Fig 2: A TOP1 gene harboring a frameshift-inducing deletion retains catalytic activity. a Exclusion of exon 6 (a symmetric exon) produces an internally truncated TOP1 protein (TOP1 ΔE6) with altered subcellular distribution. b The TOP1 ΔE6 protein can induce relaxation of supercoiled DNA. Camptothecin (TOP1 inhibitor) prevents DNA relaxation. c Summary of novel mRNAs or proteins observed in 13 CRISPR-edited commercial HAP1 cell lines (Horizon Discovery). Source data are provided as a Source Data file

Fig 3: Tdp1 deficiency causes strong accumulation of endogenous and chemically induced Top1-DPCs in embryos and cells. (a) Western blot showing zebrafish Top1-DPCs in tdp1 mutant embryos before and after camptothecin (CPT) (10 µM, 1 h) and formaldehyde (FA) treatment (5 mM, 30 min) (DPC equivalent of 1 µg DNA was loaded per well) and (b) corresponding quantification (n = 4). (c) Western blot showing zebrafish Top1-DPCs in tdp1 mutant embryos before and after sprtn silencing and CPT (10 µM, 1 h) and FA treatment (5 mM, 30 min) and (d) corresponding quantification. (e) Dot blots showing human TOP1-DPCs detected with TOP1-specific antibody before and after CPT treatment of RPE1 cells (50 nM, 1 h) with corresponding DNA loading controls (DPC equivalent of 500 ng DNA was loaded per well). (f) Quantification of (e) from three different biological replicates normalized to untreated WT cells. (g) Dot blots showing human TOP1-DPCs detected with TOP1-specific antibody before and after FA treatment of RPE1 cells (1 mM, 20 min) with corresponding DNA loading controls (DPC equivalent of 500 ng DNA was loaded per well). (h) Quantification of (g) (n = 3). Results represent mean fold change ± s.d. of three different experiments. Statistically significant changes as a result from unpaired Student's t-test are shown as *p < 0.05, **p < 0.01, ***p < 0.001 or #p < 0.0001.

Fig 4: Model of coordinated action of SPRTN and TDP1 in DNA–protein crosslink repair in human cells and zebrafish model. SPRTN is a general DPC protease that cleaves a wide spectrum of crosslinked proteins, whereas TDP1 removes protein residues bound to the 3′ end of the ssDNA break. Under physiological conditions, these two proteins function independently to resolve total DPCs, including specific TOP1-DPCs. Importantly, resolution of endogenous histone-DPCs originating at abasic (AP) sites depends on SPRTN-mediated proteolysis followed by TDP1 phosphodiesterase activity, by which the crosslinked peptide residue is removed from the DNA backbone. In response to the DNA-damaging agent camptothecin, an epistatic relationship between SPRTN and TDP1 is required for the successful removal of histones and TOP1-DPCs as well as other DPCs at the 3′ ends of ssDNA breaks. The model was created with BioRender.com.

Fig 5: TopI binds RNAP II and promotes RNAP II elongation on α-satellite chromatin.a TopI inhibition decreases RNAP II-pSer2 levels on α-satellite DNAs. HeLa cells treated with DMSO or CPT (2.5 µM) for 12 h were subjected to chromatin immunoprecipitation analysis with anti-RNAP II-pSer2 antibody. Biological replicates (n = 1 for beads, n = 4 for DMSO and CPT). b TopI inhibition reduces RNAP II-pSer2 levels on α-satellite DNAs in G1 cells. Stretched chromatin was prepared from thymidine-arrested HeLa cells treated with DMSO or CPT (2.5 µM) for 6 h and immunostaining was performed. n = 3 biological replicates. c Mitosis-specific inhibition of TopI reduces RNAP II-pSer2 levels on α-satellite DNAs. Mitotic HeLa cells were enriched by a brief treatment of nocodazole (5 µM). Collected mitotic cells were further treated with DMSO or CPT (2.5 µM) for 5 h and then subjected for chromosome spread followed by immunostaining. n = 3 biological replicates. d TopI physically interacts with RNAP II and phospho-RNAP II (pSer2). Lysates of HeLa cells transfected Luciferase or Top1 siRNAs (#1) were incubated with IgG or antibody against TopI. Pelleted proteins were blotted with the indicated antibodies. The physical interaction between TopI and non-phospho-RNAP II was recorded in Supplementary fig. 1e. “H” high exposure. e TopI and TopI-cleavage complex (cc) are present on the centromere in interphase. Stretched chromatin was prepared from log-phase HeLa cells and immunostaining was performed. Similar results were observed in at least two biological replicates. f TopI-cc is enriched on the centromere in mitosis. HeLa cells were briefly treated with nocodazole (5 µM) and mitotic cells were collected for chromosome spread followed by immunostaining. Similar results were observed in at least two biological replicates. Validation of TopI and Top-cc fluorescence signals were recorded in Supplementary Figs. 1e, f. All data here are presented as mean values +/− SEM. Two-sided Student’s T-test for (a–c). ns, not significant (P > 0.1). Numeric values for P < 0.1 are shown. The source data are provided as Source Data file.