1. Volume 11, Issue 6, Pages 1635-1646 (June 2003)
Basal Transcription Defect Discriminates between Xeroderma Pigmentosum and Trichothiodystrophy in XPD Patients 
Sandy Dubaele, Luca Proietti De Santis, Rachelle J Bienstock, Anne Keriel, Miria Stefanini, Bennett Van Houten, Jean-Marc Egly 
Molecular Cell 
Volume 11, Issue 6, Pages (June 2003)
DOI: /S (03)

2. Figure 1
XPD Mutations
The diagram shows the 760 amino acids XPD protein with the seven (I–VI) helicase motifs (Gorbalenya and Koonin, 1993). Amino acid changes resulting from mutations found in the XP, XP/CS, and TTD patients are depicted in orange, purple, and blue, respectively. Shared alleles between XP and TTD are colored in black. Subscripts 1 and 2 after the patient code denote the two alleles.
Molecular Cell  , DOI: ( /S (03) )

3. Figure 2 XPD/p44 Interaction
(A) Infected H5 cell lysates containing wild-type p44 and either wild-type or mutated XPD as indicated at the top of the panel, were immunoprecipitated with anti-p44 antibodies crosslinked to agarose beads and further washed with 0.4 M KCl. The immunoprecipitated fractions were then resolved on SDS-PAGE followed by immunoblotting using anti-p44 and anti-XPD antibodies. Δ refers to deletion of amino acids 716–730.
(B) Similarly, XPD proteins (as indicated at the bottom of each panel) were tested for their ability to remain associated with immobilized p44 after 150–300 mM KCl wash. TFIIH refers to HeLa TFIIH HPLC heparin fraction. Note that the ratio between p44 and XPD proteins varies as a function of the Western blot transfer and should be compared inside each blot panel.
Molecular Cell  , DOI: ( /S (03) )

4. Figure 3 Helicase and ATPase Activities of XPD Proteins
(A) H5 cell lysates containing wild-type or mutated XPD subunits were immunoprecipated on agarose beads linked to anti-XPD antibodies in presence or absence of purified p44 as indicated at the top of the panel. After 0.4 M KCl washing, the immunoprecipitated proteins were tested for their 5′ to 3′ helicase activity. Δ (lane C), heat-denatured DNA substrate; (−), helicase assay in absence of TFIIH.
(B) ATPase activity of XPD proteins in presence of DNA. The diagram represents the relative ATPase activity of the mutated XPD proteins compared to wild-type XPD.
(C and D) Helicase activity of XPD-G47R, XPD-K48R, or XPD-D234N was respectively monitored at different ATP or MgCl2 concentrations as indicated in inserted boxes.
Molecular Cell  , DOI: ( /S (03) )

5. Figure 4 Purification of the Recombinant IIH9s
Insect cells were infected with baculoviruses overexpressing the nine subunits of TFIIH including either wild-type or mutated XPD (as indicated at the top of each panel), and complexes were purified using mild (A) or high- (B) salt conditions as depicted on the purification schemes. Equal amounts of purified TFIIHs were then resolved by SDS-PAGE and blotted with antibodies against XPB, XPD, p62, p44, cdk7, and cyclinH subunits of TFIIH. HeLa TFIIH (TFIIH) was used as a reference for stoichiometric analysis.
Molecular Cell  , DOI: ( /S (03) )

