Structural basis of TFIIH activation for nucleotide excision repair

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.

Single molecule analysis reveals monomeric XPA bends DNA and undergoes episodic linear diffusion during damage search

“…MSD analysis revealed that the long-range mode of linear diffusion had a significantly higher diffusion coefficient than the short-range mode. Although XPA has been reported to bind DNA as a homodimer on short DNA substrates 9 , volumes of XPA on a 538 bp DNA substrate measured by AFM in this study are consistent with the size of a monomer, in support of previous reports 15,21,36 . We also observed formation of a "dimer band" at high concentrations by EMSA, but this band is indistinguishable from a complex containing one XPA bound at the lesion and one bound at the DNA end (or two otherwise distinct monomeric binding events).…”

“…XPA binds non-damaged DNA and dG-C8-AAF as a monomer. Previous reports suggest that XPA may bind DNA as a homodimer 9,35 while others indicate monomeric binding 15,21,36 . AFM offers the unique ability to distinguish between distinct complexes (e.g., one protein at a DNA end and one protein at a lesion) and true dimerization.…”

“…Early literature on the function of XPA in NER suggests that it is involved in initial steps of damage recognition 17,18 . Additional data place XPA at the damage verification step, interacting with and enhancing damage specificity of TFIIH [19][20][21] , or even later in the pathway 22 , acting as a scaffold and interacting with other NER proteins 23 , including RPA 24 . Importantly, it has been suggested that damage recognition in NER, which needs to accommodate a diverse range of structures, is accomplished via a "discrimination cascade" involving multiple proteins with imperfect selectivity 7,25,26 .…”

“…30 ), covering about half of the total protein length and a zincfinger motif. Available XPA structures include early solution NMR studies of human XPA DBD 31,32 , a recent crystal structure of the extended human DBD 33 , co-crystal structures of yeast Rad14 (XPA homolog) DBD on damaged DNA 10,34 , and a cryoelectron microscopy structure of the complex formed between XPA, TFIIH, and DNA 21 . At present, there is no structural information regarding the N-and C-termini of XPA or its homologs in the RCSB Protein Data Bank.…”

“…This result supports previous studies which concluded that XPA binds DNA as a homodimer 8,9,35 . XPA stoichiometry both on and off of DNA remains controversial as others have reported monomeric binding 15,21,36 , or that stoichiometry is dependent on the DNA substrate 16 .…”