1. p8/TTD-A as a Repair-Specific TFIIH Subunit
Frédéric Coin, Luca Proietti De Santis, Tiziana Nardo, Olga Zlobinskaya, Miria Stefanini, Jean-Marc Egly 
Molecular Cell 
Volume 21, Issue 2, Pages (January 2006)
DOI: /j.molcel
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1 p8 Is Part of a TFIIH Active in Repair and Transcription
(A) TFIIH (Heparine Ultrogel [Gerard et al., 1991]) was immunoprecipitated with Ab-p44 and washed with increasing salt concentrations. TFIIH/Ab-p44 was eluted with a competitor peptide, and the samples were resolved by SDS-PAGE. The blot was silver stained (Staining) or immunostained (WB). The subunits of TFIIH are indicated.
(B) TFIIH/Ab-p44 ([A], lane 2) was incubated with Ab-p8 antibody and eluted with a competitor peptide. The input (lane 1), the supernatant of the IP (lane 2), and the TFIIH/Ab-p8 (lane 3) were resolved by SDS-PAGE and immunostained (top). Fifty and 100 ng of the fractions were tested either in a dual incision (NER) or transcription assay (Tx). Sizes of the incision products or transcripts are indicated.
(C) Bacterially expressed core TFIIH subunits (lanes 1–4) were tested for their ability to interact with GST-tagged p8 (lanes 6, 8, 10, and 12) or GST alone (lanes 5, 7, 9, and 11) coupled to glutathione-agarose beads. Proteins on the resin were resolved by SDS-PAGE and immunostained.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

3. Figure 2 p8 Specifically Engages TFIIH in Repair
(A) Top, TFIIH/Ab-p44 (lane 1) or IIH9 (lane 2) was resolved by SDS-PAGE and immunostained. Bottom, 100 ng of TFIIH/Ab-p44 (lane 1) or IIH9 (lanes 2–4) was tested in an NER or in a Tx in the presence of 1 and 3 ng of recombinant p8 (lanes 3 and 4). Sizes of the incision products or transcripts are indicated.
(B) Top, IIH9 and IIH10 containing the nine (lane 1) or the ten (lane 2) subunits of TFIIH were purified and resolved by SDS-PAGE and immunostained. Bottom, 50 and 100 ng of IIH9 (lanes 1 and 2 and 3 and 4) or IIH10 (lanes 5 and 6) was tested in an NER or a Tx. Three nanograms of recombinant p8 was added to the reaction mixtures in lanes 3 and 4.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

4. Figure 3 p8 and CAK Interplay in Transcription and Repair
(A) Left, IIH6 and IIH9 containing the six subunits of the core without (lane 1, IIH6) or with (lane 2, IIH9) the subunits of the CAK complex were resolved by SDS-PAGE and immunostained. Right, 100 ng of IIH9 (lanes 1–3 and 9) or IIH6 (lanes 4–8 and 10) was incubated with p8 (1 and 3 ng) and/or CAK (50 and 100 ng) in an NER or a Tx assay.
(B) Transcription initiation complex containing RNA Pol II, TFIIA, IIB, TBP, IIE, IIF, and when indicated, recombinant IIH9 and p8 on an immobilized AdML template was incubated 15 min at 30°C with ATP (Incub 1). Beads were washed to remove unbound proteins and further incubated in a reconstituted dual incision assay containing XPC-hHR23B, XPA, RPA, XPG, XPF, and a Pt-DNA template (Incub 2). TFIIH/Ab-p44, lane 1, has been purified in Figure 1A.
(C) Incision complex containing XPC-hHR23B, XPA, RPA, XPG and TFIIH/Ab-p44, IIH6, p8, and/or CAK as indicated was assembled on an immobilized Pt-DNA template and incubated 15 min at 30°C with ATP (Incub 1). Beads were washed and further incubated in a reconstituted transcription assay containing RNA Pol II, TFIIA, IIB, TBP, IIE, IIF, and the AdMLP template (Incub 2).
(D) Wt human primary fibroblasts (FB789) were UV irradiated with 70 J/m2 through a 3 μm pore filter and fixed 30 min later. Immunofluorescent labeling was performed with a rabbit polyclonal antibody, anti-XPB (panels a and e), a monoclonal mouse anti-CPD (panel b), or a monoclonal mouse anti-cdk7 (panel f). Nuclei were counterstained by DAPI, and slides were merged.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

