1. Volume 26, Issue 2, Pages 231-243 (April 2007)
XPG Stabilizes TFIIH, Allowing Transactivation of Nuclear Receptors: Implications for Cockayne Syndrome in XP-G/CS Patients 
Shinsuke Ito, Isao Kuraoka, Pierre Chymkowitch, Emmanuel Compe, Arato Takedachi, Chie Ishigami, Frédéric Coin, Jean-Marc Egly, Kiyoji Tanaka 
Molecular Cell 
Volume 26, Issue 2, Pages (April 2007)
DOI: /j.molcel
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1 XPG Forms a Protein Complex with TFIIH
(A) Experimental procedure for purification of the XPG-TFIIH complex.
(B) Silver staining of the XPG complex. As a control, a mock purification was performed with nuclear extracts from nontransfected HEK293 cells. Purified polypeptides were resolved by SDS-PAGE and visualized by silver staining.
(C) The XPG complex was immunoblotted with antibodies against XPG and eight subunits of TFIIH.
(D) Nuclear extracts of HeLa cells were immunoprecipitated with anti-p44 antibody in the presence of 0.4 M KCl.
(E) Insect cells were infected with baculoviruses expressing either the core TFIIH (IIH6) (lane 1) or IIH6 plus XPG (lanes 2 and 3). The cell extracts were immunoprecipitated with antibody against p44 (lanes 1 and 2) or XPG (lane 3). The immunoprecipitants were resolved by SDS-PAGE, and XPG and TFIIH subunits were detected by immunoblotting using each antibody.
(F) The XPG complex was further separated on a Superose 6 column. After fractionation, each fraction was resolved by SDS-PAGE and visualized by silver staining (upper panel) or immunoblotted with anti-XPG and anti-XPB antibodies (bottom panels). Estimated molecular sizes are depicted in the upper panel.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
The XPG-TFIIH Complex Possesses Both 3′ Endonuclease and TFIIH Activities In Vitro
(A) The structure of the bubble substrate BS-A (90-mer) bearing a 30 nt unpaired region as described (Evans et al., 1997a). The DNA substrate was 5′-labeled with 32P on one strand.
(B) Autoradiograph after denatured 12% PAGE of the cleaved products, demonstrating a structure-specific endonuclease activity of the XPG-TFIIH complex. BS-A (100 fmol) was incubated with the XPG-TFIIH complex at 30°C for the period indicated. M, size markers (MspI digest of pBR322 3′-labeled with 32P).
(C) BS-A (100 fmol) was incubated at 30°C for 30 min with the XPG-TFIIH complex fractionated by Superose 6 gel filtration (Figure 1F).
(D) Structure of the DNA substrate pBSII KS-GTG containing a specifically located 1,3-intrastrand d(GpTpG)-cisplatin crosslink. The expanded region illustrates the DNA sequence flanking the cisplatin crosslink. Seven BstNI restriction sites are indicated. Digestion of the closed circular pBSII KS-GTG DNA containing a cisplatinated adduct with BstNI generates a 29 nt fragment encompassing the DNA adduct and, to a lesser extent, larger fragments surrounding the adduct (140 and 241 nt).
(E) Autoradiograph after denatured 14% PAGE, demonstrating DNA repair synthesis in the BstNI fragment containing a cisplatinated adduct (29 nt). DNA was incubated with [α-32P]dCTP and whole-cell extracts from HeLa (lane 1), XP11BE (XP-B) (lanes 2 and 3), XP17BE (XP-D) (lanes 4 and 5), or XP125LO (XP-G) (lanes 6 and 7) in the presence or absence of the XPG-TFIIH complex, and subsequently digested with BstNI before electrophoresis.
(F) Autoradiograph after SDS-PAGE, demonstrating CAK activity. GST-CTD was incubated without (lane 1) or with increasing amounts of the XPG-TFIIH complex (lanes 2–4).
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4. Figure 3 Truncated XPG Cannot Form an XPG-TFIIH Complex
(A) Schematic representation of the different XPG proteins examined in this study. Hatched N and I boxes represent the conserved regions required for nuclease activity. Box C, conserved C-terminal region; NLS, nuclear localization signal. A792V mimics the mutant XPG protein presumably expressed in a patient with mild XP-G, XP125LO, while Δ926–1186 mimics the mutant XPG with a C-terminal truncation derived from XP-G/CS, XPCS1RO.
(B) Each mutant XPG as well as WT XPG was fused with a FLAG-V5-6xHis tag at the C terminus and expressed in HEK293 cells. Whole-cell extracts were immunoprecipitated with anti-FLAG M2 antibody. Purified XPG complexes were then analyzed by immunoblotting with the antibodies indicated.
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5. Figure 4
XPG and CAK Were Dissociated from the Core TFIIH in the Extracts from XP-D, XP-D/CS, XP-G, and XP-G/CS Cells
(A) Schematic representation of the mutant XPG presumably expressed in XP3BR, XPCS1RO, and XPCS1LV. Hatched N and I boxes and NLS represent the same region as described in Figure 3A. Black boxes indicate non-XPG residues. The numbers on the right side of the boxes indicate the total length of the WT and mutant XPG proteins.
