TEX264 coordinates p97- and SPRTN-mediated resolution of topoisomerase 1-DNA adducts

Abstract

Eukaryotic topoisomerase 1 (TOP1) regulates DNA topology to ensure efficient DNA replication 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 hydrolyses 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.

Fig. 1. The p97 ATPase promotes TOP1cc repair.

a RADAR assay to assess TOP1cc accumulation after short interfering (si)RNA-mediated depletion of p97. Treatment with 1 μM CPT for 1 hour was used as a positive control for TOP1cc induction. Double-stranded (ds)DNA is used as a loading control. b Immunoblot to confirm p97 depletion. c Quantification of A (error bars represent mean ± SEM; n = 2 for CPT (1 μM); n = 3 for siLuc, sip97 #1 and #2; *P < 0.05; ns, not significant; Student’s t-test). d Left: immunoblots of anti-Strep-tag immunoprecipitates prepared from HEK293 transiently expressing wild-type (WT) or ATPase-defective (E578Q/EQ) p97-myc-Strep. EV denotes empty vector. LE and SE denote long and short exposure, respectively. Right: quantification of three independent experiments (error bars represent mean ± SEM; *P < 0.05; Student’s t-test). e RADAR assay to assess TOP1ccs in doxycycline (Dox)-inducible HEK293 Flp-In TRex cells expressing the indicated p97 variants. Where indicated, cells were treated with Dox for 36 h. f Quantification of e (error bars represent mean ± SD; n = 2; *P < 0.05; Student’s t-test). g Immunoblots of whole cell extracts prepared from the same cells as those subjected to RADAR in e. Arrowheads indicate endogenous p97 (lower band) and induced p97-myc-Strep (upper band). Source data are available online.

Fig. 2. TEX264 bridges p97 to TOP1.

a Above: schematic diagram of the TEX264 protein. LRR denotes leucine-rich repeat; GyrI-like, Gyrase inhibitory-like domain; SHP, SHP box. Below: sequence alignment of TEX264^280–286 with the SHP box of other p97 cofactors. Conserved residues are highlighted in black. b FLAG immunoprecipitates prepared from HEK293 cells transiently expressing TEX264^WT-FLAG cDNA or EV, treated with CPT (25 nM) or DMSO for 1 h. Right: quantification of three independent experiments (error bars represent mean ± SEM; **P < 0.01; Student’s t-test). c Immunoblots of anti-Strep-tag immunoprecipitates prepared from wild-type (WT) or CRISPR-Cas9 TEX264 knockout (ΔTEX264) HEK293 cells expressing p97-Strep-Myc. Right: quantification of two independent experiments (error bars represent mean ± SD; **P < 0.01; Student’s t-test). d Immunoblots of anti-Strep-tag immunoprecipitates prepared from wild-type (WT) or ΔTEX264 HEK293 cells expressing p97-Strep-Myc. Cells were treated with DMSO, the p97 inhibitor (p97i), CB-5083 (10 µM), for 90 min, CPT (50 nM) for 60 min, or both, as indicated. e In vitro p97 pulldown experiments after incubation of recombinant p97-S tag with His-tagged TEX264^WT or TEX264^ΔSHP. Source data are available online.

Fig. 3. TEX264 counteracts TOP1ccs.

a Immunofluorescent detection of TOP1ccs (green) in RPE-1 cells transfected with the indicated siRNAs. DAPI 4′,6-diamidino-2-phenylindole. Scale bar, 10 µm. b Quantification of a. Red line indicates median. Data are representative of two independent experiments. Significance determined by Mann–Whitney test. c Slot blot analysis of TOP1ccs prepared from WT or ΔTEX264 HEK293 cells using the RADAR assay. d Quantification of c (error bars represent mean ± SD; n = 2; **P < 0.01, *P < 0.05; Student’s t-test). e Representative images of cells analysed by alkaline comet assay in f. f Alkaline comet assay performed in RPE-1 cells treated with the indicated combinations of siRNAs. Cells were treated with CPT (100 nM) for 1 or 6 h(s) and allowed to recover as indicated. Quantification of >100 cells per condition from one representative experiment is shown, and the experiment was repeated two times. Whisker box plots show median values and data within the 10–90 percentile. Statistical analysis was performed using Kruskal–Wallis ANOVA with multiple comparisons, with Benjamini–Hochberg post-test. g Colony forming assay to assess the viability of HeLa cells transfected with the indicated siRNAs. Cells were treated for 24 h with the indicated doses of CPT, released into normal media for 7 days, then fixed and stained. Viability represents the number of colonies in each sample expressed as a percentage of the number of colonies formed in the corresponding untreated sample (error bars represent mean ± SD; n = 2). h Schematic diagram of the TEX264 protein indicating the location of residue E194. i Left: Immunoblots of FLAG immunoprecipitates prepared from ΔTEX264 HEK293 cells transiently expressing the indicated versions of FLAG-tagged TEX264. Right: Corresponding quantifications of four independent experiments (mean ± SEM; ****P < 0.0001; ***P < 0.0005; Student’s t-test). Source data are available online.