5. Figure 4 p8 Stimulates DNA Opening
(A) One-hundred nanograms of IIH9 (lanes 3–7) was tested either in the dual (top) or in the 5′ (bottom, 77 nt) incisions of the Pt-DNA in the presence of 1–8 ng of p8 (lanes 4–7). Lane 1 contains TFIIH/Ab-p44. Lane 2 contains all the NER factors except TFIIH. The 3′ incision being made first, 3′ uncoupled incision by XPG (107 nt product) can be detected with the 5′ labeled probe (Coin et al., 2004).
(B) Pt-DNA was incubated with 10 ng of XPC-hHR32B (lanes 1 and 3–14) and 300 ng of either TFIIH/Ab-p44 (lanes 3 and 4), IIH9 (lanes 5–8), IIH9/XPB(K346R) (lanes 9–11), or IIH9/XPD(K48R) (lanes 12–14). XPA (25 ng) and p8 (2 ng) were added when indicated. Lane 2, Pt-DNA with BSA only. Residues are numbered with the central thymine of the crosslinked GTG sequence designated T0. Arrows indicate KMnO4-sensitive sites. Adducted strand residues to the 3′ and 5′ of T0 are denoted by positive and negative integers (+N, −N).
(C) One-hundred nanograms of either IIH9 (lanes 2–9), IIH9/XPB(K346R) (lanes 12 and 13), or IIH9/XPD(K48R) (lanes 14 and 15) was tested in an ATPase assay in the presence of 50 ng of XPC-hHR23B, 2 ng of p8, and 120 ng of double-strand circular DNA as indicated at the top of the figure. Lanes 1 and 16 contain ATP only. A quantification of the inorganic phosphate (Pi) release and ATP was done by a Bio-imaging analyzer, and the ratio presented at the bottom of the figure shows the percentage of release of Pi.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

6. Figure 5 Recruitment of XPA at Sites of UV Damage
(A) TFIIH from MRC5 or TTD1BR-SV total cell extracts was immunoprecipitated with Ab-p44 and eluted with a competitor peptide. The eluates were loaded on a 11% SDS-PAGE gel for immunostaining (WB) or on a 15% SDS-PAGE gel for silver staining (Staining).
(B) MRC5 or TTD1BR-SV fibroblasts were UV irradiated as indicated in Figure 3D and labeled with a polyclonal anti-XPB (panels b and j), a monoclonal mouse anti-CPD (panels a, e, i, and m), or a polyclonal rabbit anti-XPA (panels f and n).
(C) Immobilized damaged DNA was incubated with XPC/hHR23B, XPA, IIH9, and p8 as indicated (Preinc) for 15 min at 30°C with ATP. DNA was further washed, supplemented with p8, RPA, ERCC1-XPF, and XPG, and subjected to dual incision assay in which one of the XPA, XPC, or IIH9 factors was omitted per lane as indicated (Inc). In lanes 1 and 5, no NER factor was added in the incubation.
(D) XPC-HR23B, IIH9, and p8 were incubated with the Pt-DNA as indicated (Preinc) for 15 min at 30°C with ATP. Beads were washed, and reactions were further supplemented with XPA, RPA, XPG, and ERCC1-XPF with or without IIH9 and p8 (Inc). Incision was allowed to continue for an additional 90 min.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

7. Figure 6 Analysis of N- and C-Terminal Deletions of p8
(A) Top, schematic representation of the different p8 constructs. Bottom, IIH9 (100 ng) was tested in a dual incision assay with XPC-hHR23B, XPA, RPA, XPG, ERCC1-XPF, and Pt-DNA in the presence of 1 and 3 ng of p8 (lanes 3 and 4), p8NΔ10 (lanes 5 and 6), p8NΔ20 (lanes 7 and 8), and p8CΔ15 (lanes 9 and 10).
(B) Plasmids expressing p8 or p8NΔ10 were microinjected in TTD1BR fibroblasts. UDS (number of radioactive grains in the nucleus) is measured after UV irradiation and incubation with radioactive thymidine and expressed as grains/nucleus (mean of >50 nuclei/sample ± SEM).
(C) A KMno4 assay, as described in Figure 4B, was performed with 300 ng of either TFIIH/Ab-p44 (lane 2) or IIH9 (lanes 3–5) in the presence of 3 and 6 ng of either p8 (lanes 4 and 5) or p8NΔ10 (lanes 6 and 7).
(D) One-hundred nanograms of TFIIH/Ab-p44 (lane 3) or IIH9 (lanes 2 and 4–7) was assayed in an ATPase assay in the presence of 50 ng of XPC-hHR23B and 120 ng of double-strand circular DNA in each lanes as well as 1 and 2 ng of either p8 (lanes 4 and 5) or p8NΔ10 (lanes 6 and 7). In lanes 8 and 9, 2 ng of p8 and p8NΔ10 was tested as control. A quantification of the Pi release was performed as described in Figure 4C.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

8. Figure 7 p8 Stabilizes the TFIIH Complex
(A) Expression vector encoding an antisense RNA toward p8 mRNA was transfected in MRC5 fibroblasts, previously labeled by latex blue fluorescent beads. The TFIIH level was measured 60 hr after transfection, using antibodies directed toward the XPB subunit (right) in either untransfected MRC5 (MRC5), transfected MRC5 (MRC5 p8−/−), or TTD-A (TTD1BR-SV) cells spotted on the same slide. Nuclei were counterstained by DAPI, and slides were merged (left).
(B) Expression vector encoding both p8 and green fluorescent protein (GFP) was either transfected (panels a, b, e, f, I, and j) or microinjected (panels d, h, and l) into TTD1BR (top panels), TTD8PV (TTD-XPD) (middle panels), and TTD1BEL (TTD-XPD) (bottom panels) cells. The level of TFIIH was measured in transfected cells by using Ab-XPB, and the level of repair was measured on cells after incubation with [3H]thymidine and autoradiography. Graphs show the amount of XPB (gray) or TBP (white) on the various transfected or untransfected cells as measured by prism spectrophotometer system. Nuclei were counterstained by DAPI, and slides showing XPB, GFP, and DAPI were merged.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions