Transcriptional Activators Stimulate DNA Repair - Cell Press

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Molecular Cell, Vol. 10, 1391–1401, December, 2002, Copyright 2002 by Cell Press

Transcriptional Activators Stimulate DNA Repair

Philippe Frit,2,3 Kyungrim Kwon,2 Fre´de´ric Coin, Je´roˆme Auriol, Sandy Dubaele, Bernard Salles,3 and Jean-Marc Egly1 Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire CNRS/INSERM/ULP B.P.163 67404 Illkirch Cedex France

Summary To counteract the deleterious effects of genotoxic injury, cells have set up a sophisticated network of DNA repair pathways. We show that Gal4-VP16 and RAR transcriptional activators stimulate nucleotide excision repair (NER). This DNA repair activation is not coupled to transcription since it occurs in Cockayne syndrome cells (which are transcription-coupled repair deficient) and is observed in vitro in the presence of ␣-amanitin and in the absence of the basal transcription factors. Using a reconstituted dual incision assay, we also show that binding of activators to their cognate sequences induces a local chromatin remodeling mediated by ATP-driven chromatin remodeling and acetyltransferase activities to facilitate DNA repair. Introduction Genome integrity is continuously threatened by the occurrence of DNA damage arising from the cellular metabolism or following genotoxic attack. These potentially lethal or mutagenic DNA lesions induce various cellular responses, including cell cycle arrest, transcription alteration, and processing by different DNA repair mechanisms, a major one being the nucleotide excision repair (NER) pathway (De Laat et al., 1999). The importance of NER is emphasized by the existence of autosomal recessive diseases, such as xeroderma pigmentosum (XP), Cockayne syndrome (CS), and trichothiodystrophy (TTD) (de Boer and Hoeijmakers, 2000; Lehmann, 1998), associated with mutations in several NER genes resulting in UV sensitivity and, in the case of XP, in a highly increased incidence of skin cancers. A functional link between transcription and NER corresponds to the socalled transcription-coupled repair (TCR) subpathway, as opposed to global genome repair (GGR). TCR is characterized by faster damage removal from the transcribed strand of genes (Bohr et al., 1985; Mellon et al., 1987). Although the current hypothesis for TCR relies on the targeted recruitment of the repair machinery to an RNA polymerase II (RNA pol II) stalled at a damage site on 1

Correspondence: [email protected] These authors contributed equally to this work. 3 Present address: Institut de Pharmacologie et de Biologie Structurale, CNRS, 31077 Toulouse Cedex, France. 2

the transcribed strand, transcription might also affect DNA repair through nucleosome rearrangements that accompany RNA pol II progression (Meijer and Smerdon, 1999; Wellinger and Thoma, 1997). This hypothesis concerns elongating RNA pol II and does not explain why, near the transcription initiation site, a preferential repair is observed on both the transcribed and the nontranscribed strands (Tu et al., 1996). Transcriptional activators are thought to operate in part by binding to DNA and negating the repressive effect of chromatin in order to ensure the formation of transcription initiation complexes (Bjorklund et al., 1999; Buratowski, 2000; Ptashne and Gann, 1997). Conceivably, such activators might also facilitate DNA repair by disengaging chromatin and recruiting factors that would stimulate DNA repair. This point has been supported by acidic transactivators, such as human p53 and herpes simplex virus activator VP16, as well as retinoic acid receptor (RAR) and estrogen receptors that interact with TFIIH and RPA, DNA repair factors which are also involved in transcription and replication, respectively (Chen et al., 2000; Cheng et al., 1992; Li and Botchan, 1993; Rochette-Egly et al., 1997; Xiao et al., 1994). The above observations prompted us to investigate the role of the transcriptional activators in DNA repair. We found that the Gal4-VP16 and RAR activators stimulate NER in the surroundings of their binding site and independently of transcription. In a reconstituted dual incision assay with a chromatinized DNA substrate, purified chromatin remodeling factors, and DNA repair factors, we show that upon binding to their target sites, activators induce a local chromatin remodeling to further allow the DNA repair machinery to proceed. Results A Template for Both Transcription and DNA Repair To investigate the connections between transcription and DNA repair, we set up an assay in which the same DNA molecule can be used in both reactions (Figure 1A). The pBluescript phagemid was modified by inserting first, the adenovirus major late promoter, second, either the GAL4 binding sites or a DR5-type retinoic acid response element (RARE) (Dilworth et al., 2000) at position ⫺85, and third, a single GTG 1,3-intrastrand cisplatin DNA adduct (at position ⫹105 on the transcribed strand). This template (105.TS), when used in an in vitro transcription system with HeLa nuclear extracts (NE), leads to 128 nt run-off RNA transcripts (Figure 1B). The synthesis of RNA is blocked by the cisplatin adduct (Pt) on 105.TS, whereas the damage on the nontranscribed strand (101.NTS) has no effect on transcript elongation. To further establish our experimental system, we used a chromatinized substrate by preincubating the damaged 105.TS-GAL4, with Drosophila embryo extracts (DmEE) as a source for chromatin assembly factors and purified core histones (Becker et al., 1994; Dilworth et al., 2000). Following the NER reaction, the repaired plas-

