Transcriptional Activators Stimulate DNA Repair Philippe Frit, Kyungrim Kwon, Frédéric Coin, Jérôme Auriol, Sandy Dubaele, Bernard Salles, Jean-Marc Egly  Molecular Cell  Volume 10, Issue 6, Pages 1391-1401 (December 2002) DOI: 10.1016/S1097-2765(02)00732-3

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.ΔGAL4 and 105.TS.ΔTATA 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 TFIIH-immunodepleted (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. Molecular Cell 2002 10, 1391-1401DOI: (10.1016/S1097-2765(02)00732-3)

Figure 2 Gal4-VP16 and RARα 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 Gal4-VP16 (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 93-mer fragment. (B) Increasing amounts (0.5 and 1 nM) of Gal4-VP16 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 RARα/RXRα heterodimer (lanes 1–4) and in the presence of the trans-retinoic acid ligand tRA (10−6 M) (lanes 5–7). Molecular Cell 2002 10, 1391-1401DOI: (10.1016/S1097-2765(02)00732-3)

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.ΔGAL4) 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. Molecular Cell 2002 10, 1391-1401DOI: (10.1016/S1097-2765(02)00732-3)

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/μl of α-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.ΔTATA, 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. Molecular Cell 2002 10, 1391-1401DOI: (10.1016/S1097-2765(02)00732-3)

Figure 5 Chromatin Remodeling by Gal4-VP16, Gal4-DBD, and RARα/RXRα 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 Gal4-VP16, Gal4-DBD (1 nM), or RARα/RXRα (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 Gal4-VP16 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. Molecular Cell 2002 10, 1391-1401DOI: (10.1016/S1097-2765(02)00732-3)

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), RARα/RXRα (1 nM), ATP (0.1 mM), and acetyl CoA (1 μM). 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. Molecular Cell 2002 10, 1391-1401DOI: (10.1016/S1097-2765(02)00732-3)

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 β-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 RARα expression vector (pSG5-RARα, 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. Molecular Cell 2002 10, 1391-1401DOI: (10.1016/S1097-2765(02)00732-3)