1,3-intrastrand cisplatin adduct and tested the changes in footprinting patterns upon incubation together with the highly purified factors XPC/HR23B, XPA, RPA, TFIIH, XPG, and XPF/ERCC1 (Fig. 1A) (7, 26). This set of factors is both necessary and sufficient to remove the damaged region, and the omission of any one of the factors prevents the dual incision reaction around the cisplatinated adduct (Fig. 1B). Together, they can open the DNA in the absence of XPF/ERCC1 and form the reconstituted opening system (ROS) (Fig. 1C). We thus observed a modification of the pattern of the single-stranded damaged DNA with an increase of the sensitivity around the cisplatin lesion at positions C−3, G−1, and T0 and the appearance of distinctively TFIIH-promoted bands at positions T−5, C−6, T−8, and T−10, (Fig. 1C, left panel, and as summarized in Fig 7A). In the absence of any NER factors, permanganate-sensitive sites were found at positions C−11, C−3, G−1, T0, G+1, C+2, and A+3, arising from the distortion induced by the damage itself (lane 1). We also noticed the appearance of breaks induced by the permanganate treatment in the undamaged strand upon the addition of the ROS factors at positions A0, C−1, C−16, T−17, C−18, and G−19 (lane 5). In the absence of TFIIH and XPC/HR23B, KMnO4 sensitive sites were not detected (lanes 6 and 7).

XPC Facilitates DNA Opening and Recruits the NER Factors

We next investigated the ability of each factor to separately target the damaged DNA. XPC/HR23B induces local changes in the footprinting pattern at positions G-1 and T-0 (Fig. 2A, lane 7 and C, lane 2). Upon longer exposure, minor changes are also detected over an extensive DNA region from about −20 to +20 (data not shown), further suggesting some “breathing” of the double helix in the region where other factors associate with the DNA (see below). The addition of XPF/ERCC1 to the damaged DNA generates a strong band at position C−3, as well as at positions G−14/G−16 and T+18/G+20 on each side of the lesion, respectively (lane 6), the specificity of which will be discussed below. We noticed, however, that the intensity of the G−1 band is slightly lower, whereas the C−11 band is slightly higher in the absence of NER factors (compare lanes 1–5); when considering weaker bands from A+3 to T+15 for example, they have the same intensity in both conditions.

We addressed the question of whether any combination of the TFIIH, XPA, RPA, and XPG factors was able to contribute to helix opening in the absence of XPC/XR23B. No significant change in the footprinting pattern was observed using the combination of these factors under standard conditions (Fig. 2B). Only upon longer exposure did we observe a minor change at positions T−5 and C−6 in the presence of XPA and TFIIH (data not shown); the XPA-TFIIH complex was found to exhibit some affinity for damaged DNA (31). Altogether, the above results are fully consistent with an early role for the XPC/HR23B complex in recognition of the damage and recruitment of the other NER factors suggested by other studies (9, 10).

We also analyzed the contribution of each component of the XPC/HR23B complex (24, 32). The addition of either XPC or HR23B to the damaged DNA provides similar permanganate footprinting patterns, as indicated by the opening at positions G−1 and T+6 (Fig. 2C, lanes 2–5) when the preformed XPC/HR23B complex is used. Upon the addition of XPC/HR23B, the G-1 band is increased 5.5-fold, whereas with either XPC or HR23B G−1 is increased only 2-fold (Fig. 2C; compare lane 2 with lanes 3 and 4). Although either XPC or HR23B similarly target the damaged DNA (Fig. 2C, left panel, lanes 3 and 4), the addition of XPA, RPA, XPG, and TFIIH to HR23B alone does not promote the opening (lane 8), suggesting that HR23B did not allow the recruitment of the unwinding factors and/or their proper positioning. However when XPA, RPA, XPG, and TFIIH are added together with XPC, either alone or in combination with HR23B, permanganate-induced bands are detected at positions T−10, T−8, C−6, T−5, C−3, G−1, T0, and T+6 (Fig. 2C, lanes 7 and 9, and also Fig. 7A), emphasizing the essential role of XPC in recruiting other NER factors. In agreement with the footprint data, the addition of HR23B alone to XPA, RPA, XPG, TFIIH, or XPF/ERCC1 does not allow the removal of the damaged oligonucleotide unless XPC is added (Fig. 2C, right panel).