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Figure 1. DNA Templates (A) The standard template (105.TS) contains GAL4 binding sites (or RARE; 105.TS-RARE) upstream of the AdMLP, which includes the TATA box. The GTG site targeted for platination (Pt) is located on the transcribed strand (TS) at position ⫹105. The positions of EcoRI (⫹61) and NdeI (⫹157) restriction sites are indicated. In the 101.NTS template, the GTG site is located on the nontranscribed strand (NTS) at position ⫹101. The 16.TS, 37.TS, 487.TS, and 1068.TS templates contain the platinated GTG site on the transcribed strand. The 105.TS.⌬GAL4 and 105.TS.⌬TATA templates lack the GAL4 binding sites and the AdMLP, respectively. In the 105.TS.TATAmut template, the TATAA sequence was replaced by a TCGAG sequence. (B) The GTG cisplatin adduct inhibits transcription elongation. Untreated or platinated 105.TS, as well as platinated 101.NTS. templates, was linearized by XhoI at position ⫹128 for a run-off transcription assay with HeLa NE (Gerard et al., 1991). RNAs were resolved on 8% urea PAGE. (C) DNA repair: undamaged or platinated (Pt) 105.TS-GAL4 templates containing GAL4 binding sites were chromatinized with calf thymus histones and DmEE extracts and incubated with either untreated (NE) or TFIIHimmunodepleted (NE-ID-IIH) HeLa nuclear extracts for DNA repair resynthesis. When indicated at the top of the panel, purified HeLa TFIIH and recombinant Gal4-VP16 (1 nM) were added. The 95-mer EcoRI-NdeI fragment containing the GTG site is indicated by an arrow.

mid is cleaved by restriction enzymes to generate a 95 nt fragment containing the radiolabeled repair patch. HeLa NE is able to repair the damaged 105.TS template (Figure 1C, lane 7), whereas DmEE extract alone exhibits no repair activity (lanes 3–4). We also noticed that DNA repair is increased upon addition of the Gal4-VP16 activator (lanes 7–8 and 11–12; see also below). The radioactivity incorporated in the fragment is highly specific and results from NER, since first, using TFIIH-immunodepleted NE in the context of preincubation with DmEE, there is no repair unless TFIIH is added (lanes 9–12), and second, in the absence of cisplatin damage, there is no signal (lane 6). DNA Repair Is Stimulated by Transcriptional Activators Addition of Gal4-VP16 to a chromatin-packed 105.TS template activates RNA synthesis (Figure 2A). Similarly, we observed a stimulation of DNA repair upon addition of increasing amounts of Gal4-VP16 (Figure 2B, upper panel, and Figure 1C). Since NER is a multistep process, including damage recognition, elimination of the damaged fragment, DNA synthesis, and ligation (Wood, 1997), we wondered which step of the DNA repair reaction was affected by activators. Thus, we monitored, in parallel to the DNA repair resynthesis assay, an in vitro dual incision assay (Araujo et al., 2000) in which the chromatinized cisplatinated 105.TS-GAL4 template is preincubated with HeLa NE and, upon addition of ATP,

the damaged fragment is excised. When increasing amounts of Gal4-VP16 were added, the dual incision was stimulated (Figure 2B, middle panel). When a naked DNA is used as a template, Gal4-VP16 has no significant effect on the dual incision (Figure 2B, lower panel). To rule out the possibility that NER activation relied on a peculiar feature of Gal4-VP16, we tested the effect of RAR, the retinoic acid nuclear receptor. When increasing amounts of the RAR␣/RXR␣ heterodimer complex are added to HeLa NE together with the chromatinized platinated 105.TS-RARE template (Figure 1A), we observe a significant increase of the dual incision reaction that parallels the DNA repair synthesis activity (Figure 2C, lanes 1–4, lower and upper panels); this occurs in the absence of the trans-retinoic acid tRA ligand (Figure 2C, lanes 5–7). The transcriptional activators usually contain a DNA binding domain and an activating domain interacting with the transcription machinery (Brivanlou and Darnell, 2002). The oligo(⫹GAL4) oligonucleotide competitor, containing a GAL4 binding site, specifically inhibits the GAL4-VP16-activated DNA repair of the chromatinized 105.TS, as compared to oligo(⫺GAL4), which lacks the Gal4 binding sites (Figure 3A, lanes 4 and 2, respectively). Similar observations were made when an oligonucleotide containing RARE was used as a competitor in a reaction containing the 105.TS-RARE template as well as RAR␣/RXR␣ heterodimer (our unpublished data).

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Figure 2. Gal4-VP16 and RAR␣ Activate DNA Repair In Vitro (A) Transcription was performed with chromatinized undamaged 105.TS template in the absence (lane 6) or in the presence of Gal4VP16 (0.1, 0.25, 0.5, 0.75, and 1 nM) (lanes 1–5). Transcription activity was analyzed by S1 nuclease protection assay leading to a 93mer fragment. (B) Increasing amounts (0.5 and 1 nM) of Gal4VP16 stimulate both DNA repair resynthesis and dual incision from a chromatinized 105.TS-GAL4 template in the presence of HeLa NE. The size of the restricted 95-mer EcoRI-NdeI fragment containing the GTG site and the excised DNA fragments is indicated at the right of the panel. The incision reaction was also conducted with the naked template (lowest panel). (C) DNA repair resynthesis and a dual incision reaction were conducted using the chromatinized 105.TS-RARE template and increasing amounts (0.25, 0.5, and 1 nM) of the RAR␣/ RXR␣ heterodimer (lanes 1–4) and in the presence of the trans-retinoic acid ligand tRA (10⫺6 M) (lanes 5–7).

