Fig 1: CHK1-mediated TOP1 S320 phosphorylation acts as a kinetic regulator of TOP1 catalytic activity.(A, B) Plasmid relaxation assay with crude nuclear extracts from U2-OS cells, untreated, or treated with CHK1i (250 nM) for 2 h (data from two independent replicates). (C, D) Plasmid relaxation assay with immunoprecipitated EGFP-TOP1WT. U2-OS cells were transiently transfected with EGFP-TOP1WT. 48 h post transfection, cells were treated with CHK1i (250 nM) for 2 h, followed by immunoprecipitation of EGFP-TOP1 with GFP-trap magnetic agarose beads (from untreated or CHK1i-treated cells). Plasmid relaxation assays were performed with the isolated immunocomplexes (data from two independent replicates). (E, F) Plasmid relaxation assay with EGFP-TOP1WT and EGFP-TOP1S320A. U2-OS cells were transiently transfected with EGFP-TOP1WT and EGFP-TOP1S320A, followed by immunoprecipitation 48 h post transfection. Plasmid relaxation assay was performed with the isolated immunocomplexes. All plasmid relaxation assays were performed with supercoiled pUC19 substrate (data from two independent replicates). (G) Schematic representation of TOP1 cleavage and religation assays. The CL-14 oligonucleotide was FITC labeled at the guanine residue highlighted in green. The CP-25 oligonucleotide was phosphorylated at 5’ end. See “Methods” for more details. (H, I) Time kinetics of TOP1 cleavage assay with immunoprecipitated EGFP-TOP1WT and EGFP-TOP1S320A. EGFP-TOP1WT and EGFP-TOP1S320A were ectopically expressed in U2-OS cells and immunoprecipitated as above. The first lane (from left) shows the unreacted 14-mer (data from two independent replicates). (J, K) Time kinetics of TOP1 religation assay with immunoprecipitated EGFP-TOP1WT and EGFP-TOP1S320A. EGFP-TOP1WT and EGFP-TOP1S320A were ectopically expressed in U2-OS cells and immunoprecipitated as above. The first lane (from left) shows the unreacted substrate (14-mer). The oligonucleotide species represented in each band are schematically shown along the gel images (data from two independent replicates). Source data are available online for this figure.
Fig 2: CHK1 suppresses TOP1cc persistence and R-loop accumulation on long genes.(A) Analysis of gene lengths from transcriptomic profiling of U2-OS cells treated with CHK1i (250 nM) or CPT (1 µM). Quantification shown from one replicate. The experiment was performed in two replicates. ****P < 0.0001. (B) Experimental workflow of ChIP and DRIP assays. (C) Relative enrichment of TOP1ccs (arising from EGFP-TOP1WT or EGFP-TOP1S320A) in transcription start sites (TSS) and gene bodies. U2-OS cells were transiently transfected with EGFP-TOP1WT and EGFP-TOP1S320A, followed by chromatin immunoprecipitation with GFP-trap magnetic agarose beads from un-crosslinked nuclei. P values (EGFP-TOP1WT vs. EGFP-TOP1S320A) are: 0.04113 (β-Actin TSS), 0.156727 (MYO3A TSS), 0.511635 (γ-Actin TSS), 0.018755 (β-Actin gene body), 0.010610 (MYO3A gene body), 0.014291 (γ-Actin gene body), 0.014334 (Geminin gene body), 0.014369 (IL4), 0.015524 (PTB), 0.938827 (intergenic) (data from two independent experiments; significance was determined using two-way ANOVA with Tukey test). Error bars represent SEM. (D) Relative enrichment of R-loops at TSS and gene bodies. U2-OS cells were transiently transfected with EGFP-TOP1WT and EGFP-TOP1S320A, followed by immunoprecipitation with S9.6 monoclonal antibody. Specificity of the signal was determined using RNase H sensitivity. P values (EGFP-TOP1WT vs. EGFP-TOP1S320A) are: 0.291128 (β-Actin TSS), 0.03 (MYO3A TSS), 0.110789 (γ-Actin TSS), 0.009672 (β-Actin gene body), 0.002608 (MYO3A gene body), 0.002977 (γ-Actin gene body), 0.143912 (Geminin gene body), 0.002618 (IL4), 0.007655 (PTB), 0.873055 (intergenic) (data from two independent experiments; significance was determined using two-way ANOVA with Tukey test). Error bars represent SEM. Source data are available online for this figure.
