Distinct Roles for the XPB/p52 and XPD/p44 Subcomplexes of TFIIH in Damaged DNA Opening during Nucleotide Excision Repair  Frédéric Coin, Valentyn Oksenych, Jean-Marc Egly  Molecular Cell  Volume 26, Issue 2, Pages 245-256 (April 2007) DOI: 10.1016/j.molcel.2007.03.009 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 The F99S Mutation Impairs Damaged DNA Opening (A) (Left) Schematic representation of XPB. The dark gray boxes indicate the helicase domains. The light gray box indicates the conserved N-terminal domain. Mutations found in XP-B patients (F99S and T119P) and mutation in the ATPase A Walker I motif (K346R) are depicted. (Right) Two established clones derived from the XPCS2BA cell line (mutation F99S) and expressing either the F99S (XP) or T119P (TTD) XPB (Riou et al., 1999) were used together with the MRC5 control cell line for TFIIH purification. TFIIH/XPB(WT), TFIIH/XPB(F99S), and TFIIH/XPB(T119P) were immunoprecipitated with antibody toward p44, a subunit of the core TFIIH, from whole-cell extracts and eluted with a competitor peptide (Coin et al., 1999). The samples were resolved by SDS-PAGE and western blotted (WB) with anti-TFIIH antibodies. The subunits of TFIIH are indicated. (B) Fifty and one hundred nanograms of TFIIH/XPB(WT) (lanes 3 and 4), TFIIH/XPB(F99S) (lanes 5 and 6), or TFIIH/XPB(T119P) (lanes 7 and 8) were tested in a dual incision assay (NER) containing the recombinant XPC-HR23b, XPA, RPA, XPG, ERCC1-XPF factors and a closed-circular plasmid containing a single 1,3-intrastrand d(GpTpG) cisplatin-DNA crosslink (Pt-DNA) as a template (Frit et al., 2002) or in a reconstituted transcription assay (Tx) composed of recombinant TFIIB, TFIIF, TBP, TFIIE factors, the purified RNA polymerase II, and the adenovirus major late promoter template (Gerard et al., 1991). Sizes of the incision products or transcripts are indicated. (C) TFIIH (100 ng) was incubated with a radiolabeled linear DNA fragment from the Pt-DNA plasmid and 40 ng of XPC-HR32b. XPA (25 ng) was added when indicated. Lane 1, 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′ ends of T0 are denoted by positive and negative integers (+N, −N). (D) The recombinant IIH6/XPB(WT), IIH6/XPB(K346R) (mutated in the ATPase A Walker I site), IIH6/XPB(F99S), and IIH6/XPB(T119P) lacking CAK and p8/TTDA were produced in baculovirus-infected insect cells (Tirode et al., 1999). TFIIH (100 ng) was tested in dual incision in the presence of 3 ng of recombinant p8/TTDA when indicated (lanes 2, 4, 6, and 8). (E) A KMnO4 assay was performed as described in Figure 1C with 100 ng of recombinant IIH6 complex incubated with a radiolabeled linear DNA fragment from the Pt-DNA plasmid and XPC-HR32b. XPA (25 ng) and p8/TTDA (6 ng) were added when indicated. Lane 1, Pt-DNA with BSA only. Lane 2, positive control with TFIIH purified from HeLa. Molecular Cell 2007 26, 245-256DOI: (10.1016/j.molcel.2007.03.009) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 Recruitment of TFIIH and XPA at Sites of UV Damage XPCS2BA(F99S) and wild-type MRC5 (labeled with blue beads) cells were plated on the same slide. Cells were UV irradiated with 70 J/m2 through a 3 μm pore filter and fixed 30 min later. Immunofluorescent labeling was performed using a rabbit polyclonal anti-XPB (A), a mouse monoclonal anti-CPD (B and F) or a rabbit polyclonal anti-XPA (E). Nuclei were counterstained with DAPI (C and G), and slides were merged (D and H). Molecular Cell 2007 26, 245-256DOI: (10.1016/j.molcel.2007.03.009) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 The F99S Mutation Thwarts the Interaction between XPB and p52 and the Stimulation of XPB ATPase Activity (A) Increasing amounts (50, 100, 200, and 400 ng) of either IIH6/XPB(WT), IIH6/XPB(F99S), or IIH6/XPB(T119P) were tested in an ATPase assay. The graph represents the percentage of phosphate released (Pi/[ATP+Pi]) from three independent experiments. (B) Increasing amounts (20, 40, 80, and 160 ng) of either XPB(WT), XPB(F99S), or XPB(T119P) were tested in an ATPase assay. The graph represents the percentage of phosphate released (Pi/[ATP+Pi]) from three independent experiments. (C) Purified XPB (50 ng) (lanes 2–9) was tested in an ATPase assay in the presence of 50 and 100 ng of purified p52 (lanes 3 and 4), 10 and 20 ng of purified p8/TTDA (lanes 5 and 6), or 50 and 100 ng (lanes 8 and 9) of purified p44 subunits of TFIIH. (D) XPB(WT) (lanes 1–3), XPB(F99S) (lanes 4–6), or XPB(T119P) (lanes 7–9) from baculovirus-infected insect cell extracts were immunoprecipitated with anti-XPB antibody. Following washes, beads were incubated with baculovirus-infected insect cell extracts expressing