When the platinated 105.TS.⌬GAL4 template lacking the GAL4 binding sites (Figure 1A) was used, Gal4-VP16 did not stimulate the DNA repair activity (Figure 3B). We then investigated the role of both the DNA binding domain of the yeast Gal4 activator (Gal4-DBD) and the acidic activating domain of the herpes simplex virus VP16 activator (VP16-AD) (Goding and O’Hare, 1989). Purified Gal4-DBD, VP16-AD proteins, as well as Gal4-VP16 (Figure 3C, left panel) were first tested in a gel shift assay. Both Gal4-DBD and Gal4-VP16 are able to form nucleoprotein complexes (NC) with labeled oligo (⫹GAL4), whereas VP16-AD does not (Figure 3C, right panel). Only oligo(⫹GAL4), and not oligo(⫺GAL4), can compete for the formation of the NC complexes, showing the specificity of the interaction (lanes 5, 6, 10, and 11). We then demonstrated that neither VP16-AD nor Gal4-DBD proteins stimulate DNA repair (Figure 3D, compare lane 2 with lanes 6, 10, and 11). However, at higher doses, Gal4-DBD exhibited a slight stimulatory effect (data not shown) that could depend on cryptic activation domains present in the N-terminal of Gal4-DBD, as observed in transcription (Figure 3E) (Pazin and Kadonaga, 1997; Koh et al., 1998). Consequently, both domains of Gal4VP16 are required for DNA repair stimulation. To test the effect of distance on repair activation, we generated several templates derived from the platinated 105.TS (Figure 1A). Gal4-VP16 stimulates repair resynthesis when the cisplatin adduct is located in the vicinity of its binding site, at positions ⫹16, ⫹37, and ⫹105 (Figure 3F, lanes 1–6); its stimulatory effect is strongly reduced or even disappears when the cisplatin lesion switches from position ⫹105 to position ⫹487 or ⫹1068 (lanes 5–10).

Gal4-VP16 Stimulates DNA Repair Independently of Transcription We then used several approaches to address the question as to how transcription activators could be involved in DNA repair. First, addition of ␣-amanitin, which inhibits RNA pol II, did not prevent the stimulation of DNA repair by Gal4-VP16 (Figure 4A). Second, chromatinized template, either containing point mutations in the TATA box (105.TS.TATAmut) that abolish RNA synthesis (Davison et al., 1983) or lacking the TATA box (105.TS.⌬TATA) (Figure 1A), allowed NER activation (Figure 4B, lanes 3–6), suggesting that the proper assembly of a preinitiation transcription complex is not a prerequisite for DNA repair activation. Third, when the damage was located on the nontranscribed strand (101.NTS-GAL4; Figure 1A), Gal4-VP16 also stimulated DNA repair (Figure 4B, lanes 1, 2, 7, and 8). Finally, since activators interact with basal transcription factors such as TBP/TFIID, TFIIB, or TFIIH (Rochette-Egly et al., 1997; Verrijzer and Tjian, 1996), their role in mediating DNA repair cannot be ruled out. The TBP/TFIID-immunodepleted (ID-TBP) extract was unable to support RNA synthesis (Figure 4D, lanes 1–4), but still retained its ability to activate DNA repair (Figure 4C, lanes 1–4). Addition of either TBP or TFIID, which restored the transcription activity (Figure 4D, lanes 5 and 6), had no significant effect on the repair activity (Figure 4C, lanes 5 and 6). As a control, we observed that an XPG-immunodepleted NE (XPG being an essential NER factor), which works in transcription (Figure 4D, lanes 7–10), lost its DNA repair activity. This activity can be restored upon addition of XPG (Figure 4C, lanes 9–12). Taken together, these results indicate that stimulation

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Figure 3. Both Domains of Gal4-VP16 Are Required for DNA Repair (A) DNA repair was conducted on the chromatinized 105.TS-GAL4 in the absence or in the presence of Gal4-VP16 (1 nM) and, when indicated, 8 pmol of either oligo(⫹GAL4) or oligo(⫺GAL4). (B) DNA repair was carried out using templates containing (105.TS) or lacking (105.TS.⌬GAL4) the GAL4 binding sites, in the absence or the presence of Gal4-VP16 (1 nM) when indicated. (C) Electrophoretic mobility-shift assay (EMSA). Various amounts (⫹, 25 ng; ⫹⫹, 50 ng; ⫹⫹⫹, 100 ng) of purified E. coli-produced recombinant Gal4-VP16, Gal4-DBD, and VP16-AD proteins (see the Coomassie blue-stained SDS-PAGE) were incubated with a radiolabeled oligonucleotide (100 fmol) containing a single GAL4 site in the absence or in the presence of 100 pmol of either oligo(⫺GAL4) or oligo(⫹GAL4) competitor. NC, nucleoprotein complexes; FP, free probe. (D) DNA repair was performed with either undamaged or cisplatinated (Pt) templates in the absence or the presence of Gal4-VP16, Gal4-DBD, or VP16-AD proteins (0.5 and 1 nM), as indicated above. (E) Transcriptions were performed in the absence or the presence of Gal4-VP16, Gal4-DBD, or VP16-AD (1 nM) using the undamaged chromatinized 105.TS template. (F) DNA repairs were performed in the absence or the presence of Gal4-VP16 (1 nM) using different chromatinized templates containing the GAL4 binding sites, in which the platinated damage was positioned on the transcribed strand at ⫹16 (16.TS), ⫹37 (37.TS), ⫹105 (105.TS), ⫹487 (487.TS), and ⫹1068 (1068.TS). The sizes of the digested fragments containing the GTG site are indicated at the right of the panel.

of NER by transcriptional activators occurs in a transcription-independent manner. Activators Stimulate Chromatin Modification around Their Binding Sites To further investigate how activators regulate DNA repair, the 105.TS-GAL4 chromatinized plasmid lacking the TATA box was incubated with either Gal4-VP16 or Gal4-DBD, digested with micrococcal nuclease (Becker et al., 1994), and analyzed for its nucleosomal organization using an ethidium bromide-stained agarose gel and by Southern blot (Figure 5A). Upon addition of Gal4VP16, Southern blots revealed some changes of the nucleosome structure proximal (⫹105) to its cognate DNA binding site (Figure 5A, left panel, lanes 1–6, probe a), but there was no distal (⫹1068) disturbance (right panel, lanes 1–6, probe b). In the presence of the trun-

cated Gal4-DBD, there is no obvious nucleosomal rearrangement (compare lanes 7–9 with lanes 1–3). Since we did not observe significant nucleosomal displacement upon addition of RAR␣/RXR␣ to the chromatinized 105.TS-RARE template (data not shown), we investigated its restriction enzyme accessibility. Indeed, upon binding to its target site, RAR␣/RXR␣ facilitates the XhoI restriction enzyme cut generating the PvuI-XhoI fragment detected by probe a (Figure 5B, lanes 2 and 3), evidencing some local nucleosomal reorganization. Similarly, addition of Gal4-VP16 to the chromatinized damaged 105.TS-GAL4 template makes the XhoI site accessible, allowing the detection of the PvuI-XhoI fragment (lanes 8 and 9). In contrast, neither activator allowed ScaI digestion, the restriction site of which is far away from the RARE and GAL4 responsive element (lanes 5 and 6 and lanes 11 and 12, respectively). To-

