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

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.


Plasmid preparations
The Tfb1-6His/pRS314 plasmid contains the TFB1 ORF and its endogenous 400 bp upstream promoter region and a 6His coding tag on the C-terminal part of the protein, all of which were inserted into the SpeI cloning site of pRS314. A small correction was made to this plasmid to respect the TFB1 Saccharomyces cerevisiae Genome Database sequence. A QuikChange (QuikChange Site-Directed Mutagenesis Kit from Stratagene) mutagenesis procedure was done to switch the amino acid 102 from a threonine to a methionine with the primers TFB1-T102m-F and TFB1-T102m-R (see Supplementary Table S2 for primers used in the study). This corrected version was used throughout the paper and referred to as pRS314Tfb1WT-6His. The tfb1-ΔPH mutant was built by overlapped PCR with TFB1(1-115).1F, TFB1(1-115).1R, TFB1(1-115). .2R, using the Tfb1WT plasmid as template. The PCR product was inserted SpeI/SpeI in pRS314. The tfb1-1 mutant was also obtained by an overlapped PCR inserting a STOP codon followed by a 6His tag to replace the glutamine 532. The primers TFB1GFP-BlpI-F, pRSTFB1(1-531)6His-R, pRSTFB1(1-531)6His-F and T7+ were used on the Tfb1WT plasmid. The PCR product was inserted BlpI/SacII in the Tfb1WT plasmid.
The pRS316RAD2cmyc plasmid (for the RAD2 strain) was generated by amplification of the RAD2 ORF and 400pb upstream of the coding sequence on genomic DNA with the primers pRS-RAD2-XhoI-F(c) and pRS-RAD2-R. The PCR product was inserted XhoI/BamHI into pRS316. Then was added the 366 bp downstream of the endogenous ORF by amplification on genomic DNA with the primers pRS-RAD2(aval)-BamHI-F and pRS-RAD2(aval)-SpeI-R. The insertion was done by digesting BamHI/SpeI. Finally, the cmyc tag was inserted in between MscI/SpeI right before the STOP codon of RAD2 by overlapped PCR using RAD2-2905-MscI-F, pRS-RAD2(cmyc)-R, pRS-RAD2(cmyc)-F and pRS-RAD2(aval)-SpeI-R. The previous intermediate plasmid was used as template. The mutant pRS316Rad2(Δ367-378)cmyc plasmid (for rad2-ΔD2) was constructed by overlapped PCR with the primers RAD2-SnaBI-F, RAD2(m367-378)I-R, RAD2(m367-378)II-F and RAD2-BlpI-R. The template used was the pRS316RAD2cmyc plasmid and the product was inserted SnaBI/BlpI into that same plasmid.

NMR samples
For the NMR chemical shift titration studies of Rad2 segments with Tfb1PH, either unlabeled Rad2 359-383 or unlabeled Rad2 642-690 was added to a sample containing 0.5 mM of 15 Nlabeled Tfb1PH in NMR buffer to a final ratio of 1:1. These studies were performed in 20 mM sodium phosphate buffer pH 6.5, 1mM EDTA, 1mM DTT (NMR buffer) with

Protein expression levels in yeast
To determine the expression level of Tfb1 and the Tfb1-∆PH mutant proteins, yeast cells were grown in 15 mL of minimal media to OD 595 = 0.8-1.0 and centrifuged. The pellets were resuspended and total protein extractions were carried out as previously described (Elagoz et al.,1999). The protein extracts (20 µg) were then migrated on a 10% (wt/vol) SDS-PAGE gel and transferred onto a nitrocellulose membrane according to the manufacturer's instructions. For detection of the Tfb1 protein and its Tfb1-∆PH mutant, we used an anti-Tfb1 rabbit polyclonal antibody, a gift from Dr. Larry Myers (Dartmouth), at a dilution of 1/1,000. The secondary antibody used was the commercially available Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch Laboratories) at a dilution of 1/5,000.
For the Rad2 protein and its mutants, normal protein levels were too low to detect when expressed from their endogenous promoters. Therefore, Rad2 and all mutants were cloned into the overexpression vector pVT-101u, which is under the control of the ADH promoter. The genes were inserted XhoI/BamHI and a myc-tag was fused on their C-terminus. The Rad2 and mutants proteins levels were determined with the anti-cmyc mouse mAb 9E10 at a dilution of 1/1,000.
The Peroxidase AffiniPure Goat Anti-Mouse IgG + IgM (H+L) (Jackson ImmunoResearch Laboratories) served as the secondary antibody, used at a dilution of 1/1,000. Loading was verified with the anti-actin mouse monoclonal antibody (Abcam) at a dilution of 1/1,000.

Supplementary Tables
Supplementary

Supplementary Figures
Supplementary Figure S1. Supplementary Figure S4. Tfb1PH binds to Rad2 642-690 . Overlay of the 1 H-15 N HSQC spectra for 15 N-labeled Rad2 642-690 in its free form (red), in the presence of 0.25 equivalents (orange), 0.5 equivalent (green) and 1 equivalent (blue) of unlabeled Tfb1PH. Arrows highlight several signals that undergo significant changes in 1 H and 15 N chemical shifts upon formation of the Rad2 642-690 -Tfb1PH complex.