6. Figure 5 DNA Repair Activities of the Recombinant IIH9s
Equal amounts of recombinant TFIIHs were added either to a TFIIH immunodepleted HeLa nuclear extract (TFIIH-ID NE) to test their ability to restore DNA repair synthesis of a platinated plasmid (A) or to an incision/excision assay using recombinant NER factors (B).
(A) Repair synthesis. The 95 nucleotides fragment (95 nt) containing the repair patch is generated by EcoRI-NdeI cleavage of the Pt-plasmid. NE, HeLa nuclear extracts. TFIIH-ID NE, TFIIH immunodepleted extracts. (−), assay performed in absence of TFIIH.
(B) Dual incision. The area of the gel containing the excision products is shown. (+) and (++) correspond to 15 and 30 ng of rIIH9 added to the reaction. (−) represents the reaction carried out in absence of TFIIH.
(C) Helicase activity of the recombinant IIH9s. Equivalent amounts of rIIH9 (adjusted according to Western blotting) were tested for their 5′ to 3′ XPD helicase activity. (−), assay performed in absence of TFIIH; Δ, heat-denatured template; TFIIH, qualitative control; probe, nondenatured template. The 5′→3′ and 3′→5′ polarity of the helicase is indicated on the left side of the panel.
Molecular Cell  , DOI: ( /S (03) )

7. Figure 6 Transcription Activity of Recombinant IIH9s
Complexes were tested for basal (A) and Gal4-VP16 activated (B) transcription activity.
(A) The rIIH9 were added to an in vitro reconstituted transcription system lacking TFIIH. The length of the corresponding transcript is indicated on the left side. (−), reaction performed in absence of TFIIH. HeLa TFIIH, control used as a reference. The transcription activity of wt, R112H (TTD), D234N (XP), R685C (TTD), and R722W (TTD) TFIIH was assessed using various amounts of rIIH9. The results are depicted in the bottom left panel and represent the mean of three independent experiments. The bottom right diagram indicates (mutated/wild-type TFIIH) ratios.
(B) rIIH9s were incubated with a TFIIH-immunodepleted nuclear extract (TFIIH-ID NE) and tested for their ability to restore activated transcription of a chromatinized DNA template in presence of Gal4-VP16. Activated transcription is measured using a S1 nuclease assay that generates a 115 nt oligonucleotide as indicated on the left of the panel. NE, nuclear extracts. + and ++ indicate the addition of 100 and 150 ng of recombinant TFIIH.
(C) The retinoic acid (light gray bars) and vitamin D (dark gray bars) mediated transactivations were assayed in wild-type MRC5 (wt), TTD8PV (R112H) XPJCLO (R683W), and TTD1VI (R722W) fibroblasts using a luciferase reporter gene. Positions of the relevant mutation in each XPD patient are shown on the bottom of the panel. The histogram represents the (with/without t-RA or VitD3) ratios calculated from three independent experiments.
Molecular Cell  , DOI: ( /S (03) )

8. Figure 7 TTD Fibroblasts Exhibit Reduced Amounts of TFIIH
Normal and mutated XPD cells were labeled with fluorescent beads and spotted on the same slide. Wild-type fibroblasts (wt, 0.79 μm, green beads), XPJCLO (2 μm, green beads), TTD8PV (1.75 μm, blue beads), TTD1RO (1.75 μm, red beads), and TTD12PV (no beads) were fixed and immunoassayed with monoclonal antibodies directed against XPB (TFIIH), TBP, or XPA, before being revealed with the secondary antibody (goat anti-mouse Cy3 conjugated). Nuclei were counterstained by DAPI.
Molecular Cell  , DOI: ( /S (03) )

9. Figure 8
Molecular Model of XPD Helicase Based on the UvrB Crystal Structure
(A) XPD mutations located in the helicase motifs. The helicase motifs are colored as follow: I, dark blue; Ia, light blue; II, orange; III, red; IV, yellow; V, green; and VI, magenta.
(B) Mutations preventing the interaction with p44.
(C) Mutations corresponding to XP and TTD are presented in orange and blue respectively.
(D) Causative mutations giving rise to XP and TTD phenotypes. Null alleles are hatched. Biochemical activities of the corresponding TFIIH are depicted at the top and at the bottom of the picture. XPD/p44 interaction in green; TFIIH level, cellular TFIIH concentration; Basal T, basal transcription; NR-Act T, nuclear receptor activated transcription.
Molecular Cell  , DOI: ( /S (03) )