The XBP-Bax1 Helicase-Nuclease Complex Unwinds and Cleaves DNA

Abstract

XPB helicase is an integral part of transcription factor TFIIH, required for both transcription initiation and nucleotide excision repair (NER). Along with the XPD helicase, XPB plays a crucial but only partly understood role in defining and extending the DNA repair bubble around lesions in NER. Archaea encode clear homologues of XPB and XPD, and structural studies of these proteins have yielded key insights relevant to the eukaryal system. Here we show that archaeal XPB functions with a structure-specific nuclease, Bax1, as a helicase-nuclease machine that unwinds and cleaves model NER substrates. DNA bubbles are extended by XPB and cleaved by Bax1 at a position equivalent to that cut by the XPG nuclease in eukaryal NER. The helicase activity of archaeal XPB is dependent on the conserved Thumb domain, which may act as the helix breaker. The N-terminal damage recognition domain of XPB is shown to be crucial for XPB-Bax1 activity and may be unique to the archaea. These findings have implications for the role of XPB in both archaeal and eukaryal NER and for the evolution of the NER pathway. XPB is shown to be a very limited helicase that can act on small DNA bubbles and open a defined region of the DNA duplex. The specialized functions of the accessory domains of XPB are now more clearly delineated. This is also the first direct demonstration of a repair function for archaeal XPB and suggests strongly that the role of XPB in transcription occurred later in evolution than that in repair.The superfamily 2 helicase XPB (Rad25 in Saccharomyces cerevisiae) is an essential component of transcription factor TFIIH. The ATPase activity of XPB is required for both nucleotide excision repair (NER) 2 and transcription initiation from RNA polymerase II promoters (1, 2). The NER pathway is a highly flexible system that is required for the detection and removal of a wide variety of bulky and helix-distorting lesions, including photoproducts. Mutations in the xpb or xpd genes in humans can cause the serious genetic diseases xeroderma pigmentosum, trichothiodystrophy, and Cockayne's syndrome, due to defects in both transcription and repair (reviewed in Ref.3). Examples of xpb mutations in humans are much more rare than those seen in the xpd gene, probably due to the crucial role of the XPB protein in basal transcription (4).Although the ATPase activities of both proteins are required for NER (5), the respective roles of the XPB and XPD helicase components of TFIIH are still a matter of debate. XPD is the more robust helicase (6), and it has been suggested to bind 5Ј of the DNA lesion and translocate in a 5Ј to 3Ј direction toward the damage site, potentially acting as a sensor or proofreader of DNA damage for the NER pathway either by jamming directly on DNA lesions (7) or perhaps through damage sensing by its iron-sulfur cluster binding domain (8, 9). However, little direct evidence exists in support of these possibilities at present, and indeed it is not yet clear whether XPD or XPB binds first at repair sites or whether they bi...

Structural basis of the XPB helicase–Bax1 nuclease complex interacting with the repair bubble DNA

“…Furthermore, the ThM motif of XPB intrudes between the two ssDNA tails like a wedge with the 3′-overhang extending through the channel formed by the HD2/ThM of XPB (Figures 1D and 2A and B ) and the 5′-overhang extending into the space between two N-terminal β-hairpins of Bax1 ΔC (Figures 1D and 2A ). These observations are consistent with the 3′–5′ helicase polarity of archaeal XPB ( 7 ) (moving along the 3′-overhang strand toward the fork junction) and the nuclease activity of Bax1 on the DNA substrate containing a 5′-overhang ( 33 ) in vitro . Neither XPB nor Bax1 ΔC interacts with the remaining nucleotides of the two ssDNA tails further away from the fork, leading to poor electron density for this portion of the DNA.…”

The complete structure of the human TFIIH core complex

“…Our structure assigns XPB residues 165–300 to a DRD-like domain that connects the NTD to the RecA-like domain ( Figure 3A , Figure 3—figure supplement 1A ), the deletion of which is lethal in yeast ( Warfield et al, 2016 ). The DRD is a DNA-binding domain found in DNA repair enzymes and chromatin remodelers ( Mason et al, 2014 ; Obmolova et al, 2000 ) and has been implicated in DNA damage recognition in archaeal XPB ( Fan et al, 2006 ; Rouillon and White, 2010 ). Our 3.7 Å-resolution map of TFIIH reveals that in eukaryotic XPB, one β-strand of the DRD of archaeal XPB is replaced by an insertion of approximately 70 residues that exhibits relatively low sequence conservation ( Figure 3E , Figure 3—figure supplement 1B ) and shifts the domain boundaries of the human XPB DRD-like domain with respect to previous sequence alignments ( Fan et al, 2006 ; Oksenych et al, 2009 ).…”

XPB and XPD helicases in TFIIH orchestrate DNA duplex opening and damage verification to coordinate repair with transcription and cell cycle via CAK kinase

“…Furthermore, GFP tagged mutants were defective in recruitment to sites of UV damage in CHO cells, suggesting that recruitment of XPB to damage is an active process requiring ATP hydrolysis coupled with structural stabilization of XPB on DNA by the ThM domain and RED motif [108]. In vitro experiments using mutant Sulfolobus solfataricus XPB (SsoXPB) proteins, found that the RED motif is involved but not essential for SsoXPB function while both the ThM and DRD domains were essential for XPB function [109], confirming the importance of these accessory domains and motifs for XPB function.…”