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Figure 4. Activation of DNA Repair Occurs Independently of Transcription (A) DNA repair analysis with chromatinized platinated 105.TS template using HeLa NE in the absence or the presence of Gal4-VP16 (1 nM) and, when indicated, 5 ng/␮l of ␣-amanitin. (B) DNA repair analysis with templates platinated either on the transcribed strand (105.TS; 105.TS.TATAmut, template with a point-mutated TATA box; 105.TS.⌬TATA, template without TATA box) or on the nontranscribed strand (101.NTS). (C and D) DNA repair (C) and transcription (D) analyses were performed with platinated 105.TS templates in the absence or the presence of Gal4-VP16 (1 nM). HeLa NEs were either untreated (control) or TBP- (ID-TBP) or XPG- (ID-XPG) immunodepleted. Purified TBP, TFIID, or XPG was added as indicated.

Figure 5. Chromatin Remodeling by Gal4VP16, Gal4-DBD, and RAR␣/RXR␣ The chromatin was assembled with Drosophila embryo extracts and calf thymus histones on either 105.TS-GAL4 or 105.TS-RARE platinated closed circular DNA. (A) After 30 min of incubation with either Gal4VP16, Gal4-DBD (1 nM), or RAR␣/RXR␣ (1 nM) as indicated, chromatinized templates were submitted to a time course Mnase digestion (1 U/assay). Following deproteinization, separation on a 1.2% agarose gel in 0.5⫻ TBE, and ethidium bromide staining (EtBr; upper panel), DNA was electro transferred, and the chromatin structure at specific regions was examined by Southern blot analysis using 32P-labeled oligonucleotides. Probe a overlaps the GTG cisplatinated damaged sequence, and probe b corresponds to a distal region (⫹1068) of the plasmid. (B) Upon addition of either RAR/RXR or Gal4VP16 and treatment either by XhoI or by ScaI, the damaged and chromatinized templates were phenol/chloroform extracted and further treated by PvuI and BspHI to generate either a 560 bp or a 620 bp fragment, respectively. The restricted fragments were then analyzed by Southern blot using 5⬘ end-labeled probes a and b, respectively. Lanes 1, 4, 7, and 10 show the digested naked plasmid.

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Figure 6. DNA Repair Activation Is Mediated by Chromatin Remodeling Activities The purified chromatinized cisplatinated 105.TS-GAL4 (A and C) and 105.TS-RARE templates (B and D) were preincubated, when indicated, with ACF (500 pM), p300 (500 pM), Gal4-VP16 (1 nM), RAR␣/RXR␣ (1 nM), ATP (0.1 mM), and acetyl CoA (1 ␮M). In (A) and (B), HeLa NE was added, and DNA repair synthesis activity was measured. The HAT activity (A) was tested upon addition of H3-CoA-20 HAT inhibitor (0.5 mM). In (C) and (D), the dual incision products were monitored by adding purified NER factors.

gether these data show that the subtle nucleosomal modifications generated by activators are not only due to a simple occupancy (as shown by the truncated Gal4DBD) of their DNA binding sites but require also the activating domain that will attract cofactors for additional rearrangements. p300 and ACF Chromatin Remodeling Activities Are Involved in DNA Repair We then investigated the relationship among ATPdependent chromatin remodelling, histone acetyltransferase activities (that also play a role in transcription activation; Kingston and Narlikar, 1999; Strahl and Allis, 2000), and DNA repair (Hasan et al., 2001; Ura et al., 2001). The chromatinized template was first applied to a size exclusion chromatography column to eliminate low molecular weight components including ATP and acetyl CoA (Pazin and Kadonaga, 1997; Dilworth et al., 2000). In such conditions, addition of either acetyl CoA or ATP together with HeLa NE and Gal4-VP16 does not significantly stimulate DNA repair, unless they are concomittantly added (Figure 6A, lanes 1–6). Similarly, significant stimulation of DNA repair also occurred when both acetyl CoA and ATP were added together with RAR␣/RXR␣ to a damaged 105.TS-RARE template (Figure 6B). Moreover addition of H3-CoA-20, an inhibitor of histone acetyl transferase activity (HAT), prevents DNA repair activation (Figure 6A, lanes 7–9). In an attempt to dissect the molecular events that involve activators, we established a “purified” chromatin

template-based dual incision system, containing the cisplatinated 105.TS template, Gal4-VP16 or RAR␣/RXR␣, ACF (ATP utilizing chromatin assembly and remodeling factor), p300 (HAT), HeLa TFIIH, XPC/HR23B, XPA, RPA, ERCC1/XPF, and XPG recombinant NER factors (Araujo et al., 2000). Both ACF and p300 are required for optimal activation of the dual incision reaction triggered by either Gal4-VP16 or RAR␣/RXR␣ (Figures 6C and 6D). Note that ACF together with ATP enhances the removal of the damaged fragment (lanes 1 and 2); this stimulation is higher when the activator is present (lane 5). In these conditions, p300 also stimulates slightly, but significantly, the removal of the damage in the presence of the activators (lanes 4 and 7). Together, our data suggest that binding of activators to their proper target sites induces a local ATP- and acetyl CoA-dependent chromatin remodeling that is sufficient to stimulate the removal of the DNA lesion, the first step of the NER reaction. In Vivo Stimulation of DNA Repair by Activators The effect of transcriptional activators on DNA repair was then investigated in vivo. A eukaryotic expression vector was modified by inserting a cassette containing either the GAL4 or RAR binding sites as well as a cisplatin 1,3-GTG adduct (or the unmodified GTG as a control), resulting in the p(GAL4/RARE)-GTG(⫾Pt) constructs (Figure 7A). The adduct was localized at position ⫹120 on the transcribed strand so that luciferase expression was expected to be impaired in transfected

