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Human XPG Nuclease Structure, Assembly, and Activities with Insights for Neurodegeneration and Cancer from Pathogenic Mutations

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Human XPG Nuclease Structure, Assembly, and Activities with Insights for Neurodegeneration and Cancer from Pathogenic Mutations Human XPG nuclease structure, assembly, and activities with insights for neurodegeneration and cancer from pathogenic mutations Susan E. Tsutakawaa,1,2, Altaf H. Sarkerb,1, Clifford Ngb, Andrew S. Arvaic, David S. Shina, Brian Shihb, Shuai Jiangb, Aye C. Thwinb, Miaw-Sheue Tsaib, Alexandra Willcoxd,3, Mai Zong Hera, Kelly S. Tregob, Alan G. Raetzb,4, Daniel Rosenberga, Albino Bacollae,f, Michal Hammela, Jack D. Griffithd,2, Priscilla K. Cooperb,2, and John A. Tainera,e,f,2 aMolecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; bBiological Systems and Engineering, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; cIntegrative Structural & Computational Biology, The Scripps Research Institute, La Jolla, CA 92037; dLineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599 eDepartment of Cancer Biology, University of Texas MD Anderson Cancer Center, Houston, TX 77030; and fDepartment of Molecular and Cellular Oncology, University of Texas MD Anderson Cancer Center, Houston, TX 77030 Contributed by Jack D. Griffith, March 19, 2020 (sent for review December 9, 2019; reviewed by Alan R. Lehmann, Wim Vermeulen, and Scott Williams) Xeroderma pigmentosum group G (XPG) protein is both a func- containing an unpaired bubble (bubble DNA) through its direct tional partner in multiple DNA damage responses (DDR) and a interactions with TFIIH, the XPD helicase activity of which is pathway coordinator and structure-specific endonuclease in nucle- strongly stimulated by the interaction (2). Requiring the phys- otide excision repair (NER). Different mutations in the XPG gene ical presence of XPG, the XPF-ERCC1 heterodimer is ERCC5 lead to either of two distinct human diseases: Cancer-prone recruited and incises the double-stranded (ds)DNA 5′ to the xeroderma pigmentosum (XP-G) or the fatal neurodevelopmental lesion (3–6). XPG incises the dsDNA on the 3′ side of the le- disorder Cockayne syndrome (XP-G/CS). To address the enigmatic sion at a position 1 nt from the bubble junction (3–6). Defects structural mechanism for these differing disease phenotypes and in XPG’s NER nucleolytic and scaffolding roles are linked to for XPG’s role in multiple DDRs, here we determined the crystal the hereditary and highly skin cancer-prone disease xeroderma structure of human XPG catalytic domain (XPGcat), revealing XPG- pigmentosum (XP) (7). BIOCHEMISTRY specific features for its activities and regulation. Furthermore, XPG Different mutations in ERCC5 cause a particularly severe DNA binding elements conserved with FEN1 superfamily members form of the fatal developmental disorder Cockayne syndrome enable insights on DNA interactions. Notably, all but one of the (CS), which (in contrast to XP) features pronounced neuro- known pathogenic point mutations map to XPGcat, and both XP-G degeneration and very early death, but no cancer predisposition and XP-G/CS mutations destabilize XPG and reduce its cellular protein levels. Mapping the distinct mutation classes provides Significance structure-based predictions for disease phenotypes: Residues mu- tated in XP-G are positioned to reduce local stability and NER ac- DNA repair is essential to life and to avoidance of genome in- tivity, whereas residues mutated in XP-G/CS have implied long- stability and cancer. Xeroderma pigmentosum group G (XPG) range structural defects that would likely disrupt stability of the protein acts in multiple DNA repair pathways, both as an active whole protein, and thus interfere with its functional interactions. enzyme and as a scaffold for coordinating with other repair Combined data from crystallography, biochemistry, small angle proteins. We present here the structure of the catalytic domain X-ray scattering, and electron microscopy unveil an XPG homo- responsible for its DNA binding and nuclease activity. Our dimer that binds, unstacks, and sculpts duplex DNA at internal analysis provides structure-based hypotheses for how XPG unpaired regions (bubbles) into strongly bent structures, and sug- recognizes its bubble DNA substrate and predictions of the gest how XPG complexes may bind both NER bubble junctions and structural impacts of XPG disease mutations associated with replication forks. Collective results support XPG scaffolding and two phenotypically distinct diseases: xeroderma pigmentosum DNA sculpting functions in multiple DDR processes to maintain (XP, skin cancer prone) or Cockayne syndrome (XP/CS, severe genome stability. progressive developmental defects). endonuclease | ERCC5 | electron microscopy | crystallography | crystal Author contributions: S.E.T., A.H.S., D.S.S., J.D.G., P.K.C., and J.A.T. designed research; structure A.H.S., C.N., B.S., S.J., A.C.T., A.W., M.Z.H., K.S.T., A.G.R., D.R., M.H., and J.D.G. performed research; A.S.A., M.-S.T., A.W., D.R., M.H., J.D.G., P.K.C., and J.A.T. analyzed data; and S.E.T., M.