Fig. 5: Phosphorylation is required for enhanced TopoIIα decatenation. | Nature Communications

Fig. 5: Phosphorylation is required for enhanced TopoIIα decatenation.

From: The Aurora B specificity switch is required to protect from non-disjunction at the metaphase/anaphase transition

Fig. 5

a DLD1 cells induced to express GFP-TopoIIa wild type or mutants were assessed for the metaphase catenation response. Cells were treated ±1 μM ICRF193 immediately prior to time-lapse imaging and scored for the time to progress from metaphase to anaphase. The mean ± SEM of three independent experiments is represented by the graph, ≥100 cells per condition were analysed. (one-way ANOVA; WT + ICRF193 55.6 ± 2.23 min S29A + ICRF193 44.18 ± 6.69 min, T1460A + ICRF193 46.11 ± 2.77 min, S29/T1460A + ICRF193 45.52 ± 1.71 min). b In vitro decatenation assay. Quantification of the amount of decatenated kDNA present after addition of TopoIIα and recombinant Aurora B (wild type or S227A). Graph represents the mean ± SD of five independent experiments. (two-tailed Students’ t test, *P = 0.012, **P = 0.0025; Control 0.024 ± 0.009, WT 0.208 ± 0.06, S227A 0.132 ± 0.05). c Representative immunofluorescence images of DLD1 cells after 1 h treatment with ICRF193 (1 μM) showing TopoIIα pS29 (magenta), a-tubulin (green) and DNA (blue). Images are representative of three independent experiments. Scale bar = 5 μm. d, e Catenation spread assay analysis. d DLD1 cells induced to express GFP-TopoIIα wild type or mutants were scored for the per cent joined (catenated) chromatids. The mean ± SEM of three experiments where ≥15 cells per experiment were analysed is represented by the graph. (two-tailed Students’ t test; *P = 0.047; siControl 43.38% ± 8.22, siTopoIIa 78.64% ± 6.61, WT 55.88% ± 4.58, S29A 69.24% ± 0.36, T1460A 72.07% ± 7.94, S29/T1460A 83.17% ± 12.49). e HEK293 cells induced to express GFP-TopoIIa wild type or mutants were scored for the per cent joined (catenated) chromatids. The mean ± SEM of three experiments where ≥15 cells per experiment were analysed is represented by the graph (two-tailed Students’ t test; ****P = ≤0.0001; siControl 25.66% ± 6.17, siTopoIIa 99.57% ± 0.26, WT 25.62% ± 3.63, S29A 90.68% ± 4.44, T1460A 83.21% ± 8.47, S29/T1460A 90.46% ± 4.25).

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