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Figure 7. DNA Repair Stimulation by Transcriptional Activator In Vivo (A) The pGAL4-GTG or pRARE-GTG plasmids contain five GAL4 binding sites or a single RARE, respectively, upstream of the SV40 promoter and a cisplatinated or undamaged GTG site located on the transcribed strand (TS) of the luciferase gene in a region corresponding to the 5⬘-UTR of the transcript. (B) MRC5, XP-A, XP-At (derived from the XP-A cell line by transfection with a retroviral vector bearing the XPA cDNA), or CS-B fibroblasts were cotransfected with platinated or undamaged p(⫾GAL4)GTG vectors, together with a ␤-galactosidase expression vector, and either a Gal4-VP16 expression vector (pSG5-Gal4-VP16, black bars) or the empty vector as a control (pSG5, gray bars). (C) Transfections were carried out as in (B) using platinated or undamaged p(⫾RARE)-GTG template and either a RAR␣ expression vector (pSG5-RAR␣, black bars) or the corresponding empty vector (pSG5, gray bars). Results from at least three triplicated experiments are plotted as mean ratios of normalized luciferase activities obtained with damaged versus nondamaged plasmid ⫾ SD.

cells by the persistence of the damage, blocking RNA pol II progression (i.e., in the absence of repair). Human fibroblasts were transfected by either damaged or undamaged pGAL-GTG reporter and either the pSG5Gal4-VP16 overexpressing the Gal4-VP16 protein or the empty pSG5 vector as a control together with a ␤-gal expression vector. In each case, we used a series of plasmid concentrations so as to undertake our analyses in the linear response range and in conditions where transcription activities were not saturated. As the capac-

ity to activate transcription might be different in the various cell lines, and in order to have a comparison of the effect of the activator with or without lesions, results are plotted as mean ratios of luciferase activities (normalized with the ␤-gal control) obtained with platinated versus undamaged plasmid. In MRC5 cells, the cisplatin damage results in 80% inhibition of luciferase gene expression (Figure 7B, lane 1, gray bar), which is overcome when cells are cotransfected with pSG5-Gal4-VP16 (Figure 7B, lane 1, black bar). Similar experiments using the pRARE-GTG reporter also showed a 2- to 3-fold increase of the luciferase expression upon cotransfection of MRC5 cells by the pSG5-RAR␣ expression vector whether or not tRA is added (Figure 7C, lane 1). The DNA repair activation is highly specific, since the relative luciferase activity from platinated constructs lacking either the GAL4 or the RAR binding sites is unchanged whether or not MRC5 cells are cotransfected with pSGGal4-VP16 or pSG-RAR␣, respectively (Figures 7B and 7C, lanes 2). In NER-deficient XP-A cells, the activators could not exert their stimulatory effect (Figures 7B and 7C, lanes 3) unless the cells were rescued with the wild-type XPA cDNA (lane 4), indicating that recovery of luciferase expression in MRC5 relies on the ability of the activators to stimulate NER; reintroducing XPA repair factor (which is not a transcription factor) restores the luciferase gene expression, thus validating our in vivo experiments. Since the cisplatin adduct is located on the transcribed strand of the luciferase gene, its faster removal upon activator binding could be accounted for by TCR resulting from transcription activation. To examine this hypothesis, we transfected TCR-deficient CSB fibroblasts (from Cockayne syndrome patients). As for MRC5 cells, cotransfection of CS-B cells with either pGAL4GTG or pRARE-GTG and either the Gal4-VP16 or RAR␣ expression vector significantly facilitates DNA repair (Figures 7B and 7C, lanes 5), suggesting that activatormediated NER stimulation does not rely on increased TCR but rather on global genome activation in accordance with the in vitro data (Figure 4). Discussion The preferential repair of the transcribed strand of genes (Mellon et al., 1987) as well as promoter surrounding sequences (Tu et al., 1996) has established a functional link between transcription and DNA repair. Using chromatin templates and purified NER factors, we show that transcriptional activators stimulate the removal of DNA damage in promoter regions. However, contrary to what was expected, we show that the DNA repair activation is not mediated by the transcription machinery but rather results from a specific function of activators in local chromatin remodeling to give access to DNA repair factors. Activators Stimulate the NER Pathway Gal4-VP16 and RAR stimulate the removal of lesions located in the surroundings of their respective responsive elements. A gradient of stimulation (from their binding sites to position ⫹500) is observed, in which a higher activation occurs when the damage is close to the acti-

