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TEX264 Coordinates P97- and SPRTN-Mediated Resolution of Topoisomerase 1-DNA Adducts

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TEX264 Coordinates P97- and SPRTN-Mediated Resolution of Topoisomerase 1-DNA Adducts This is a repository copy of TEX264 coordinates p97- and SPRTN-mediated resolution of topoisomerase 1-DNA adducts. White Rose Research Online URL for this paper: http://eprints.whiterose.ac.uk/158341/ Version: Published Version Article: Fielden, J., Wiseman, K., Torrecilla, I. et al. (14 more authors) (2020) TEX264 coordinates p97- and SPRTN-mediated resolution of topoisomerase 1-DNA adducts. Nature Communications, 11 (1). 1274. ISSN 2041-1723 https://doi.org/10.1038/s41467-020-15000-w Reuse This article is distributed under the terms of the Creative Commons Attribution (CC BY) licence. This licence allows you to distribute, remix, tweak, and build upon the work, even commercially, as long as you credit the authors for the original work. More information and the full terms of the licence here: https://creativecommons.org/licenses/ Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing [email protected] including the URL of the record and the reason for the withdrawal request. [email protected] https://eprints.whiterose.ac.uk/ ARTICLE https://doi.org/10.1038/s41467-020-15000-w OPEN TEX264 coordinates p97- and SPRTN-mediated resolution of topoisomerase 1-DNA adducts John Fielden1,7, Katherine Wiseman1,7, Ignacio Torrecilla 1, Shudong Li1, Samuel Hume 1, Shih-Chieh Chiang2, Annamaria Ruggiano1, Abhay Narayan Singh1, Raimundo Freire 3,4,5, Sylvana Hassanieh1, Enric Domingo 1, Iolanda Vendrell1,6, Roman Fischer 6, Benedikt M. Kessler 6, Timothy S. Maughan1, ✉ Sherif F. El-Khamisy 2 & Kristijan Ramadan 1 1234567890():,; Eukaryotic topoisomerase 1 (TOP1) regulates DNA topology to ensure efficient DNA repli- cation and transcription. TOP1 is also a major driver of endogenous genome instability, particularly when its catalytic intermediate—a covalent TOP1-DNA adduct known as a TOP1 cleavage complex (TOP1cc)—is stabilised. TOP1ccs are highly cytotoxic and a failure to resolve them underlies the pathology of neurological disorders but is also exploited in cancer therapy where TOP1ccs are the target of widely used frontline anti-cancer drugs. A critical enzyme for TOP1cc resolution is the tyrosyl-DNA phosphodiesterase (TDP1), which hydro- lyses the bond that links a tyrosine in the active site of TOP1 to a 3’ phosphate group on a single-stranded (ss)DNA break. However, TDP1 can only process small peptide fragments from ssDNA ends, raising the question of how the ~90 kDa TOP1 protein is processed upstream of TDP1. Here we find that TEX264 fulfils this role by forming a complex with the p97 ATPase and the SPRTN metalloprotease. We show that TEX264 recognises both unmodified and SUMO1-modifed TOP1 and initiates TOP1cc repair by recruiting p97 and SPRTN. TEX264 localises to the nuclear periphery, associates with DNA replication forks, and counteracts TOP1ccs during DNA replication. Altogether, our study elucidates the existence of a specialised repair complex required for upstream proteolysis of TOP1ccs and their subsequent resolution. 1 Cancer Research UK and Medical Research Council Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford OX3 7DQ, UK. 2 The University of Sheffield Neuroscience Institute and the Healthy Lifespan Institute, Department of Molecular Biology and Biotechnology, Firth Court, University of Sheffield, S10 2TN Sheffield, UK. 3 Unidad de Investigación, Hospital Universitario de Canarias, Ofra s/n, La Cuesta, 38320 La Laguna, Tenerife, Spain. 4 Instituto de Tecnologías Biomédicas, Universidad de La Laguna, 38200 La Laguna, Tenerife, Spain. 5 Universidad Fernando Pessoa Canarias, 35450 Las Palmas de Gran Canaria, Spain. 6 TDI Mass Spectrometry Laboratory, Target Discovery Institute, Nuffield Department of Medicine, University of ✉ Oxford, Oxford, UK. 7These authors contributed equally: John Fielden, Katherine Wiseman. email: [email protected] NATURE COMMUNICATIONS | (2020) 11:1274 | https://doi.org/10.1038/s41467-020-15000-w | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15000-w opoisomerase 1 (TOP1) resolves DNA topological stress relevant doses of TOP1 poisons, and exhibit DNA replication Taccumulated during DNA replication and transcription. As stress. Our data suggest that p97 and TEX264 enable the repair part of its catalytic cycle, TOP1 forms an intermediate that of TOP1ccs by facilitating their proteolysis via SPRTN upstream is covalently bound to DNA, known as a TOP1 cleavage complex of TDP1. This discovery is important for preserving genome (TOP1cc). TOP1ccs are usually transient but can become trapped stability from endogenous TOP1ccs and could be relevant for if TOP1 cleaves near a DNA alteration