TFIIH Is Recruited to the XPC/HR23B-DNA Complex and Further Advances DNA Opening

Having shown that XPC has an early role in open complex formation, we next addressed the contribution of RPA, XPA, XPG, and TFIIH to DNA opening by incubating the damaged DNA with the ROS system in which one NER factor was systematically omitted. The absence of TFIIH resulted in the loss of the extension of the footprinting pattern induced by XPC/HR23B (Fig. 3A, compare lane 7 with lanes 2 and 3), whereas the omission of RPA or XPG did not significantly affect the overall footprinting pattern (Fig. 3A, lanes 4, 6, and 3, respectively). We especially noticed that, in the absence of XPA, the footprinting intensity at positions T−10, T−8, C−6, and T−5 as well as at positions near the damage site is 2-fold weaker (Fig. 3A, lane 5). Once XPC/HR23B and TFIIH are bound to the damaged DNA, additional NER factors are no longer required to extend the footprinting pattern on the 5′ side. Only on the 3′ side are all NER factors imperative (Fig. 3A, lanes 2–6).

We next addressed the role of ATP, which is required for the activity of the XPB and XPD helicases of TFIIH (33–35) in modulating the footprinting pattern of the damaged DNA around the lesion (36). We first observed that ATP does not modify the XPC/HR23B footprinting pattern of the damaged DNA (Fig. 3B, lanes 2 and 3). The addition of TFIIH in the absence of ATP did not allow modification of the XPC/HR23B-induced DNA footprint (Fig. 3B, lanes 3 and 7). In the presence of ATP, TFIIH opens the DNA according to the typical footprinting pattern in which permanganate breaks are found at both positions T−5 and C−6 and near the damage (Fig. 3B, lanes 4 – 6). The footprinting pattern induced by XPC/HR23B and TFIIH is even increased in the presence of XPA at positions T0, G−1, C−3, T−5, and C−6, and additional bands appeared at positions T−8 and T−10 (Fig. 3B, lanes 8–11). The addition of either RPA or XPG in the absence of XPA only slightly modified the opening profile around the damage and at positions T−5 and C−6 (lanes 15–16). Even if all of the factors are present, the addition of ATP is indispensable for dual incision (e.g. complete ROS) (Fig. 3B, lanes 12 and 13). The addition of RPA to a reaction containing XPC, TFIIH, and XPA did not modify the footprinting pattern of the damaged strand (lanes 14 and 17), whereas the addition of XPG increased the KMnO₄ sensitivity pattern detected in the presence of XPA. Indeed, scanned T−5 and C−6 bands are increased 2.5–3-fold (Fig. 3B, lanes 14 and 18). When the double combination RPA+XPG is tested, we observed a slight increase at positions T−5 and C−6 (Fig. 3B, lanes 15, 16, and 19). Although RPA was shown to crosslink damaged DNA (37, 38), under our experimental conditions it does not modify the single-stranded structure induced by XPC and TFIIH (Fig. 3C, lanes 4 and 6). Indeed RPA, either alone or in combination, does not modify the permanganate footprinting pattern of both the damaged and the undamaged DNA strand (Fig. 3, B and C).

The XPG-XPF/ERCC1 Connection for Incision

Upon the addition of all five NER core factors, including XPC-HR23B, TFIIH, XPA, RPA, and XPG, we detected an optimal (both quantitatively and qualitatively) footprinting pattern (Fig. 3B, lane 20). In such a case, T−5 and C−6 are 3-fold higher (compare Fig. 3B, lanes 18 and 20). In the presence of XPF/ERCC1 we observed a 2-fold decrease of the bands at positions T−5, C−6, T−8, T−10, and near the lesion (mainly at positions G−1 and T0), as well as the appearance of other bands at positions T+12, T+15, and A+20 (Fig. 3B, lane 20 and 21; see also below and Fig. 7A).

At this stage of our investigation we were wondering whether the additional bands induced by XPG and XPF/ERCC1 endonucleases, either alone or in combination, were the result of either an extension of the DNA opening or incisions in the damaged DNA. Therefore, the ability of either XPG or XPF/ERCC1 to incise the DNA on each side of the cisplatin lesion was tested. Both experiments were conducted in parallel under the same experimental conditions, except that after the incision reaction only one set of samples was KMnO₄-treated. When XPG is added to the intermediate complex containing XPC/HR23B, TFIIH, XPA, and RPA, we observe some cut at position +12 (at t = 10 min) (Fig. 4A, lanes 4, 6, and 8). When added to the same intermediate complex, XPF/ERCC1 endonuclease did not significantly hydrolyze the damaged DNA in the presence of the four NER factors (Fig. 4A, lanes 10–18). We did, however, notice some incision at position −3 that probably results from an inappropriate positioning of XPF/ERCC1 on the intermediate complex in the absence of XPG (Fig. 4A, lanes 16–17; see also Fig. 2A, lane 6). We should mention that, in such a case, both XPG and XPF endonuclease activities are rather weak (compare, for example, Fig. 4, A and B, the relative intensity of C−11 and T+12 at t = 10 min).

