Architecture of the Human XPC DNA Repair and Stem Cell Coactivator Complex

Architecture of the human XPC DNA repair and stem cell coactivator complex

Elisa T. Zhanga,b,c, Yuan Hed,1, Patricia Groba,b, Yick W. Fonga,b,2, Eva Nogalesa,b,d, and Robert Tjiana,b,c,e,3

aDepartment of Molecular and Cell Biology, University of California, Berkeley, CA 94720; bHoward Hughes Medical Institute, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720; cLi Ka Shing Center for Biomedical and Health Sciences, CIRM Center of Excellence, University of California Berkeley, CA 94720; dLife Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94710; and eHoward Hughes Medical Institute, Chevy Chase, MD 20815-6789

Contributed by Robert Tjian, October 16, 2015 (sent for review August 20, 2015; reviewed by Montserrat Samso and Ning Zheng) The Xeroderma pigmentosum complementation group C (XPC) involved in base excision repair (BER). BER is responsible for complex is a versatile factor involved in both nucleotide excision removing primarily non-helix-distorting lesions from the ge- repair and transcriptional coactivation as a critical component of nome (2). In BER, the XPC complex helps repair oxidative the NANOG, OCT4, and SOX2 pluripotency gene regulatory net- damage by stimulating the activities of DNA glycosylases such work. Here we present the structure of the human holo-XPC com- as OGG1 and TDG (3) to target lesions including 8-oxoguanine, plex determined by single-particle electron microscopy to reveal a independently of other downstream GG-NER factors (17). flexible, ear-shaped structure that undergoes localized loss of order More recently, the XPC complex has also been found to upon DNA binding. We also determined the structure of the complete perform crucial duties in the regulation of gene transcription, the yeast homolog Rad4 holo-complex to find a similar overall architec- second primary function of the genome. In embryonic stem cells ture to the human complex, consistent with their shared DNA repair (ESCs), the XPC complex acts as a coactivator to enhance the functions. Localized differences between these structures reflect an expression of OCT4- and SOX2-driven pluripotency genes, most intriguing phylogenetic divergence in transcriptional capabilities that notably NANOG (4), buttressing the gene regulatory network we present here. Having positioned the constituent subunits by tag- that establishes and maintains the unique self-renewal and plu- ging and deletion, we propose a model of key interaction interfaces ripotency properties of ESCs. The XPC complex performs its