or is exposed to TOP1 clinical resistance to TOP1 poisons. poisons. Due to their bulky structure, TOP1ccs hinder the pro- gression of DNA replication and transcription, and are therefore Results highly cytotoxic1–3. The ATPase p97 promotes TOP1cc repair. As TOP1ccs are Endogenous TOP1ccs pose a constant threat to genome sta- common endogenous DNA lesions, we reasoned that factors that bility, as illustrated by the numerous neurodegenerative diseases – promote their repair should interact with the TOP1 protein even associated with defective TOP1cc repair2 8. The cytotoxicity of in the absence of TOP1 poisons1,26. To identify modulators of TOP1ccs is also exploited in cancer therapy by the widely used TOP1cc repair, we isolated chromatin from YFP-TOP1-expressing class of anti-cancer drugs, known as camptothecins (CPT), which human embryonic kidney (HEK) 293 cells and subjected YFP stabilise TOP1ccs by binding the TOP1–DNA interface. immunoprecipitates to liquid chromatography–tandem mass CPT derivatives are routinely used to treat ovarian, colon, and spectrometry (LC–MS/MS; Source Data). This analysis identified lung cancers but resistance is common, underscoring the need to the ATPase p97 as an abundant interacting partner of TOP1 on identify molecular biomarkers and determinants of resistance to chromatin, which we confirmed by immunoblotting (Supple- improve patient stratification and outcomes9. mentary Fig. 1A). By using energy generated from ATP hydrolysis, A key enzyme in TOP1cc repair is TDP1, a phosphodiesterase p97 remodels its substrates and extracts them from macro- that directly hydrolyses the phosphotyrosyl bond that covalently – molecular structures such as chromatin27 33. Given this known links TOP1 to the 3′ end of an ssDNA break10. This step is role of p97, and since Cdc48 has been implicated in TOP1cc necessary to allow re-ligation of the broken DNA strand and repair, we investigated whether p97 contributes to TOP1cc pro- ensure genome stability. Mutations in TDP1 give rise to the cessing in human cells. neurodegenerative disease, SCAN12,4,5. However, TDP1 alone To assess if p97 counteracts TOP1cc accumulation in cannot resolve TOP1ccs since its substrate bond is protected unchallenged conditions, we employed a modified version of within the bulky TOP1cc structure and is therefore inaccessible to the recently described rapid approach to DNA adduct recovery the TDP1 active site10. TDP1 is unable to resolve full-length, (RADAR) assay to analyse the abundance of proteins covalently recombinant TOP1ccs in vitro, however, its activity is enabled if – attached to DNA34. By lysing cells in chaotropic salts (6 M the TOP1ccs are heat-denatured or proteolytically digested11 15. guanidium isothiocyanate), detergents (4% Triton X-100 and 1% This raises the question of how TOP1cc processing upstream of N-lauroylsarcosine), and a reducing agent (1% DTT), all TDP1 occurs in vivo. A deeper understanding of this process molecular interactions, other than covalent interactions, are could unveil mechanisms of clinical resistance to TOP1 poisons disrupted. Depletion of p97 in HEK293 cells with either of two and potential targets of therapeutic intervention. different siRNA sequences resulted in substantial TOP1cc The proteasome and the metalloproteases SPRTN (in accumulation, to a similar extent as a short treatment with metazoans) and Wss1 (in yeast) are thought to digest the protein – 1 µM CPT (Fig. 1a–c). In co-immunoprecipitation experiments, component of TOP1ccs16 19. In humans, mutations in SPRTN we found that TOP1 bound ~3-fold more strongly to a substrate- that impair its proteolytic activity cause Ruijs–Aalfs syndrome trapping, ATPase-defective p97 variant (E578Q; EQ) than to (RJALS), which is characterised by hepatocellular carcinoma and wild-type p97 (Fig. 1d). This suggested that the TOP1 protein is premature ageing20. SPRTN-deficient human cells accumulate subject to p97’s ATPase activity. endogenous TOP1ccs and are sensitive to TOP1cc-inducing To test if p97 ATPase activity contributes to TOP1cc repair, we agents19. SPRTN hypomorphic mice accumulate TOP1ccs from treated human retinal pigmented epithelial (RPE-1) cells with CB- an early age, particularly in the liver, and ultimately develop liver 5083, a potent, selective inhibitor of p97 ATPase activity, and tumours18. This suggests that SPRTN plays a critical role in visualised TOP1cc foci formation by immunofluorescence using an processing TOP1ccs. antibody which specifically recognises TOP1ccs (Supplementary Both the proteasome and SPRTN are highly pleiotropic and Fig. 1B)35,36. We observed that acute chemical inhibition of p97 preferentially cleave pre-unfolded substrates and/or unstructured caused TOP1cc accumulation (Supplementary Fig. 1C, D). To protein regions19,21,22.
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