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vator binding site. This DNA repair activation, which was also seen in vivo, is specific, since in the absence of its responsive element, the activator protein is unable to stimulate DNA repair. It is remarkable to notice the extent to which both activators enhance in vivo DNA repair (2- to 5-fold as a function of their nature and their concentration), thus allowing the cell to recover its normal transcription activity. This observation highlights how a “faster repair” at the promoter is physiologically important in cases of genotoxic injury. The fact that transcriptional activators stimulate NER might correspond to a role for TCR as an important backup for the removal of damage in the transcribed strand of genes for which GGR is too slow. However, this hypothesis is unlikely since (1) the DNA repair stimulation occurs on both the transcribed and nontranscribed strands and is seen in TCR-deficient CS-B cells upon overexpression of either Gal4-VP16 or RAR␣; (2) the ␣-amanitin RNA pol II inhibitor does not affect activator-mediated NER stimulation; and (3) the components of the basal transcription machinery are not required. How Do Activators Stimulate the Removal of DNA Damage? It is generally accepted that upon binding to a chromatinized template, activators remove barriers that restrict the access of the transcription machinery (Kornberg and Lorch, 1999). Chromatin also represses DNA repair (Gaillard et al., 1997; Tuteja and Tuteja, 1996; Wang et al., 1991; Ura et al., 2001). We show here that upon binding to their target sites, Gal4-VP16 and RAR␣ activators, together with ATP-driven remodeling complexes and histone acetyltransferases, induce a local chromatin remodeling to preferentially repair the damaged surrounding sequences. However, activators could also act more directly by recruiting DNA repair factors, in a manner reminiscent of the situation in transcription activation (Brivanlou and Darnell, 2002). Indeed, we and others have demonstrated that Gal4-VP16 and RAR interact with RPA and TFIIH, respectively, two essential NER factors (He et al., 1993; Li and Botchan, 1993; Rochette-Egly et al., 1997), which are recruited once XPC/HR23B is bound to the damaged DNA to initiate the GGR subpathway of NER (Volker et al., 2001). A Model to Explain the Preferential Repair One of the main consequences of genotoxic injuries is the physical hampering of gene expression. Cells thus have to turn to sophisticated DNA repair systems to counteract the deleterious effects of DNA damage. This point is illustrated by TCR, in which RNA pol II was shown to play a major role. However, this scenario only takes into account the elongating RNA pol II and not the (pre)initiation step. Here, we demonstrate that transcriptional activators stimulate the removal of DNA lesions on both DNA strands and in the vicinity of their DNA binding sites, which usually include the transcription initiation site. Due to their positioning in the genome, activators keep a close eye on the surrounding sequences. Following DNA damage, activators would locally derepress chromatin to facilitate the repair process and to further allow accurate transcription initiation.

Such a hypothesis is supported by the present report as well as previous studies demonstrating that DNA repair was much faster and without strand selectivity near the transcription start site of the human JUN gene that is surrounded by up to five activator DNA binding sites (Tu et al., 1996). This, in addition to the fact that the formation of the transcription complex requires both the coding and the noncoding strands (Kim et al., 2000; Robert et al., 1998), emphasizes the importance of such a transcription initiation associated repair (TIAR) that operates without strand bias. To integrate into this model the preferential repair observed on the transcribed strand, we propose that once RNA synthesis is initiated, the elongating RNA pol II and its accompanying factors would fulfill the role previously assumed by the activator in surveying the integrity of the gene to be transcribed, this transcription elongation coupled repair (TECR) pathway targeting specifically the transcribed strand. This scenario is supported by the finding that RNA pol II is associated not only with transcription factors but also with DNA repair factors such as RPA, XPG, and XPF (Maldonado et al., 1996; Tantin et al., 1997). The present study raises the question of the real function of transcriptional activators. Their role seems not to be restricted to transcription, since the recruitment of chromatin remodeling activities (Ikeda et al., 1999; Ikura et al., 2000; Ito et al., 2000; Kraus et al., 1999; Kundu et al., 2000; Shen et al., 2000; Tumbar et al., 1999) and the interaction with factors can also modulate DNA repair (the present study) and DNA replication (He et al., 1993; Li and Botchan, 1993; Pazin and Kadonaga, 1997). We show here that chromatin remodeling can be dissociated from the transcriptional function of activators. Contrary to what was observed in transcription, NER stimulation is not triggered by liganded RAR. It is likely that the function of activators is dual: (1) they help to derepress chromatin, a step common to several processes, and (2) concomitantly or subsequently, in concert with the factors bound to the surrounding DNA sequences (including promoter, DNA lesion, or replication origin), they may participate in the recruitment of specific factors involved in transcription, repair, or replication. Is such a recruitment dictated by the activators through ligand binding, posttranslational modification, or cofactor targeting? The mechanism by which the transcription mode versus DNA repair or replication is selected remains to be established. Experimental Procedures Oligonucleotides, DNA Probes, and Plasmids A 25-mer oligonucleotide, 25-GTG, containing a unique GTG sequence at an ApaLI restriction site (5⬘-TCTCTTCTTCTGTGCAC TTCTTCCT-3⬘) was allowed to react with cisplatin and was further HPLC purified (Araujo et al., 2000). Oligo(⫹GAL4), a 40 bp doublestranded oligonucleotide (5⬘-CTCGGGTGCCTCGGAGGATTGTCC TCCGAACAATCTCGGG-3⬘) containing a single GAL4 binding site, as well as oligo(⫺GAL4), a nonspecific competitor (5⬘-CGATAGGA GGAAGAAGTGCACAGAAGAAGAGAGGCCTC-3⬘), were used. For S1 nuclease analysis, 10 pmol of 5⬘-radiolabeled 5⬘-CCTATC GATTGATCCCCCGGGCTGCAGGAA-3⬘ oligonucleotide, corresponding to the ⫹64/⫹93 sequence of the nontranscribed strand of the 105.TS template (Figure 1A), was used as a primer in PCR reaction (40 cycles) with the KpnI-digested 105.TS phagemid, the 143-mer probe was recovered from the 5% urea PAGE gel, and transcription was performed according to Dilworth et al. (2000).