-S.T., A.B., J.D.G., P.K.C., and J.A.T. wrote the paper. eroderma pigmentosum group G (XPG) protein acts in Reviewers: A.R.L., University of Sussex; W.V., Erasmus University Medical Center; and S.W., Xmultiple DNA damage response (DDR) pathways, and National Institutes of Health. mutations in its ERCC5 (excision repair cross-complementing The authors declare no competing interest. rodent repair deficiency, complementation group 5) gene are This open access article is distributed under Creative Commons Attribution License 4.0 associated with two distinct diseases. Functionally, it has a (CC BY-NC-ND). structure-specific endonuclease activity (1) and interacts with Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, multiple DNA processing proteins in nucleotide excision repair http://www.pdb.org (PDB ID code 6VBH). (NER), transcription-coupled repair (TCR), base excision repair 1S.E.T. and A.H.S. contributed equally to the work. (BER), and homologous recombination (HR). In NER, XPG 2To whom correspondence may be addressed. Email: [email protected], jdg@med. enzymatic activity cleaves the damaged DNA strand 3′ to the unc.edu, [email protected], or [email protected]. lesion. NER is initiated by lesion recognition requiring XPC, 3Present address: School of Medicine, University of Washington, Seattle, WA 98915. opening of the DNA around the lesion by the helicase activities 4Present address: Department of Chemistry, University of California, Davis, CA 95616. of the TFIIH repair/transcription complex, and binding the This article contains supporting information online at https://www.pnas.org/lookup/suppl/ resulting single-stranded (ss)DNA by replication protein A doi:10.1073/pnas.1921311117/-/DCSupplemental. (RPA). XPG is recruited to the NER complex on DNA First published June 10, 2020. www.pnas.org/cgi/doi/10.1073/pnas.1921311117 PNAS | June 23, 2020 | vol. 117 | no. 25 | 14127–14138 Downloaded by guest on September 30, 2021 (7, 8). The hallmark of CS is loss of the preferential repair of to cut DNA, making it distinct from other nuclease classes where DNA damage in transcribed strands by TCR. While the mech- substrate validation is at the binding step. Furthermore, for XPG anistic connection of XPG loss to CS is not entirely clear, it is some substrates are bound but not incised (4, 28). As incision evident that it extends beyond the enzymatic function of XPG in appears not to be required for all XPG functions, could some NER. The requirement for XPG in TCR likely involves its DDR pathways involve its structured DNA binding capability? demonstrated interactions with RNA polymerase II (RNAPII), To address questions regarding XPG structural mechanisms in TFIIH, and CS group B (CSB) proteins, and biochemical evi- the DDR, we solved the crystal structure of the human XPG dence suggests that XPG may participate in allowing lesion re- catalytic domain (XPGcat). We furthermore combined bio- moval in the presence of stalled RNAPII (3, 4, 9). chemical, small angle X-ray scattering (SAXS), and electron XPG also plays important roles in other DDR pathways microscopy (EM) analyses of full-length protein with experi- through multiple protein interactions distinct from its NER mental data on disease-causing mutations both in XPG in patient functions. XPG depletion causes DNA double-strand breaks, cells and in bacterially expressed XPGcat. We show that XPG chromosomal abnormalities, cell-cycle delays, defective HR re- structure shares key metal-binding and basic residues with FEN1 pair (HRR), sensitivity to poly(ADP-ribose) polymerase in- and is likely to work through an analogous disorder-to-order hibition, inability to overcome replication fork stalling, and transition requiring the properly folded XPGcat structure for an replication stress (10). These phenotypes reflect its function in analogous multistep process to that associated with specificity in HRR mediated by its direct interactions with BRCA1, BRCA2, FEN1 and EXO1. We furthermore found that human XPG PALB2, and the RAD51 recombinase, which likely promote unexpectedly formed a stable dimer in solution and on DNA that loading of RAD51 onto RPA-coated ssDNA. In BER, XPG appears to be required for catalytic activity and for protein sta- – stimulates glycosylase activity of NTH1 in vitro (11 13). XPG bility. Rotary shadowing EM data showed that XPG bound interacts with and stimulates Werner syndrome helicase (WRN) specifically to bubble DNA and replication forks and strongly in vitro, and the two proteins colocalize during replication (14). bent bubble DNA. We examine XP-G and XP-G/CS missense ’ XPG s incision activity is implicated in cleavage of R-loops, in- mutations and characterize the impact of these mutations on ex- cluding those generated by loss of WRN in the premature aging pression and endonuclease activity, prompting a reexamination of disorder Werner syndrome (15–17). While the multiple roles of – how these mutations result in the different disease phenotypes. XPG in DDR depend on its many protein protein interactions, The collective findings provide fundamental mechanistic implica- some require its enzymatic activity but others apparently do not.
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