Fig. 4. TEX264 can bind SUMO1-modified TOP1.

a Analysis of TOP1ccs isolated by RADAR from the indicated cell lines. Where indicated, cells were treated with 1 μM CPT for 30 min. b Quantification of a (error bars represent mean ± SD; n = 2). c Above: schematic diagram of TEX264, indicating the location of its putative SUMO-interacting motifs (SIMs). Below: structural model of the GyrI-like domain of TEX264 generated using Phyre2. Residues 158–184 are highlighted in pink. Putative SIMs are highlighted in yellow. d Immunoblot of GFP & SUMO1 after incubation of purified GFP-tagged WT & SIM mutant TEX264 with free SUMO1. e Above: tandem-affinity purifications (TAP) procedure to identify SUMO1–modified proteins that interact with TEX264. Below: immunoblots of TAP experiment performed in ΔTEX264 HEK293 expressing the indicated variants of TEX264 and HA-SUMO1. SE and LE denote short and long exposure, respectively. Asterisks indicate SUMO1-modified versions of TOP1. f Immunofluorescent detection of TOP1ccs (green) and TEX264-FLAG (red) in U2OS cells transfected with the indicated siRNAs and cDNAs. Scale bar, 10 μm. g Quantification of experiments represented in f. Red line indicates median. Data are representative of two independent experiments. Statistical analysis was performed using Kruskal–Wallis ANOVA with multiple comparisons, with Dunn's post-test. Source data are available online.

Fig. 5. TEX264 cooperates with the metalloprotease SPRTN in TOP1cc repair.

a Left: analysis of anti-Strep-tag immunoprecipitates prepared from HEK293 cells expressing SPRTN-SSH, transfected with the indicated siRNAs. Right: corresponding quantification of two independent experiments (mean ± SD; ***P < 0.0005). b Immunoblots to confirm SPRTN depletion/induction in doxycycline (Dox)-inducible HEK293 Flp-In T-REx cells used in c and d. Cells were transfected with siRNA targeting the 3′UTR of SPRTN 3 days prior to collection. Dox was added for the final 24 h. c TOP1cc detection by RADAR in same cells as b. d Quantification of c (error bars represent mean ± SEM; Student’s t-test; n = 3; *P < 0.05; **P < 0.01; ns, not significant). e Total DNA–protein crosslinks isolated by RADAR from HEK293 cells treated with the indicated siRNAs. DPCs were resolved by SDS–PAGE and visualised by silver staining. f Quantification of e (error bars represent mean ± SD; n = 2; **P < 0.01; ns, not significant; Student’s t-test). Source data are available online.

Fig. 6. TEX264 acts at replication forks.

a Immunoblots of anti-Strep-tag immunoprecipitates prepared from doxycycline (Dox)-inducible TEX264-SSH HEK293 Flp-In TRex cells, incubated with and without Dox. b Measurement of EdU incorporation rates in four different cell lines treated with the indicated siRNAs. EdU incorporation is plotted as a percentage of the corresponding control cells. Cells were incubated with 10 µM EdU for 30 min prior to collection (error bars represent mean ± SEM; ****P < 0.0001; ***P < 0.0005; **P < 0.01, *P < 0.05; Student’s t-test; experiments were performed at least three times). c Confocal images of U2OS cells transfected with the indicated cDNAs encoding SSH-tagged variants of TEX264 and stained with an anti-HA antibody. TEX264^ΔLRR image was deconvolved. Cells were treated with 10 µM EdU for 30 min before fixation. EdU was labelled using a Click-iT™ Alexa Fluor imaging kit. White arrowheads indicate examples of co-localisation. Scale bars, 5 μm (large panels) and 2 μm (magnified panels). See also Supplementary Fig. 7C. d Same as in c, except cells were co-transfected with FLAG-tagged SPRTN and co-stained with an anti-FLAG antibody. e Representative images of HeLa cells transfected with the indicated siRNA and stained with an antibody against γH2AX (phosphorylated on Ser139). f Quantification of the mean nuclear γH2AX intensity for experiment in e (****P < 0.0001; one-way ANOVA; red line indicates mean values). At least 100 nuclei were measured per condition. Scale bar, 20 μm. g, Immunoblots to confirm the efficacy of TEX264 and TOP1 depletions. h Proposed model: TEX264 is tethered at the nuclear periphery by its LRR. TEX264 binds to unmodified and SUMO1-modified TOP1 and counteracts TOP1cc accumulation by recruiting p97-SPRTN sub-complexes to TOP1ccs. S1 denotes SUMO1. Source data are available online.