p52, washed at 0.2 or 0.4 M KCl as indicated, and then resolved by SDS-PAGE and western blotted. HC, Ab heavy chain. (E) XPB(WT) (lanes 2–4) or XPB(F99S) (lanes 5–7) from baculovirus-infected insect cell extracts were immunoprecipitated with anti-XPB antibody. Following washes, beads were incubated with increasing amounts of baculovirus-infected insect cell extracts expressing p52, washed at 0.4 M KCl, and then tested in an ATPase assay or resolved by SDS-PAGE and western blotted as indicated. HC, Ab heavy chain. Molecular Cell 2007 26, 245-256DOI: (10.1016/j.molcel.2007.03.009) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 Mapping the Domain of p52 Involved in the Stimulation of the XPB ATPase (A) Schematic representation of p52. The stretches of highly conserved residues in eukaryotes are indicated in black, and XPB binding regions are delimited. (B) Purified FLAG-tagged p52(WT) (25, 50, and 100 ng) (lanes 3–5), p52(1–304) (lanes 6–8), and p52(305–462) (lanes 9–11) were incubated with 50 ng of recombinant XPB (lanes 2–11) and then resolved by SDS-PAGE and western blotted against XPB and the FLAG tag (WB) or incubated in an ATPase assay (ATPase). Molecular Cell 2007 26, 245-256DOI: (10.1016/j.molcel.2007.03.009) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 Mutations in Helicase Domains of XPB Spare TFIIH Repair Activity (A) Schematic representation of XPB. Mutations introduced in conserved helicase domains are indicated. (B) (Upper panel) Immunoprecipitated recombinant XPB (50 and 200 ng) (expressed in baculovirus-infected insect cells) were tested in helicase assay using a bidirectional probe (Coin et al., 1998) at either 37°C or 4°C as indicated. Lane 1 contains highly purified TFIIH from HeLa cells. Δ, probe has been heated 5 min at 100°C. Lane 11 is a control without XPB. Values under the autoradiograph represent the percentage of probe (3′→5′) released relative to control lane 12. (Lower panel) Immunoprecipitated XPB (50 and 200 ng) was tested in an ATPase assay at 30°C or 4°C when indicated. Values under the autoradiograph represent the percentage of phosphate released (Pi/[ATP+Pi]). (C) One hundred nanograms of either IIH6/XPB(WT) (lanes 1–3), IIH6/XPB(K346R) (lanes 4–6), IIH6/XPB(T469A) (lanes 7–9), or IIH6/XPB(Q638A) (lanes 10–12) was tested in a dual incision assay in the presence of increasing amount of p8/TTDA (1.5 and 3 ng) as indicated. (D) The TFIIH (50, 100, and 200 ng) tested in (C) was assessed in a reconstituted transcription assay as in Figure 1B, in the presence of 50 ng of recombinant CAK complex (Rossignol et al., 1997). The size of the transcript is indicated. (E) Fifty and one hundred nanograms of IIH6/XPB(WT) (50 and 100 ng) (lanes 3–5), IIH6/XPB(K346R) (lanes 6–8), or IIH6/XPB(T469A) (lanes 9–11) were incubated with p8/TTDA and XPC-HR32b, with or without ATP, in a KMnO4 assay. Lane 1, Pt-DNA with BSA only. Lane 2, positive control with TFIIH purified from HeLa. (F) CHO27-1 cells were transfected with pLuc plasmid expressing the luciferase gene previously irradiated (lanes 3–6) or not (lane 2) in combination with pcDNA expressing either XPB(WT) (lane 4), XPB(T469A) (lane 5), or XPB(fs740) (lane 6). Repair complementation was assessed by monitoring luciferase activity in cell lysates (48 hr posttranfection) normalized with the internal β-galactosidase standard. Results are expressed as relative luciferase activity. The error bars were calculated on the basis of three independent experiments. Fifty micrograms of total extract was resolved by SDS-PAGE and western blotted (WB) with mouse anti-human XPB antibody (Coin et al., 2004). Molecular Cell 2007 26, 245-256DOI: (10.1016/j.molcel.2007.03.009) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 6 The XPD Helicase Activity Opens Damaged DNA in NER (A) Schematic representation of XPD. The helicase domains are indicated by dark gray boxes. The region of interaction with the regulatory subunit p44 is highlighted (Coin et al., 1998; Dubaele et al., 2003). (B) A KMnO4 assay, as described in Figure 1C, was performed with 100 ng of TFIIH incubated with the Pt-DNA, XPC-HR32b, and XPA. Eight nanograms of p8/TTDA was added when indicated. Lane 1, Pt-DNA with BSA only. (C) One hundred nanograms of IIH6/XPD(WT) (lanes 3–5), IIH6/XPD(R658H) (lanes 6–8), or IIH6/XPD(R658H)/XPB(T469A) (lanes 9–11) was tested in a dual incision assay with increasing amounts of p8/TTDA (1.5 and 3 ng). Values under the autoradiograph represent the repair activity (RA) calculated from three independent experiments. Molecular Cell 2007 26, 245-256DOI: (10.1016/j.molcel.2007.03.009) Copyright © 2007 Elsevier Inc. Terms and Conditions