Importantly, the addition of XPF/ERCC1, together with XPG, modified the KMnO₄ footprinting pattern (Fig. 4B). An increase of the 3′ incision cuts were obtained at positions +12, +15, and +20 with a progressive decrease of the KMnO₄-induced cuts on the 5′ side of the lesion and the appearance of new bands at positions −20, −18, and −17. To discriminate between bands produced by KMnO₄-induced hydrolysis and bands resulting from the XPG and/or XPF/ERCC1 nucleases activities, DNA products were analyzed on a sequencing gel after 1′, 10′, 20′, and 30′ of incision reaction in the absence of permanganate treatment. Upon the addition of XPF/ERCC1, we observed the formation of incision-specific bands on both sides of the opened DNA bubble at positions −20, −18, −17, +12, +15, and +20 (Fig. 4C, lanes 4, 6 and 8, and Fig. 7A). Our data show that both XPG and XPF work in concert and that the hydrolysis occurs at the limit of the opened DNA structure.

We next investigated whether the XPG endonuclease activity was regulated by either the physical presence of XPF or the enzymatic activity of XPF/ERCC1. We thus analyzed whether XPF-D676A and XPF-D720A, two XPF mutants impaired in nuclease activity and the dual incision reaction (Fig. 5A) but not DNA binding, can affect the activity of XPG to cut damaged DNA (39). When KMnO₄ assays were carried out in the presence of XPF-D676A and XPF-D720A/ERCC1, we detected a 5′ footprinting profile very similar to the one observed in the absence of XPF/ERCC1 (Fig. 5B, left panel). However, we should notice that the presence of the XPF mutants significantly increases the bands at positions C+7, T+8, C+10, C+11, and T+12, with the latter one being an XPG-induced sensitive site as shown in the incision assay (Fig. 5B, right panel). Except for this one site, it is interesting to note that the other XPG-sensitive sites (at positions +15 and +20) require the presence of an active XPF, showing once more the synergy between both nucleases in the elimination of the damaged oligonucleotide.

Photo Cross-linking of XPC and XPB around the Damaged DNA

We have used site-specific protein-DNA photo cross-linking to determine the location of XPC/HR23B and TFIIH subunits, the two factors that initiate the formation of the pre-incision complex between nucleotides −30 and +30 around the cisplatin damage. Four photoprobes placing the photoreactive nucleotide AB-dUMP at specific locations around the damage were designed (Fig. 6A). The DNA repair pre-incision complex containing either XPC/HR23B alone or XPC/HR23B and TFIIH was incubated with the various photoprobes in the presence of ATP and further UV-irradiated. Under those conditions, the photoreactive nitrene of AB-dUMP can be covalently crosslinked to the polypeptides located at a distance of ~10 A from the DNA backbone (40, 41, 59). The samples were then digested with exonucleases to remove non-crosslinked DNA tails, and the crosslinked polypeptides were analyzed by SDS-PAGE. To ensure the specificity of the crosslinked bands, all of the assays were carried out in parallel with photoprobes lacking the cisplatin adduct, which did not result in the formation of covalent protein-DNA complexes (data not shown). A crosslinking signal was considered specific when its intensity was significantly higher in reactions containing the damaged photoprobe as compared with the undamaged photoprobe.

The interaction of the DNA repair factors XPC/HR23B and TFIIH with the damaged DNA is much weaker that the interaction of the basal transcription factors (including TFIIH) and RNA polymerase II with promoter DNA (28), which explains the low intensity of the cross-linking signals in Fig. 6B. When XPC/HR23B alone was used in the reactions, we repeatedly obtained cross-linking of XPC at all the positions tested (Fig. 6B). When TFIIH and ATP were added to reactions containing XPC/HR23B, we obtained cross-linking of XPB at all three positions (cross-link at position +21/+22 is always weak) as well as cross-linking of XPC at the two positions located upstream of the damage (at position −23/−20 and −8/−7). Fig. 6C shows the relative intensity of the XPC and XPB cross-linking signals obtained in three independent experiments using our four photoprobes in the presence of both XPC/HR23B and TFIIH. Whereas the cross-linking of XPB is weaker than that of XPC at positions −23/−20, both factors cross-link with similar efficiencies at positions −8/−7. Notably, only XPB cross-linked to both photoprobes +12/+15 and +21/+22. XPC did not crosslink to photoprobes +12/+15 and +21/+22 in the presence of TFIIH. Together, these results indicate that XPC makes extensive DNA contacts around the lesion in the absence of TFIIH but is displaced from the 3′ side of the lesion when TFIIH associates with the XPC/HR23B-DNA complex, suggesting that the entry of TFIIH induces a relocalization of XPC in the complex. According to SDS-PAGE, our results also indicate that XPB is the TFIIH subunit that comes in closest contact with the open DNA.