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 transcrip- tion 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).