that reveals the structural basis for this difference in functional con- BIOCHEMISTRY servation. Together, our findings establish a framework for under- coactivator functions independently of DNA binding (4, 18), standing the structure-function relationships of the XPC complex in presumably by bridging interactions between the sequence-spe- the interplay between transcription and DNA repair. cific transcription factors OCT4 (octamer-binding transcription factor 4; also known as POU5F1) and SOX2 [SRY (sex- transcription | stem cells | DNA repair | structure | biochemistry determining region Y)-box 2] and the general transcriptional machinery, such as TFIID and RNA pol II, thus following a enomes of living organisms serve two primary functions: as mechanism reminiscent of that of other coactivator complexes Gvehicles for hereditary information and as the template for gene products involved in an organism’s development and re- Significance sponses to environmental stimuli. Vital to maintaining the health of genomes in the face of intrinsic and extrinsic sources of DNA Embryonic or pluripotent stem cells are unique in their ability to damage are a suite of DNA repair pathways, each dedicated to self-renew in culture and to generate all lineages of an adult or- handling specific lesions. Similarly, proper use and expression of ganism, making them valuable tools for modeling early de- this essential genomic information is regulated by a host of tran- velopmental processes and for developing regenerative medicine scription factors, chromatin remodelers, and epigenetic modifiers technologies. An important factor in controlling the expression of and readers (1). The Xeroderma pigmentosum complementation pluripotency genes is the Xeroderma pigmentosum complemen- group C (XPC) protein complex performs crucial roles in both tation group C (XPC) DNA repair complex. This study presents, to of these capacities by participating in nucleotide excision repair our knowledge, the first complete structures of different XPC complexes by electron microscopy to establish an important (NER) (2) and base excision repair (BER) (3), as well as transcrip- ’ tional regulation (4) and other processes (5). framework for a molecular understanding of XPC stwoprimary The XPC complex is one of seven XP complementation groups functions. In conjunction with our biochemical findings, we syn- thesize a model of how XPC performs both its evolutionarily A–G and is composed of the 125-kDa XPC, the 58-kDa RAD23B conserved DNA repair function and its evolutionarily noncon- (Rad23 homolog B; also known as HHR23B), and the 18-kDa served transcription function. CETN2 (Centrin2) subunits (2). RAD23B and CETN2 associate – tightly with XPC and stabilize both its DNA repair (6 10) and Author contributions: E.T.Z., E.N., and R.T. designed research; E.T.Z., Y.H., and Y.W.F. stem cell coactivator functions (4). The XPC complex is the ini- performed research; E.T.Z. and Y.W.F. contributed new reagents/analytic tools; E.T.Z., tiator and main DNA damage sensor in global genome nucleotide Y.H., P.G., and Y.W.F. analyzed data; and E.T.Z., E.N., and R.T. wrote the paper. excision repair (GG-NER), one of two branches of the nucleotide Reviewers: M.S., Virginia Commonwealth University; and N.Z., University of Washington. excision repair pathway that repairs a wide array of bulky, helix- The authors declare no conflict of interest. distorting lesions (2, 11); the second form of NER or transcrip- Data deposition: Atomic coordinates and structure factors have been deposited in the tion-coupled repair (TC-NER) targets lesions blocking tran- Electron Microscopy Data Bank (EMDB), https://www.ebi.ac.uk/pdbe/emdb/ (accession scription to reactivate proper gene expression (2, 12). Defects nos. EMD-6495–EMD-6498). 1 in GG-NER lead to photosensitivity and a predisposition to cer- Present address: Department of Molecular Biosciences, Northwestern University, Evanston, IL 60208. tain cancers in animal models and in human patients with Xero- 2Present address: Brigham Regenerative Medicine Center, Cardiovascular Division, De- derma pigmentosum (13). In conjunction with the UV-damage partment of Medicine Brigham and Women’s Hospital, Harvard Medical School, Cam- DNA-binding protein (UV-DDB) (2, 12), the XPC complex bridge, MA 02139. recruits >30 downstream factors, such as XPA (14), TFIIH (15, 3To whom correspondence should be addressed. Email: [email protected]. 16), and the endonucleases XPF and XPG to remove these ad- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ducts (2, 11). In addition to its role in GG-NER, XPC is also 1073/pnas.1520104112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1520104112 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 such as Mediator and p300/CBP (19). In a separate study, XPC TFIIH-BD1 DNA-BD A 124 144 607 742 was found to localize to active but not to inactive RNA pol II- XPA- & OGG1-BD OCT4-BD SOX2-BD 1 154 331 511 808 940 dependent gene promoters in the absence of exogenous geno- XPC TGD TGD BH1 BH2 BH3 204 223 496 734 814 toxic stress (11, 20). RAD23B-BD2 TFIIH-BD2 RAD23B-BD1 847 863 Although both the DNA repair and transcriptional functions 1 276 334 419 CETN2-BD RAD23B UbL of mammalian XPC complexes have been characterized bio- UBA1 XPC-BD UBA2 chemically and genetically, 3D structural information of the 1 172 CETN2 EF EF holo-complex has been unavailable. The partial crystal structure 105 166 XPC-BD of Rad4, the yeast homolog of the human XPC subunit, in B D R C complex with the Rad4-interaction domain of yeast Rad23, has XPC provided information helpful toward an understanding of Rad4/ XPC’s biochemical behavior and of certain phenotypic outcomes 180° 180° ’

