Structural basis of TFIIH activation for nucleotide excision repair

Goran Kokic,Aleksandar Chernev,Henning Urlaub,Dimitry Tegunov,Christian Dienemann,Patrick Cramer

doi:10.1038/s41467-019-10745-5

Abstract

Nucleotide excision repair (NER) is the major DNA repair pathway that removes UV-induced and bulky DNA lesions. There is currently no structure of NER intermediates, which form around the large multisubunit transcription factor IIH (TFIIH). Here we report the cryo-EM structure of an NER intermediate containing TFIIH and the NER factor XPA. Compared to its transcription conformation, the TFIIH structure is rearranged such that its ATPase subunits XPB and XPD bind double- and single-stranded DNA, consistent with their translocase and helicase activities, respectively. XPA releases the inhibitory kinase module of TFIIH, displaces a ‘plug’ element from the DNA-binding pore in XPD, and together with the NER factor XPG stimulates XPD activity. Our results explain how TFIIH is switched from a transcription to a repair factor, and provide the basis for a mechanistic analysis of the NER pathway.

Highlights

Nucleotide excision repair (NER) is the major DNA repair pathway that removes UV-induced and bulky DNA lesions
A mechanistic dissection of the NER machinery was far hampered because transcription factor IIH (TFIIH) was not available in large quantities
Translocase activity was due to XPB because it was sensitive to triptolide (Supplementary Fig. 1c), a drug that targets XPB10

Summary

Results

The purified TFIIH core comprised seven subunits including the ATPases XPB and XPD, whereas the kinase module contained CDK7, cyclin H, and MAT1. Core TFIIH showed 5′–3′ helicase activity, which was lost upon mutation of the XPD active site (Fig. 1a, Supplementary Fig. 1b). To test whether other NER factors affect TFIIH activities, we purified human XPA, XPG, RPA, and XPF-ERCC1 complex (Supplementary Fig. 1d). In our structure DNA is held in a positively charged DNA duplex tunnel that is formed between the extended helix of XPA and the XPB ATPase (Fig. 3a). Our cryoEM data revealed an alternative state of the complex with the DNA duplex disengaged from XPB but retained by the XPA extended helix (Fig. 3b, Supplementary Fig. 2d). Previous studies of the yeast XPA counterpart suggested that XPA may detect DNA lesions[21]

Helix bundle p62

Discussion

Methods