Fig 3: Characterization of S320 phosphorylation as a regulator of TOP1 dynamics.(A, B) Representative RADAR assay in U2-OS cells transfected with EGFP-TOP1WT, EGFP-TOP1S320A, EGFP-TOP1S394A, or EGFP-TOP1T570A without depletion of endogenous TOP1. Cells were transfected with plasmids expressing EGFP-TOP1WT, EGFP-TOP1S320A, EGFP-TOP1S394A, or EGFP-TOP1T570A followed by treatment with CHK1i (250 nM) for 2, 4, or 6 h, 48 h post transfection. Dot blots were probed with anti-GFP antibody. P values (0 h) are: 0.007925 (EGFP-TOP1WT vs. EGFP-TOP1S320A), 0.966818 (EGFP-TOP1WT vs. EGFP-TOP1S394A), and 0.076058 (EGFP-TOP1WT vs. EGFP-TOP1T570A) (data from three independent experiments; significance was determined using two-way ANOVA with Tukey test). Error bars represent SD. (C, D) Representative Proximity Ligation Assay (PLA) images (and quantification across three replicates) of EGFP and TOP1cc in U2-OS cells transfected with EGFP-TOP1WT, EGFP-TOP1S320A, EGFP-TOP1S394A, or EGFP-TOP1T570A. Cells were transfected, followed by PLA with anti-GFP and anti-TOP1cc antibody 48 h post transfection. Nuclei are marked with white dotted lines. Scale bars: 10 µm). P values are: 0.0011598 (EGFP-TOP1WT vs. EGFP-TOP1S320A), 0.114290 (EGFP-TOP1WT vs. EGFP-TOP1S394A), and 0.084425 (EGFP-TOP1WT vs. EGFP-TOP1T570A) (data from three independent experiments with 200 cells per condition; significance was determined using Kruskal–Wallis test with Dunn’s post hoc analysis). Error bars represent SEM. (E, F) In vitro kinase assay with recombinant human CHK1 and biotinylated peptides encompassing TOP1 S320 and its phosphoresistant mutant S320A. (F) Multiple sequence alignment of TOP1 orthologs from nine eukaryotic species, highlighting conservation of Serine 320. WT and S320A peptides (amino acid highlighted in green) were biotinylated at their N-termini (red). In vitro kinase reactions were performed with recombinant CHK1 and relevant peptides, followed by purification of peptides via streptavidin agarose beads (green). Peptides were then dissociated from the beads and subjected to a dot blot. (G) Structural context of Serine 320 in the TOP1-DNA complex (PDB ID:1A31). (H) Structural context of Serine 320 in TOP1 protein (PDB ID: 1A31). Positioning of Serine 320 relative to α5 and α6 “nose-cone” helices is shown in inset. Source data are available online for this figure.
Fig 4: Ablation of CHK1-mediated TOP1 S320 phosphorylation induces replication-associated genomic instability.(A) Representative microscopic images of γ-H2AX levels in U2-OSEGFP-TOP1WT and U2-OSEGFP-TOP1S320A stable cells. Cells in white dotted boxes are shown in the zoomed panel. Scale bars: 20 µm. (B, C) Quantification of γ-H2AX and 53BP1 foci in U2-OSEGFP-TOP1WT and U2-OSEGFP-TOP1S320A stable cells. ***P = 0.0005, *P = 0.0264 (data from three independent experiments with 200 cells per condition; significance was determined using unpaired t test). Error bars represent SEM. (D, E) Representative microscopic images (and quantification across three replicates) showing colocalization of γ-H2AX/53BP1 foci with EdU in U2-OSEGFP-TOP1WT and U2-OSEGFP-TOP1S320A cells. Cells were pulse-labeled with EdU (10 µM; 20 min) prior to fixation. γ-H2AX/53BP1 were visualized by immunofluorescence, while EdU was detected using click chemistry. Scale bars: 20 µm. Cells in white dotted boxes are shown in the zoomed panel. For γ-H2AX foci, P values are: 0.9980 (U2-OSEGFP-TOP1WT EdU+ vs. EdU-), < 0.0001 (U2-OSEGFP-TOP1WT EdU+ vs. U2-OSEGFP-TOP1S320A EdU+), 0.0011 (U2-OSEGFP-TOP1WT EdU− vs. U2-OSEGFP-TOP1S320A EdU−), < 0.0001 (U2-OSEGFP-TOP1S320A EdU+ vs. EdU-). For 53BP1 foci, ns P = 0.9992, *P = 0.0197, **P = 0.0030, ****P < 0.0001 (data from three independent experiments with 200 cells per condition; significance was determined using Kruskal–Wallis test with Dunn’s post hoc analysis). Error bars represent SEM. (F–H) Single-molecule DNA fiber assay in U2-OS cells transiently transfected with EGFP-TOP1WT and EGFP-TOP1S320A. Five representative DNA fibers have been shown per sample. Scale bars: 5 µm. In total, 250–300 individual fibers were scored from a total of 15 fields per sample. Quantification pertaining to the experiment shown in (G) is presented. Error bars represent median±interquartile range. ****P < 0.0001. See Appendix Fig. S11C for quantification of all replicates. (I) Proliferation kinetics of U2-OSEGFP-TOP1WT and U2-OSEGFP-TOP1S320A stable cells. Cells were seeded in 60 mm dishes and cell number was quantified 3-, 6-, or 9-days post seeding (data from two independent replicates). (J, K) Representative metaphase spreads (and quantification across three replicates, with 100 metaphases per condition) prepared from U2-OSEGFP-TOP1WT and U2-OSEGFP-TOP1S320A cells. Scale bars: 10 µm. Error bars represent SEM. **P = 0.0255 (Kruskal–Wallis test with Dunn’s post hoc analysis). Source data are available online for this figure.
Fig 5: A schematic model of the role played by CHK1 in the regulation of TOP1 catalysis, and its impact on genomic stability. (A) Under physiological conditions, CHK1-mediated phosphorylation of TOP1 at Serine 320 ensures efficient catalysis, maintaining minimal TOP1cc levels. (B) CHK1 inhibition leads to abrogation of this regulation, resulting in accumulation of TOP1ccs. These TOP1ccs are targeted by SPRTN, p97 and CtIP, while being relatively refractory to processing by the TDP1, proteasomal and autophagic machineries. Abrogation of CHK1-mediated TOP1 phosphorylation at Serine 320 eventually leads to R-loop stabilization, imapired replication and transcription, transcription-replication conflicts and genomic instability.
Supplier Page from Abcam for Recombinant human Chk1 protein