(18, 21). However, given XPC s low sequence homology with the 170 Å yeast homolog Rad4 (21, 22), the absence of key domains in the available crystal structure, and the divergence of requirements for

transcriptional vs. repair activities (4, 15, 18, 23), structural in- 100 Å formation of the complete, three-subunit, native human XPC R MBP C C E complex is much needed for an understanding of the functional XPC versatility of the XPC complex. At present, no information has 180° been reported on the overall architecture of the XPC holo- 180°

complex, the possible large-scale conformational rearrangements UbL in XPC upon DNA-binding, or the extent of structural conser- UBA1 vation of the human XPC complex with homolog complexes. UBA2 Here we address the lack of structural data on human XPC using 3D single-particle reconstruction by electron microscopy Fig. 1. 3D reconstruction of the human XPC complex and localization of (EM) to characterize the overall architecture of XPC, as well as subunits. (A) Schematic representation of the subunits and domains of the genetic tagging and computational docking to locate the relative human XPC complex. Transglutaminase homology domain (TGD), β-hairpin positions of its constituent subunits. We also assess the confor- domains 1–3 (BH), ubiquitin-like domain (UbL), ubiquitin-associated domains 1 mational changes to the complex upon binding to DNA. Given and 2 (UBA), EF-hand domains (EF), and protein- and DNA-binding domains the evolutionary conservation of GG-NER (24), we queried the (BD) are indicated accordingly. (B) Front and back views of the XPC complex extent of structural and functional conservation over evolution- reconstructed in EMAN2 (49). Estimated dimensions are indicated. (C) Positive σ ary time by solving the structure of the complete yeast homolog (yellow) and negative (purple) 3D difference densities at 4 between the Rad4 complex and testing whether the OCT4/SOX2 transcrip- complex containing MBP-CETN2 and untagged complex. (D) Positive (pink) 3D difference density at 5σ between the full complex and the XPC-RAD23B sub- tional coactivation function is supported by the yeast complex. – complex, indicating the likely position for the CETN2 subunit. No negative Together with existing biochemical data (14 16, 23, 25), we difference density was observed at this threshold. (E) Docking of the yeast sought to identify the approximate regions of contact between Rad4/Rad23 [Protein Data Bank (PDB) ID 2QSF; cyan/green] into the model the XPC complex and its partner proteins OCT4, SOX2, XPA, with the human CETN2 and XPC interaction peptide (PDB ID 2GGM; pink/cyan) and TFIIH. by Situs (31) in a manner consistent with the difference density data in A and B. Shown are predicted approximate positions of the UbL, UBA1, and UBA2 Results domains of RAD23B based on positional information of the Rad23Rad4-BD (dark Reconstitution of the Human XPC Complex. We purified the com- green) N and C termini in the crystal structure. plete, three-subunit human XPC-RAD23B-CETN2 complex (Fig. 1A) expressed in Sf9 insect cells using a two-step affinity purifi- elongated appearance. Such elongated shapes would be consistent cation procedure (Fig. S1 A and B). SDS/PAGE analysis indicated that the purified complex was nearly homogeneous and stoichio- with our observation that the XPC complex runs as a relatively broad peak centered at 275 kDa on a size-exclusion column, metric (Fig. S1B, Left). This result is consistent with previous data ∼ showing that XPC and RAD23B interact in a 1:1 ratio (26) and slightly larger than its mass of 200 kDa (Fig. 1A and Fig. S1 B that XPC and CETN2 also interact in a 1:1 ratio (27, 28). Fur- and C). thermore, a 1:1:1 stoichiometry is consistent with the size of the Ab Initio 3D Reconstruction of the Human XPC Complex by Random ∼200-kDa product we observe for the cross-linked complex (Fig. Conical Tilt and Subunit Localization. We used random conical tilt S1B, Right). – Initial attempts at negative stain EM data collection were (29) (Fig. S1 D G)togenerateanabinitio3Dreconstructionof hampered by the extremely heterogeneous appearance of the the human XPC complex (Fig. 1B and Fig. S2). Handedness and particles in both size and shape (data not shown). These results robustness of our EM reconstruction was supported by the results suggested that the complex was either unstable during EM sample of the freehand test (30) using projection-matching of particle preparation and/or suffered from extreme conformational flexi- pairs at 0° and 30° tilt, which indicated that 40% of particles fall bility. To overcome these limitations, we optimized cross-linking within 26 degrees of the expected tilt angle (Fig. S3D). The struc- ∼ ∼ ∼ conditions across temperature, incubation time, and cross-linker ture is 170 Å by 100 Å by 70 Å and roughly resembles the concentration to identify the minimum requirements for achieving shapeofahumanear(Fig.1B). To localize the position of the complete subunit incorporation as detected by Coomassie staining CETN2 subunit within the reconstruction, we followed two par- (for more details, see SI Materials and Methods, Expression and allel strategies: visualization of a complex that included a maltose- Purification of XPC/Rad4 Complexes). These particular conditions binding protein (MBP) tag at the N terminus of CETN2, and were then used for subsequent negative stain sample preparation, visualization of a complex lacking the CETN2 subunit (Fig. 1 C followed by data collection and single particle analysis (Fig. S1B, and D and Fig. S2 F and G). As seen in the 3D difference maps, the Right and Fig. S2A). MBP density is localized primarily outside the shorter end or ear- Two-dimensional reference-free class averages (Fig. S2B) show lobe of the XPC complex, whereas the CETN2 density is localized C-shaped views, multilobed structures, and some very small, primarily inside the earlobe (Fig. 1 C and D). Consistent with this compact, globular shapes, with most of the particles having an localization of CETN2, the crystal structure of Rad4/Rad23Rad4-BD