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To construct the platinated templates (Figure 1A), the pBluescript II-KS(-) phagemid (Stratagene) was modified as follows. The standard 105-TS (GAL4) template contains the adenovirus major late promoter (AdMLP) (between a KpnI site at position ⫺55 and an XbaI site at ⫹11) including the TATA box (from ⫺31 to ⫺27). A 225 bp fragment containing five GAL4 binding sites was inserted at the KpnI site, and the most 3⬘ GAL4 binding site ended at ⫺91. The plasmid was also modified to contain several restriction sites flanking the sequence of the 25-GTG oligonucleotide, the platinated GTG site being located on the transcribed strand at ⫹105. The position of the GTG site was switched to ⫹16 (16.TS), ⫹37 (37.TS), ⫹487 (487.TS), or ⫹1068 (1068.TS) by deleting or inserting a DNA fragment between the start site and the GTG site. In the 101.NTS template, the GTG site was located on the nontranscribed strand at ⫹101 and resulted from the 105.TS template by inverting, first, a short region containing the 25-GTG sequence and, second, a 515 bp BssHII fragment (two sites at ⫺317 and ⫹198) to restore the production of single-stranded phage DNA complementary to the 25-GTG oligonucleotide. The 105.TS.⌬GAL4 and 105.TS.⌬TATA templates correspond to the 105.TS template lacking the GAL4 binding sites and the AdML promoter, respectively. In the 105.TS.TATAmut template, the TATAA sequence was replaced by a TCGAG sequence. For transfection, the pGL3-promoter phagemid (Promega) was modified as follows. First, two complementary oligonucleotides (5⬘AGCTTAGGAAGAAGTGCACAGAAGAAGAGAAGCCAC-3⬘ and 5⬘CATGGTGGCTTCTCTTCTTCTGTGCACTTCTTCCTA-3⬘) were annealed and inserted between the HindIII and NcoI sites, resulting in the pGTG plasmid containing the 25-GTG sequence on the transcribed strand of the luciferase gene upstream of the coding sequence. Second, the pGAL4-GTG plasmid was derived from the pGTG plasmid by inserting into the KpnI site the five GAL4 binding sites from the 105.TS template. Similarly, the pRARE-GTG plasmid was derived from the pGTG plasmid by inserting between XhoI and KpnI a single RARE formed by annealing two complementary oligonucleotides (5⬘-CTAGGGTTCACCGAAAGTTCAC-3⬘ and 5⬘-TCG AGTGAACTTTCGGTGAACCCTAGGTAC-3⬘). In the resulting pGAL4GTG and pRARE-GTG plasmids, the cisplatinated GTG site was localized on the transcribed strand downstream of the activator binding sequence at a distance of 250 bp. Preparation of Single-Site Modified Templates An XL1-blue bacterial strain containing the appropriate phagemid (pBluescript or pGL3 derivatives) was infected with VCSM13 helper phage (Stratagene), and the corresponding phage particles were collected to obtain the single-stranded circular phage DNAs. 25 ␮g of phage DNA was annealed in 40 ␮l with a 4-fold molar excess of modified (or untreated as a control) 25-GTG oligonucleotide previously phosphorylated with T4 polynucleotide kinase. Fifty units of T7 DNA polymerase and 800 units of T4 DNA ligase (New England Biolabs) were then added, and the polymerization-ligation reaction was performed at 37⬚C for 1 hr in 200 ␮l of reaction mixture containing 20 mM Tris-HCl (pH 7.5,) 10 mM NaCl, 10 mM MgCl2, 1 mM DTT, 50 ␮g/ml BSA, 2 mM ATP, and 600 ␮M each dNTP. Closed circular DNA was purified by CsCl density centrifugation. The presence of cisplatin damage in the purified products was assessed by ApaLI digestion, which generates two fragments, one of the three ApaLI sites being uncleavable due to the adduct. Preparation of Drosophila Embryo Extracts, Nuclear Cell Extracts, and Purified Proteins Chromatin assembly extracts were prepared from postblastoderm Drosophila embryos (Becker et al., 1994). HeLa NE were prepared as described (Dignam et al., 1983), except that the nuclear protein extraction step was performed at 700 mM NaCl. TFIIH-, TBP-, and XPG immunodepletions were performed by applying HeLa NE to protein A sepharose beads coupled to monoclonal antibodies raised against either the N terminus of p44-TFIIH or TBP, or against XPG residues 701–720, respectively. His-tagged versions of Gal4-DBD (aa 1–147), VP16-AD (aa 411– 490), and Gal4-VP16 fusion protein were expressed from pET3 expression vectors in the BL-21(DE3)pLysS E. coli strain and purified by Ni2⫹ column chromatography (Novagen). XPG expressed in baculovirus-infected Sf9 cells was purified on a phosphocellulose column

