Volume 10, Issue 5, Pages 1163-1174 (November 2002) Rapid Switching of TFIIH between RNA Polymerase I and II Transcription and DNA Repair In Vivo  Deborah Hoogstraten, Alex L Nigg, Helen Heath, Leon H.F Mullenders, Roel van Driel, Jan H.J Hoeijmakers, Wim Vermeulen, Adriaan B Houtsmuller  Molecular Cell  Volume 10, Issue 5, Pages 1163-1174 (November 2002) DOI: 10.1016/S1097-2765(02)00709-8

Figure 1 Characterization of XPB-GFP-Expressing Cells (A) Immunoblot probed with anti-XPB and anti-p62 monoclonals of WCE of MRC5 (repair-proficient), XPCS2BA (lanes 1 and 2), and two FACS-sorted populations of XPCS2BA cells stably expressing XPB-GFP (XPB-GFP cells) at a relatively low and high level, respectively (lanes 3 and 4). Note that the total XPB content is similar in low- and high-expressing cells. Molecular weight markers indicated at the left are in kilodaltons. (B) Immunoblot of anti-p62 immunoprecipitated WCE of XPB-GFP-expressing cells probed with an anti-XPB antibody (lane 1, WCE; lane 2, precipitated TFIIH). Band marked with an asterisk represents the heavy IgG chains of anti-p62. (C) Survival after UV irradiation of MRC5 (red line), XPCS2BA (black line), and XPB-GFP cells (green line). The percentage of surviving cells is plotted against the applied UV-C dose. (D) Confocal and transmitted light image of two XPB-GFP cells. White arrows indicate nucleoli. Left panel, GFP-fluorescence; middle panel, phase-contrast; right panel, merged image. Scale bar is 5 μm. Molecular Cell 2002 10, 1163-1174DOI: (10.1016/S1097-2765(02)00709-8)

Figure 2 Characterization of the Nucleolar Accumulation in XPB-GFP-Expressing Cells under Different Conditions (A) Confocal image of a fixed XPB-GFP cell. Left panel, GFP-fluorescence; middle panel, anti-RNAP1 immunofluorescence; right panel, merged image. (B) Transcription after microinjection of a polyclonal antibody directed against XPB visualized by autoradiographic grains after in situ [3H]uridine incorporation. Red arrows indicate nucleoli in microinjected cells, and green arrows represent nucleoli of noninjected neighboring cells. Note that the nucleoli are much darker in noninjected cells because they are covered by grains. (C) Cells 3 hr after incubation with transcription inhibitor DRB (100 μM). (D) Cells 5 min after irradiation with 8 J/m2 UV. Confocal images: left, GFP-fluorescence; right, GFP-fluorescence merged with phase-contrast image. Scale bars are 5 μm. Molecular Cell 2002 10, 1163-1174DOI: (10.1016/S1097-2765(02)00709-8)

Figure 3 FRAP Analysis of the Dynamics of Nucleolar Stuctures by Photobleaching (A) FRAP of a nucleolar cluster. The nucleolar accumulation in the square is bleached. Left, before bleaching; middle, 1 s after bleaching; right, 30 s after bleaching. (B) FLIP of a nucleolar cluster. The region within the strip is bleached. Left, before bleaching; middle, 5 s after bleaching; right, 45 s after bleaching. (C) FRAP curve of a nucleolar cluster. Relative fluorescence is plotted against time. (D) FLIP curve of a nucleolar cluster. The time until complete recovery is longer in the FLIP than in the FRAP experiment since in FLIP bleached molecules have to diffuse from the bleached area to the nucleolar clusters. Scale bar is 5 μm. Molecular Cell 2002 10, 1163-1174DOI: (10.1016/S1097-2765(02)00709-8)

Figure 4 FRAP Analysis of TFIIH Engagement in Transcription Initiation (A) Scheme of temporal strip-FRAP analysis to determine effective diffusion coefficients. Green ellipses represent confocal images of cell nuclei, where darker regions contain fluorophores bleached by a focused laser beam; gray bars represent bleach pulses. A strip spanning the entire nucleus is photobleached for 100 ms at high laser intensity. The recovery of fluorescence in the strip is monitored at low laser intensity. (B) Temporal FRAP analysis of untreated (n = 90, green line) and transcription inhibited (DRB) cells (n = 70, red line). (C) Scheme of simultaneous FRAP/FLIP analysis used to determine the TFIIH-GFP mobility. A small area at one side of a nucleus is bleached at relatively low laser intensity for a relatively long period of time (8 s). Subsequently, fluorescence is monitored at regular time intervals in the bleached area and in a region at the other side of the nucleus. (D) Computer simulation curves of the (log) of fluorescence redistribution difference between FRAP and FLIP determined in the two areas indicated in (C). (E) Simulations of molecules with different diffusion coefficients (D = 10 [red line], 5 [closed squares], 2.5 [open squares], and 1.25 μm2/s [closed circles]). (F) Computer simulations of molecules with constant diffusion rates (10 μm2/s) and an immobile fraction of 40% with increasing binding times of 7 s (closed squares), 14 s (open squares), 28 s (closed circles), and 56 s (open circles); red line as in (E). Note that in this method, correction should be made if nuclei have different sizes. (G) Simultaneous FRAP/FLIP analysis of untreated and transcription-inhibited (DRB treated) cells at 37°C (green squares and red circles, respectively). (H) Simultaneous FRAP/FLIP analysis of untreated and transcription inhibited-cells at 32°C (green open squares and red open circles, respectively). The curves of untreated cells and DRB-treated cells at 37°C (dotted green and red lines, respectively) are shown as a reference. Molecular Cell 2002 10, 1163-1174DOI: (10.1016/S1097-2765(02)00709-8)

Figure 5 FRAP Analysis of DNA Damage-Induced TFIIH Immobilization (A) Temporal FRAP analysis of UV-irradiated cells (blue curve). DRB-treated (red curve) and untreated cells (green curve) (Figure 4B) are shown as references. (B) UV dose-dependent immobilization of TFIIH. Percentage of immobilization is plotted against UV dose. (C) Scheme of the procedure to locally irradiate living cell nuclei showing a confocal XZ-scan of a TFIIH-GFP-expressing cell covered by a UV-blocking membrane (white) containing 5 μm-wide pores. (D) Confocal images of locally UV-irradiated TFIIH-GFP cells 5 min after irradiation (UV dose applied to the membrane: 48 J/m2). Left panel, GFP-fluorescence; right panel, merged image together with phase-contrast. Black arrows indicate the accumulation of TFIIH at the sites of local UV damage. White arrows indicate the accumulation of TFIIH in the nucleoli. Scale bar is 5 μm. (E) FRAP curves of the UV-damaged area of cells cultured at 27°C and 37°C (open and closed squares, respectively). (F) FLIP curve of the locally damaged area. (G) Ratio between fluorescence in the damaged area and fluorescence in the remainder of the nucleoplasm at different temperatures (n = 35 for each temperature), showing that at lower temperatures, the amount of accumulated TFIIH is considerably increased. Molecular Cell 2002 10, 1163-1174DOI: (10.1016/S1097-2765(02)00709-8)

Figure 6 Simultaneous FRAP/FLIP Analysis in Unirradiated Areas of Locally Damaged Cells (A) Scheme to show where transcription is measured in locally irradiated cells. Blue is the locally irradiated area, and green is where transcription initiation is measured. (B) Difference in fluorescence intensity in the FRAP and FLIP areas after bleaching plotted against time (see also Figures 4C and 4D). Closed squares represent mobility measurements in the undamaged area of locally irradiated nuclei. The black line represents undamaged nuclei at 37°C (n = 20). (C) Similar graphs as in (B) of cells cultured at 27°C in the presence (open circles) and absence (open squares) of transcription inhibitors (α-amanitin) (n = 20). Dotted line, untreated cells at 37°C. Molecular Cell 2002 10, 1163-1174DOI: (10.1016/S1097-2765(02)00709-8)

Figure 7 Model for the Dynamic Switching of TFIIH between RNAP1, RNAP2 Transcription Initiation and NER The green arrows represent the equilibrium between the different kinetic pools under unchallenged (normal) conditions. After induction of UV lesions, a rapid shift in the equilibrium toward association in NER occurs (as indicated by the red arrows) in which TFIIH is engaged for a significantly longer period of time. Molecular Cell 2002 10, 1163-1174DOI: (10.1016/S1097-2765(02)00709-8)