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1520104112 Zhang et al. Downloaded by guest on September 25, 2021 docked (31) into the upper end of the “ear” in an orientation such A that the C terminus of Rad4 points downward toward the “earlobe.” In further agreement with this subunit organization, docking of the CETN2 was placed in the earlobe by objective, automatic docking using Situs (31) (Fig. 1E). Based on the orientation of 90° 90° the Rad23 termini observed in the Rad4/Rad23 crystal structure, we also indicate the predicted, approximate locations of the UbL, UBA1, and UBA2 subunits of RAD23B that are not observed in the Rad4/Rad23 crystal structure (Fig. 1E). To ensure that the cross-linking and the use of stain did not significantly compromise the integrity of the structure obtained, EM Densities: Docked structures: B we also analyzed the native, uncross-linked complex and used apo apo 3D cryo-electron microscopy (cryo-EM). The native complex in DNA-bound DNA-bound [apo] - [bound] Rad23 (apo) 2D phosphotungstate stain and the cross-linked complex under cryo [bound] - [apo] Rad23 (bound) conditions were all consistent with the cross-linked complex under negative stain conditions (Fig. 1B and Fig. S3 A–C). Al- Fig. 3. DNA binding by XPC is accompanied by large but localized confor- though the native complex appears to have better definition mational changes distal from the sites of presumed DNA contact. (A) Front, between the earlobe and the rest of the structure, this difference side, and back views of the XPC complex bound to a mismatch bubble sub- σ strate generated in RELION (32) (yellow) shown with the apo structure (mesh density is only present at 2 and not at the statistically significant − σ σ gray), with the 3D [apo] [DNA-bound] difference density at 3 (cyan), and 3 (Fig. S3C). with the [DNA-bound] − [apo] difference density (brown). Also shown is the The low resolution of our reconstruction (∼25 Å) suggested Situs-based docking (31) of the yeast Rad4/Rad23 apo (PDB ID 2QSF; purple/ that the XPC complex may be flexible and adopt multiple con- orange) structure with the DNA-bound (PDB ID 2QSH; green/yellow) structure formations. To gain an understanding of the possible range of aligned to the apo via the Rad23 subunit. (B) Comparison of 3D reprojections conformational states, we used a large data set of ∼210,000 of the model to their corresponding reference-free 2D class averages. particles and performed 3D sorting and classification using RELION (32) to produce three distinct structures representing one of the strongest affinities for the XPC complex. Comparison the range of XPC conformations (Fig. 2A), followed by Situs of the reconstructions obtained from the apo vs. DNA-bound BIOCHEMISTRY docking using available crystal structures. The XPC complex ap- samples indicates that the addition of DNA primarily affected pears to be partitioned between a more elongated (Fig. 2A, Top) the density region immediately above the “earlobe” (Fig. 3A). and more compact states (Fig. 2A, Middle and Bottom). Three- Addition of DNA to XPC did not appear to lock the structure dimensional reprojections of these three models match reference- into a single conformation because the 2D class averages (Fig. free 2D class averages (Fig. 2B). 3B) and the resolution of ∼24Åaresimilartothoseweobserved Structural Changes Following DNA Binding. To visualize possible for the apo complex. The changes observed are consistent with a structural changes in the human XPC complex upon binding to possible movement of the BH domains of XPC toward the region DNA, we used a monomeric avidin and biotinylated-DNA af- of DNA-binding. This assessment isbasedoncomparisonsbetween finity purification strategy to isolate only DNA-bound XPC mol- the changes imposed by DNA binding in the Rad4 crystal structure ecules (Fig. S4 A and B). The 48-bp DNA bubble mismatch duplex and the position of the crystal structure docked into the EM density was chosen from a validated EMSA probe (22) that demonstrated either as the intact Rad4/Rad23 crystal structure (Fig. 3A)orwith the C-terminal portion of the Rad4 TGD domain considered sep- arately from the N-terminal portion to better reflect structural homology with the human XPC, which contains an insertion in its A B TGD domain (Figs. S4C and S5 A and B). A second interesting possibility is that certain regions of RAD23B become disordered 180° 3D upon DNA-binding, thus resulting in the observed loss of density; this is consistent with the finding that some regions of Rad23 that 2D are ordered in the apo Rad4/Rad23 crystal structure becoming disordered upon binding DNA (21). The XPC complex has been demonstrated to bind other substrates as well, such as single-stranded DNA (33). Therefore, we also prepared ssDNA-bound XPC molecules using the same 3D 180° strategy of biotinylated-DNA pull-down (Fig. S4 A and B). Three- dimensional difference density analysis indicates that this structure 2D is nearly indistinguishable from the mismatch-DNA-bound XPC complex (Fig. S4D).