followed by immunopurification using a monoclonal XPG antibody. TFIIH and TFIID were purified from HeLa WCE (Gerard et al., 1991). For other NER factors, the purification procedure will be published elsewhere. The human recombinant TBP was expressed from pET11a expression vector (Novagen) in the BL21(DE3)pLysS E. coli strain and further purified. RAR␣/RXR␣ heterodimers were purified as described (Dilworth et al., 2000). Drosophila ACF (Flag tagged on the Acf1 subunit) and human His-tagged p300 were purified on a Flag-M2 resin (Sigma) or on a Ni-NTA affinity resin (Qiagen), respectively, after infection of Sf9 cells by appropriate baculoviruses and expression. DNA Repair Assays Chromatin was assembled with DmEE and calf thymus histones (Boehringer) on either platinated or undamaged closed circular DNA for 5 hr at 27⬚C (Dilworth et al., 2000). When indicated, the chromatinized templates were further purified by exclusion chromatography through S-300HR sephacryl spin columns (Pharmacia). In the transcription reaction, 50 ng of chromatin-packed DNA was incubated at 27⬚C for 15 min with 150 ␮g of HeLa NE in the presence of activator or dialysis buffer as a negative control. Preinitiation complexes were allowed to form at 27⬚C for 15 min in a 40 ␮l reaction mixture containing 20 mM HEPES/KOH (pH 7.6), 60 mM KCl, 5 mM MgCl2, 10% glycerol, 1 mM DTT, 0.3 mM EGTA, 0.4% polyvinyl alcohol, 0.4% polyethylene glycol 10000. rNTPs (500 ␮M each) and dNTPs (20 ␮M dATP, dGTP, dTTP, 4 ␮M dCTP, and 5 ␮Ci [␣-32P]dCTP when DNA repair resynthesis was analyzed) were then added for 45 min at 30⬚C. Nucleic acids were extracted for further analysis. For DNA repair analysis, the dried pellet was resuspended in 50 ␮l of appropriate digestion buffer. All DNA templates except 16.TS and 37.TS were digested with EcoRI and NdeI to generate a 95-mer fragment (88-mer for 101.NTS) containing the resynthesised patch (Moggs et al., 1996). 16.TS and 37.TS templates were digested with AgeI/EcoRI or AgeI/XhoI, and the 124- and a 105-mer fragments were further analyzed by urea PAGE. A dual incision assay was carried out (Araujo et al., 2000) in a 15 ␮l incubation mixture containing chromatinized template (25 ng) and Gal4-VP16 (1 nM) or RAR␣/RXR␣ (1 nM), when indicated, ACF (500 pM), p300 (500 pM), ATP (0.1 mM), and acetyl CoA (1 ␮M) incubated at 27⬚C for 20 min. Repair reaction was performed in 25 ␮l final volume at 30⬚C for 45 min after addition of either HeLa NE or purified XPC/HR23B (10 ng), TFIIH (0.3 ␮l of HAP fraction), XPG (5 ng), RPA (50 ng), XPA (25 ng), XPF/ERCC1 (15 ng), and ATP (2 mM). The excised fragment was detected on 14% urea PAGE after annealing with 6 ng of the complementary oligonucleotide and addition of four radiolabeled [␣-32P]dCMP residues by sequenase V2.1 (USB). Elecrophoretic Mobility-Shift Assay His-tagged Gal4-VP16, Gal4-DBD, or VP16-AD and 20,000 cpm of 5⬘-end-labeled oligo (⫹GAL4) probe were incubated at 24⬚C for 15 min in 30 ␮l of reaction mixture containing 20 mM Tris-HCl (pH 7.5), 100 mM KCl, 1 mM MgCl2, 0.1 mM DTT, 50 M ZnCl2, 10% glycerol, 30 ␮g BSA, and 1 ␮g poly dI.dC and were resolved on 5% nondenaturing PAGE in 0.5⫻ TBE buffer. Cell Culture and Transfection SV40-transformed human fibroblats MRC5 (from a normal individual), XP12RO (from a patient suffering from XP group A), XP-At (derived from the XP-A cell line by transfection with a retroviral vector bearing the XPA cDNA), and CS1AN cells (from a patient suffering from Cockayne syndrome group B) were maintained at 37⬚C, 5% CO2 in MEM supplemented with 10% fetal calf serum, 2 mM L-glutamine, 125 U/ml penicillin, and 125 ␮g/ml streptomycin. For transfections, 5 ⫻ 105 cells were seeded onto six-well multidishes and allowed to attach for 6 hr. Transfection was then carried out by adding 200 ␮l of DNA mix containing 400 ng pGAL-GTG or pRARE-GTG derivative (either unmodified or platinated), 250 ng pSG5 (Stratagene) or pSG5-Gal4-VP16 or pSG5-RAR␣, 1 ␮g pCMVLacZ, 125 mM CaCl2, 140 mM NaCl, 25 mM BES, and 0.75 mM Na2HPO4 (pH 6.9). Cells were incubated at 37⬚C for 10 hr, then fresh medium was added for 2 hr, and cells were harvested after PBS wash. The cell pellet was resuspended in 60 ␮l lysis buffer (25 mM Tris-phosphate [pH 7.8], 2 mM EDTA, 10% glycerol, 1% Triton X-100,

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and 1 mM DTT) and disrupted by three freeze-thaw cycles. Luciferase activity was determined for 30 ␮l of lysate by adding 50 ␮l of luciferin mix (20 mM Tris-phosphate [pH 7.8], 1 mM MgCl2, 2.7 mM MgSO4, 0.10 mM EDTA, 30 mM DTT, 500 ␮M ATP, 470 ␮M luciferin, and 270 ␮M coenzyme A). The emitted light was quantified with a TopCount-NXT luminescence counter (Packard). Values obtained were normalized with the corresponding galactosidase activities determined in vitro with the ONPG substrate. Acknowledgments We are grateful to J. Dilworth, G. Almouzni, A. Harrel Bellan, and J. Moggs for help in setting the chromatin system and fruitfull discussions; R. Wood, S. Clarckson, J.T. Kadonaga, and K. Hanaoka for providing baculoviruses and setting the in vitro NER; J.B. Bell and P. Calsou for critical reading of the manuscript; and C. Braun and L. Trouilh for technical expertise. This work was supported by grants from the Association pour la Recherche sur le Cancer (ARC9083) and the Human Frontier Program (RG-193/97). This work was supported by the ARC (P.F.), the Korean government (K.K.), the CNRS/ BDI fellowship (S.D.), and the Fondation pour la Recherche Medicale (J.A.). Received: March 13, 2002 Revised: September 19, 2002 References Araujo, S.J., Tirode, F., Coin, F., Pospiech, H., Syvaoja, J.E., Stucki, M., Hubscher, U., Egly, J.M., and Wood, R.D. (2000). Nucleotide excision repair of DNA with recombinant human proteins: definition of the minimal set of factors, active forms of TFIIH, and modulation by CAK. Genes Dev. 14, 349–359.

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