(B and C) Immunoprecipitation of whole-cell extracts derived from XP6BE, XPCS-2, XP8BR, XP3BR, XPCS1RO, and XPCS1LV cells as well as WI38VA13 (WT) and BJ1 (WT) cells using anti-XPB (B) and anti-XPG (C) antibodies, respectively.
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6. Figure 5
Dissociation of CAK and XPD from the Core TFIIH in the XPG-Downregulated Cells
(A) Two XPG siRNA sequences are depicted. Cells expressing luciferase (control)- or XPG-siRNA were established. To express siXPG-2-resistant V5 tagged-XPG, silent mutations were introduced into XPG cDNA (XPG-2M).
(B) Real-time quantitative PCR analysis of XPG, XPB, and GAPDH was performed with cDNA prepared from control siLuc (black)-, siXPG-1 (gray)-, or siXPG-2 (white)-transfected HeLa cells. The histograms represent the mean ± SEM of the relative expression level normalized with actin in three independent experiments.
(C) UV sensitivity of XPG-knockdown cells as determined by the colony assay. siLuc (closed circles)-, siXPG-1 (closed triangles)-, or siXPG-2 (open squares)-transfected HeLa cells were UV irradiated at the indicated dose. Results represent the mean ± SD of three independent experiments.
(D) Immunoblot of whole-cell extracts from siLuc-, siXPG-1-, or siXPG-2-transfected HeLa cells (lanes 1–3). Whole-cell extracts were then immunoprecipitated with anti-XPB antibody, and the immunoprecipitants were analyzed by immunoblotting with the antibodies indicated (lanes 4–6).
(E) Real-time quantitative PCR analysis was performed as in (B) with cDNA prepared from siLuc (black)- and siXPG-2 (white)-transfected HEK293 cells, respectively.
(F) XPG-downregulated cells were transfected with pcDNA5 vectors encoding siRNA-resistant WT XPG. Twenty-four hours later, cells were lysed, and then XPG and TFIIH were immunoprecipitated with anti-XPG (lanes 1–3) or anti-XPB (lanes 4–6) antibodies, followed by immunoblotting with the antibodies indicated.
(G) WT and mutant XPG cDNA, which avoid siRNA-induced downregulation, were transfected into XPG-downregulated HEK293 cells, respectively. Twenty-four hours later, cells were lysed, and then XPG and TFIIH were immunoprecipitated with anti-XPB antibody. The histogram in the lower panel presents the relative amount of cdk7 bound to the core TFIIH normalized by the expression level of each XPG protein.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
Nuclear Receptor-Mediated Transactivation in WT, XP-D, XP-D/CS, XP-G, XP-G/CS, and xpg−/− Cells
(A) SV40-transformed WT, XP-D, XP-D/CS, XP-G, and XP-G/CS cells were cotransfected with pERE-Luc, pCH110, and either pcDNA3-ERα or empty pcDNA3 and subsequently treated with 10−7 M 17β-estradiol (E2) for 16 hr. Luciferase activity was measured and normalized relative to β-galactosidase activity. Histograms show the ratio of the luciferase activity with or without E2 and indicate the values without (open boxes) or with ERα (closed boxes). The results are the mean ± SEM of at least three independent experiments performed in duplicate.
(B) ERα-mediated transactivation in the xpg+/+ and xpg−/− MEF, and the MEF transfected with pcDNA5-XPG. The transactivation assay was performed as described in (A). Histograms indicate the E2-induced transactivation in the xpg+/+ MEF (open boxes) or the xpg−/− MEF (closed boxes).
(C) E2-induced transactivation of ERα in WI38VA13, XPCS1RO, and XPG-GFP cDNA-corrected XPCS1RO cells. The transactivation assay was performed as described in (A).
(D) E2-induced phosphorylation of ERα in WI38VA13, XPCS1RO, and the XPG-GFP-corrected XPCS1RO cells. After transfection with pcDNA3-ERα and subsequent treatment with E2, cell lysates were prepared for immunoblot analysis using an antibody elicited against ERα specifically phosphorylated at Ser118. β-tubulin was used as an internal control. The quantitative analysis of ERα phosphorylation represents the ratio ERα phosphorylated at Ser118/ERα signals and was set up to 1 for WI38VA13 cells.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
Schematic Model for the Role of XPG in Maintaining the Integrity of TFIIH and Implications for CS Features in XP-G/CS Patients
WT XPG forms a complex with TFIIH and functions in maintaining the integrity of TFIIH (left panel). Full-length XPG with a missense mutation in the nuclease domain that was found in a patient with mild XP-G forms a complex with TFIIH as the WT XPG does (middle panel), while a mutant XPG with a C-terminal deletion that is derived from a XP-G/CS patient caused the dissociation of CAK and XPD from the core TFIIH (right panel).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions