Structural and functional characterization of interactions involving the Tfb1 subunit of TFIIH and the NER factor Rad2

Abstract

The general transcription factor IIH (TFIIH) plays crucial roles in transcription as part of the pre-initiation complex (PIC) and in DNA repair as part of the nucleotide excision repair (NER) machinery. During NER, TFIIH recruits the 3'-endonuclease Rad2 to damaged DNA. In this manuscript, we functionally and structurally characterized the interaction between the Tfb1 subunit of TFIIH and Rad2. We show that deletion of either the PH domain of Tfb1 (Tfb1PH) or several segments of the Rad2 spacer region yield yeast with enhanced sensitivity to UV irradiation. Isothermal titration calorimetry studies demonstrate that two acidic segments of the Rad2 spacer bind to Tfb1PH with nanomolar affinity. Structure determination of a Rad2-Tfb1PH complex indicates that Rad2 binds to TFIIH using a similar motif as TFIIEα uses to bind TFIIH in the PIC. Together, these results provide a mechanistic bridge between the role of TFIIH in transcription and DNA repair.

Figure 1.

tfb1-ΔPH is sensitive to UV irradiation, but not bleomycin or temperature. (a) The survival of TFB1 (blue), tfb1-ΔPH (red) and tfb1-1 (+ control; black) yeast was determined following increasing doses of UV irradiation. The y-axis represents the percentage of surviving cells (normalized to the number of viable cells not exposed to UV light) and the x-axis shows the energy level of the UV irradiation applied (J/m²). The results are the mean ± SEM of three independent experiments. (b) The survival of TFB1, tfb1-ΔPH, rad2 (− control) and rad52 (+ control) yeast was determined before (left) or after (right) exposure to bleomycin. The yeast strains were incubated on plates containing YPD ± 250 ng/ml bleomycin and survivors were determined by spotting 8 µl of cells in serial dilutions (10⁻¹–10⁻⁴). The assay was repeated three times, and a typical set of results is shown. (c) The survival of TFB1 (− control), tfb1-ΔPH and tfb1-1 (+ control) yeast was determined following growth at either 30°C (left) or 37°C (right). The survivors were assayed by spotting 8 µl of cells in serial dilutions (10⁻¹–10⁻⁴) on SC-LW plates. This assay was repeated three times and a typical set of results is shown.

Figure 2.

The Rad2 spacer region contains a high affinity Tfb1PH-binding site between residues 642 and 690. (a) Identification of amino acid segments located between residues 642–760 from the Rad2 spacer region that align with the Tfb1PH-binding sites from TFIIEαCTD, p53TAD2 and VP16C. In the alignments, the residues of TFIIEαCTD, p53TAD2 and VP16C that form the binding site with p62PH/Tfb1PH are underlined and crucial hydrophobic residues are shaded in gray. (b) Comparison of the dissociation constant (K_d) values for the binding of different Rad2 segments located between residues 642 and 760 with Tfb1PH. (c) Thermogram of the Tfb1PH titration with successive additions of Rad2_642–690. Experiments are performed at 25°C, in 20 mM NaPO₄ pH 7.5, and the results fit to a single-binding site model with 1:1 stoichiometry.

Figure 3.

Rad2_642–690 and p53TAD2 share a common binding site on Tfb1PH. (a and b) Ribbon models of the 3D structure of Tfb1PH (blue; PDB code 1Y5O). The amino acids of ¹⁵N-labeled Tfb1PH showing a significant chemical shift change {Δδ(ppm) > 0.15; Δδ = [(0.17ΔN_H)² + (ΔH_N)²]^1/2} upon formation of a complex with either Rad2_642–690 (a) or p53TAD2 (b) are highlighted in yellow and green, respectively. (c) Overlay of a selected region from the ¹H–¹⁵N HSQC spectra of ¹⁵N-labeled p53TAD2 (0.5 mM) in the free form (green) and in the presence of unlabeled Tfb1PH (0.4 mM; blue). (d) Same overlay as in (c), but after the addition of unlabeled Rad2_642–690 (0.5 mM; black). Signals of p53TAD2 that undergo significant changes in ¹H and ¹⁵N chemical shifts upon formation of the complex with Tfb1PH (c), and that return toward their original position following the addition of Rad2_642–690 (d) are indicated by arrows.

Figure 4.

NMR structure of the Rad2_642–690–Tfb1PH complex. (a) Stereo view of the 20 lowest-energy structures of the complex between Tfb1PH (blue) and Rad2_642–690 (yellow; PDB code 2LOX). The structures were superimposed using the backbone atoms C′, C^α and N of residues 4–65 and 85–112 of Tfb1PH and residues 661–680 of Rad2_642–690. (b) Ribbon representation of Tfb1PH (blue) and backbone trace of the region of Rad2_642–690 (yellow) interacting in the first groove. In this groove, Phe670 of Rad2 forms a cation–π interaction with Arg61 of Tfb1 and van der Waals interaction with Met59 and Lys57. (c) Ribbon representation of Tfb1PH (blue) and backbone trace of the region of Rad2_642–690 (yellow) interacting in the second groove. On one side of the groove Val673 of Rad2 is inserted where it interacts with Leu48, Ala50, Lys101 and Gln105 of Tfb1. On the other side of the groove, Thr675 of Rad2 interacts with Gln105, Ile108 and Lys112 of Tfb1.

Figure 5.

The Rad2 spacer region contains a second high-affinity Tfb1PH-binding site. (a) (Top) Identification of amino acid segments located between residues 363–382 from the Rad2 spacer region that align with the Tfb1PH-binding sites from TFIIEαCTD and Rad2_642–690. In the alignments, the residues of TFIIEαCTD and Rad2_642–690 that form the binding interface with p62PH/Tfb1PH are underlined and crucial hydrophobic residues are shaded in gray. (Bottom) Dissociation constant (K_d) values for the binding of Rad2_359–383 and Rad2_642–690 with Tfb1PH as determined by ITC analysis. (b) Ribbon models of the 3D structure of Tfb1PH (blue; PDB code 1Y5O). The amino acids of ¹⁵N-labeled Tfb1PH showing a significant chemical shift change {Δδ(ppm) > 0.15; Δδ = [(0.17ΔN_H)² + (ΔH_N)²]^1/2} upon formation of a complex with Rad2_359–383 are highlighted in orange. (c) Overlay of a selected region from the ¹H–¹⁵N HSQC spectra of ¹⁵N-labeled Rad2_642–690 (0.5 mM) in the free form (green) and in the presence of unlabeled Tfb1PH (0.4 mM; blue). (d) Same overlay as in c, but after the addition of unlabeled Rad2_359–383 (1.0 mM; black). Signals of Rad2_642–690 that undergo significant chemical shift changes in ¹H and ¹⁵N chemical shifts upon formation of the complex with Tfb1PH (c), and that return toward their original position following the addition of Rad2_359–383 (d) are indicated by arrows.

Figure 6.

Multiple regions of the Rad2 spacer are required for repair of UV damage. (a) The survival of RAD2 (blue), rad2-ΔD1 (red), rad2-ΔD2 (black), rad2-ΔD3 (orange) and rad2-ΔD2D3 (aqua) yeast was determined following increasing doses of UV irradiation. The y-axis represents the percentage of surviving cells (normalized to the number of viable cells not exposed to UV light) and the x-axis shows the energy level of the UV irradiation applied (J/m²). The results are the mean ± SEM of three independent experiments. (b) The survival of RAD2 (blue), rad2 (black), rad2-ΔD1 (red) and rad2-ΔD1PPPP (aqua) yeast was determined following increasing doses of UV irradiation. The y-axis represents the percentage of surviving cells (normalized to the number of viable cells not exposed to UV light) and the x-axis shows the energy level of the UV irradiation applied (J/m²). The results are the mean ± SEM of four independent experiments.

Figure 7.

The structures of the Rad2_642–690–Tfb1PH and TFIIEαCTD–p62PH interfaces are remarkably similar. (a–c) Ribbon diagrams of the lowest energy structures of the Rad2_642–690–Tfb1PH (a; PDB code 2LOX), the p53TAD2–Tfb1PH (b; PDB code 2GS0) and the TFIIEαCTD–p62PH (c; PDB code 2RNR) complexes. Tfb1PH (a and b) and p62PH (c) are shown in blue, Rad2_642–690 (a) in yellow, p53TAD2 (b) in green and TFIIEαCTD (c) in red. In panels (d–f), the 3D structure of Tfb1PH (d–e) and p62PH (f) are shown as molecular surfaces (blue) and Rad2_642–690 (d), p53TAD2 (e) and TFIIEαCTD (f) are shown as ribbons in yellow, green and red, respectively. Selected residues of Rad2_642–690 (d), p53TAD2 (e) and TFIIEαCTD (f) at the binding interface are also shown.