Conservation of Structure and Function. The human XPC complex (XPC, RAD23B, CETN2) and the yeast homolog Rad4 complex 180° 3D (Rad4, Rad23, Rad33; ref. 34) appear to function equivalently in nucleotide excision repair, given their similar binding properties 2D to damaged DNA (22). These two complexes are also thought to be structurally similar based on strong sequence homology between human RAD23B and yeast Rad23 (6) and between human Fig. 2. The XPC complex adopts highly flexible conformations. (A) Three models of the XPC complex generated in RELION with Situs-based docking (31) of the CETN2 and the purported yeast CETN2 homolog Rad33 (Fig. yeast Rad4/Rad23 (PDB ID 2QSF; cyan/green) and the human CETN2/XPCCETN2-BD S5C). Despite low sequence conservation overall between XPC (PDB ID 2GGM; pink/cyan) crystal structures. (B) Comparison of 3D reprojections and Rad4, these two proteins share sequence homology of key of the models to their corresponding reference-free 2D class averages. domains (35) and very similar predicted secondary structures

Zhang et al. PNAS Early Edition | 3of6 Downloaded by guest on September 25, 2021 (Fig. S5 A and B). To examine this question of structural con- A OCT4 servation, we obtained a 3D reconstruction at ∼23 Å resolution SOX2 of the complete yeast Rad4 complex (Fig. 4 A and B). The XPAXPA overall architecture of the complexes from the two species is OGG1 – remarkably similar at both the 3D and 2D levels (Fig. 4 B D, 180° Right vs. Fig. 1B and Fig. S2 B and E), although there are small areas of difference between the human and yeast complexes, as seen in the 3D difference maps (Fig. 4C). We posit that the region of difference density in the earlobe may be due to structural differences between the CETN2 and Rad33 homologs TFIIH (Fig. 1E and Fig. S5C). The structural similarity between the human and yeast XPC/ Rad4 complexes suggested that other functions of the XPC B complex, in particular its transcriptional roles in ES cells, might also be conserved. To our surprise, we observed that unlike the human and mouse XPC complexes, the Rad4 complex exhibited no coactivator activity in our in vitro transcription assay (Fig. 4E) and was completely incapable of forming a stable interaction with SOX2, the primary requisite activator for in vitro activation BD4 of NANOG gene transcription (4) (Fig. 4F). Using information BD3 on XPC’s interaction domains with partner proteins (14, 15, 25,

27, 36), sequence homology between yeast Rad4 and human BD5 XPC (Fig. S5), as well as the docking of the Rad4/Rad23 crystal structure, we were able to generate a model indicating the predicted locations of the interaction domains on the XPC complex EM densities: Docked crystal structures: apo XPA- and OGG1-BD (by homology) (Fig. 5A). These interaction domains are clustered on the top of [human] – [yeast] OCT4-BD (by homology) the ear. Intriguingly, when we superimpose the regions of differ- SOX2-BD (by homology) ence density between the human and yeast EM maps with the DNA-binding residues predicted interaction domains, we note that one such region is Fig. 5. Model of key interaction interfaces on the XPC complex. (A)Modelof located near the OCT4- and SOX2-binding interfaces but not the predicted interaction surfaces based on docking and sequence homology with the yeast Rad4/Rad23 crystal structure (PDB ID 2QSF). Indicated are residues involved in binding to XPA (14) (blue; yeast residues 101–296), OCT4 (25) (red; yeast residues 298–392), SOX2 (25) (orange; yeast residues 392–609), and TFIIH A glutaraldehyde B D 2D3D – – - + (15, 16, 44) (violet; human residues 847 863 and yeast residues 76 115 and – 180° 610 631). (B) Top back view of the XPC complex showing an area of positive difference density between the human and yeast structures (yellow) that co- 250 incides with the predicted OCT4- (red) and SOX2-binding domains (orange) 150 but not the DNA-binding residues (dark blue residues and circles; BD3-5). The 100 Rad4 XPA binding domain has been omitted in the enlarged view for clarity. 75

Rad23 C 50 DNA-binding residues in regions DNA-BD3-5 (Fig. 5B), sug- 37 180° gesting that these regions of difference may underlie the phylo- 25 20 Rad33 genetic divergence in transcriptional activity between the human 15 and yeast complexes. 10 Discussion

EFE yeast mouse human IP: HA-SOX2 IP: HA-RFP Our single particle analyses reveal an ear-like shape for the human - XPC Rad4 XPC Rad4 XPC complex and indicate the existence of a range of conforma- IN IP IN IP IN IP IN IP tional states for this DNA repair and stem cell coactivator complex IB: His XPC/Rad4 (Figs. 1 and 2). We show that the yeast homolog Rad4 holo-com- IB: FLAG plex has a similar overall architecture but small regions of difference RAD23(B) compared with the human XPC complex that may reflect their -140 OS OS IB: HA functional nonequivalence in biochemical assays (Fig. 4). Using ( ) NanogCAT 4x labeling, mutational, and docking strategies, we localize the indi- vidual subunits of the complex within the structure (Fig. 1). The Fig. 4. Comparative studies of the human and yeast XPC/Rad4 complexes re- binding to the two distinct DNA substrates used in this study reveal divergence in function but not structure. (A) Purification and cross-linking of the homologous yeast Rad4-Rad23-Rad33 complex. (B) Three-dimensional sulted in a similar overall conformational change in the left domain model of the yeast Rad4 complex as solved by EMAN2 (49). (C) Three- immediately above the earlobe, which is consistent with previous dimensional difference density at 3σ between the human (mesh pink) and the observations in the Rad4/Rad23 crystal structures (21) (Fig. 3). yeast (solid gray) complexes. Positive difference density, or [human]-[yeast], is Our study reveals the apparent flexibility of the XPC complex, shown in cyan; negative difference density or [yeast] − [human] is shown in in large part mirroring its functional versatility. The flexibility of purple. (D) Comparisons of 3D reprojections of the yeast Rad4 complex with 2D the complex may stem, at least in part, from RAD23B, because class averages. (E) Titrations over a fourfold concentration range of yeast, certain regions of Rad23 were found to be disordered in the Rad4/ mouse, and human XPC homolog complexes in in vitro transcription reactions of a NANOG promoter template engineered with four extra copies of the oct-sox Rad23 crystal structure (21). Part of the conformational hetero- composite binding element (bottom), performed in the presence of OCT4 and geneity seen in our EM structures may be due to variations in the SOX2 protein. Transcripts are indicated with arrowheads. (F) Coimmunopreci- interaction between the UbL and the equivalent UBA1 and pitation of human and yeast XPC/Rad4 complexes with HA-tagged SOX2 or RFP. UBA2 domains of RAD23B (Fig. 1A). Some of the conformational

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1520104112 Zhang et al. Downloaded by guest on September 25, 2021 variability may also originate from the XPC subunit. Structure relevant in yeast. The extent of structural conservation is consistent prediction analysis (14), limited proteolysis (14), and NMR (37) with their equivalence in repair (22). Regions displaying differ- identified several highly disordered regions: the N terminus (resi- ences may reflect the divergence in functional capabilities that we dues 1–154), the C terminus (residues 816–940), and a loop in- observe in a transcriptional context (Fig. 4). Indeed, one of these serted into the TGD domain comprising residues 331–517. Finally, regions at the top portion of the ear corresponds to residues ho- CETN2 may also contribute to this overall flexibility, because it can mologous to those shown to interact with OCT4 and SOX2 (Fig. adopt different conformations depending on its metal-binding state 5B, yellow). Importantly, this region is well separated from DNA- (38); however, the resolution of the XPC-RAD23B subcomplex binding residues (Fig. 5B, circled BD3-5; key residues in dark was not markedly improved, suggesting that this contribution to blue). The only close-by DNA-binding residues (BD3) are in fact complex flexibility is minor, as would be expected for its relatively not conserved and are not found in the human sequence (Fig. small mass contribution to the complex. The recently described S5B). This structural separation-of-function suggests that the requirement of RNA for the XPC complex to interact with its local degree of structural conservation, even at modest resolu- transcription partner SOX2 (25) invokes the idea of low-complexity tion, is predictive of functional convergence or divergence. From domains or regions, perhaps interspersed throughout XPC, linking an evolutionary point of view, a conserved process, such as their inherent flexibility to a critical aspect of the XPC complex’s nucleotide excision repair, would be expected to exhibit func- function. The possibility that the mammalian-specific insertion tional conservation between the yeast and human XPC homologs, within the TGD domain (residues 331–517; Fig. 1A and Fig. S5B) whereas a nonconserved process, such as regulating genes could be partly responsible for some of the observed structural expressed in mammalian embryonic stem cells, would not. Indeed, heterogeneity is particularly interesting. a number of XPC mutations that have differential effects in DNA With the use of an MBP-tag as a labeling strategy, as well as repair vs. transcription support this idea. For instance, although the exclusion of CETN2 from the complex, we were able to lo- deletion of the N-terminal UbL domain of yRad23 (42) and the calize CETN2 to the earlobe of the structure (Fig. 1 C and D). W690S mutation of XPC (18, 43) have adverse consequences on Extending these findings, we used rigid-body docking in an un- nucleotide excision repair capabilities, respective mutations in biased manner to place the Rad4/Rad23 crystal structure at the XPC did not affect the ability to coactivate transcription (4) (Y.W. top of the ear and the CETN2 crystal structure in the earlobe F., unpublished). Similarly, although the N and C termini of XPC (Fig. 1E). Attempts to tag the XPC and RAD23B subunits were are critical for recruitment and stimulation of TFIIH at sites of not successful for a variety of reasons. The absence of CETN2 damage for global nucleotide excision repair (15, 16, 44, 45), the

does not appear to impose large conformational rearrangements, removal of the N- and C-terminal TFIIH-binding domains of XPC BIOCHEMISTRY as seen by the comparison between the full complex and the (residues 1–195 and 814–940, respectively) only impacts repair but XPC-RAD23B subcomplex (Fig. 1 B and D). This observation is not transcriptional activity (4, 25). consistent with the lesser functional consequence of removing Reflecting the ever-expanding repertoire of reported XPC CETN2 than that of removing RAD23B in transcriptional coac- roles is the number of known physical and functional interactors tivation assays, as well as with the inconsequential removal of the of the XPC complex, e.g., TFIIH (15, 16), OGG1 (23), TDG (3), C-terminal CETN2-interaction domain on XPC (residues 814– SOX2 (4, 25) (Fig. 4), and OCT4 (4, 25), among others (46). It is 940) (4). possible that the XPC complex serves as a coactivator not just for The similarity between the dsDNA- and ssDNA-bound struc- OCT4 and SOX2, especially given that its transcriptional activ- tures is consistent with the fact that the XPC complex is capable of ities do not appear to be cell-type-restrained (20); therefore, the binding a large suite of different DNA structures, including UV- list of XPC’s functional and physical partners is likely to grow. induced thymine dimers (2), mismatch bubbles (22), ssDNA– Although the residues on XPC through which some of these dsDNA junctions (39), apurinic/apyrimidic (AP) sites (40), and known interactions occur have been mapped, there is a degree of even undamaged duplex and certain single-stranded DNA substrates overlap between some of these regions, suggesting the need for (33). This similarity between different DNA-bound structures is also more fine-tuned characterization and structural elucidation in consistent with recent work describing a kinetic but not structural the future (Fig. 1A). Our recent work describing the involve- means of discrimination between damaged and undamaged DNA by ment of RNA in mediating the XPC-SOX2 interaction adds an Rad4 (41). The reduction of density in our DNA-bound structures additional and potentially intriguing dimension to future struc- compared with the apo XPC complex reflects a conformational tural studies in this regard (25). Additionally, it would be in- change consistent with two phenomena observed in the Rad4 teresting to explore whether structural changes are imposed on crystal structure: (i) the C-terminal portion of Rad4 shifting the XPC complex upon binding to its partner proteins; further toward the DNA substrate, and in the context of our docking, biochemical and structural work to assemble such larger pro- away from the region of reduced density (Fig. 3A and Fig. S4 C tein assemblies is required. In summary, this work provides a and E); (ii) portions of Rad23 originally contributing to ordered structural framework for integrating biochemical and struc- density in the apo crystal structure becoming disordered upon tural information into a mechanistic understanding of the XPC binding to DNA (21). We are inclined to favor this latter ob- complex’s undoubtedly complicated roles in DNA repair and tran- servation to explain the DNA-induced changes to the XPC com- scriptional regulation. plex because the primary EM density difference we observe cannot easily be accounted for by the modest, ∼13- to 14-Å shift Materials and Methods in Rad4 observed in the crystal structure (Fig. S5E). Therefore, Detailed methods can be found in SI Materials and Methods. XPC/Rad4 we propose that DNA binding by the XPC complex induces complexes were affinity purified from Sf9 cells. DNA-bound XPC samples specific conformational changes and disorder of certain domains, were affinity purified using biotinylated DNA substrates. EM was performed possibly such as the UbL domain (Fig. 1E). using continuous carbon films and uranyl formate or phosphotungstate The overall similarities between the human XPC structure and stain for negative stain. Leginon software (47) was used to collect images in the yeast Rad4 structure, despite their divergent amino acid se- a Tecnai F20 microscope equipped with a Gatan UltraScan 4000 camera. quences, provide additional, indirect validation of the accuracies Data processing was performed primarily in the Appion pipeline (48). Three- dimensional reconstructions were performed using EMAN2 (49) and RELION of the 3D models. Indeed, it seems quite remarkable that without (32). Coimmunoprecipitation assays were performed in HEK293T cells. In requiring major changes to the overall evolutionarily conserved vitro transcription assays were performed essentially as described (50). 3D shape and structure of the mammalian XPC complex, it has nevertheless adopted entirely new transcriptional coactivator func- ACKNOWLEDGMENTS. We thank G. Kemalyan, R. Louder, S. Howes, and tions in the context of ES cell regulatory pathways that are not D. W. Taylor for microscope and data processing guidance; T. Houweling for

Zhang et al. PNAS Early Edition | 5of6 Downloaded by guest on September 25, 2021 computer support; G. Dailey for help with expression constructs; and S. Zheng Grant RB4-06016 (to R.T.) and by the National Institute of General Medical and C. Inouye for help with in vitro transcription components. We are Sciences (GM63072; to E.N.). E.T.Z. was a National Science Foundation Grad- grateful to M. Iadanza and T. Gonen for help with initial negative stain uate Research Fellow and a University of California Berkeley Distinguished analysis. We thank R. Lesch and R. Schekman for yeast cells for cloning the Fellow. Y.W.F. was a CIRM Scholar (Training Grant T1-00007). R.T. and E.N. yeast Rad4 complex. We thank C. Cattoglio, J. J. Ho, G. E. Katibah, D. C. Rio, are Howard Hughes Medical Institute (HHMI) Investigators. R.T. is the Pres- A. Martin, and D. E. Wemmer for valuable discussion. This work was sup- ident of HHMI and the Director of the Li Ka Shing Center for Biomedical and ported by the California Institute for Regenerative Medicine (CIRM) Research Health Sciences.

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