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. 2019 Mar 12;20(5):1238. doi: 10.3390/ijms20051238

Figure 1.

Figure 1

The impact of internal deletions of the CTD on the mitotic localisation of TOP2A (a) Schematic of human TOP2A showing the domain structure: the N-terminal ATPase gate (consisting of the ATPase and transducer domains); the DNA-binding gate (consisting of the TOPRIM domain, the Winged Helix Domain (with the active site tyrosine 805) and the Tower domain); the C-gate (formed by the coiled-coil domain); and the unstructured C-Terminal Domain (CTD). Shown below are the internally deleted variants analysed. In each the terminal amino acids 1447–1531, which encompass the main nuclear localisation signal (NLS) and the chromatin tether domain (ChT) are retained. (b) Western blotting of whole cell lysates from HTETOP-derived transfectants stably expressing Flag-tagged TOP2A, either full length (FT) or internally deleted variants (FTΔ2, Δ3 and Δ5). The antigen recognised by the TOP2A isoform-specific antibody is retained in all variants. Transfectants have been grown in the presence or absence, of doxycycline. The full length untagged TOP2A protein (~170 KDa.) present in the parental HTETOP cells is transcribed under a tet-regulatable promoter and is suppressed by doxycycline, while the Flag-tagged versions of the protein (expressed under a CMV promoter) are unaffected (indicated by “>”). LC = an unidentified protein detected by the TOP2A antibody and described previously [45]. (c) Immunofluorescence localisation of the internally deleted forms of TOP2A in interphase and mitotic cells of stable transfectants, detected using an anti-Flag antibody (green), shown alongside cells expressing the Flag-tagged full length protein (FT). DNA has been counterstained with DAPI (blue). FT (Flag:TOP2A WT) and FTΔ3 cells are shown grown in the presence of dox (the localisation phenotypes were unchanged in the absence of dox/presence of untagged TOP2A WT protein). The FTΔ2 cells shown had been passaged in the absence of dox (long term growth in doxycycline is lethal). FTΔ5 cells grown in both the presence or absence, of dox are shown. This variant localises normally to the nucleus but its targeting of mitotic chromatin is perturbed. This disruption is especially severe in the presence of full length TOP2A protein, where the Flag-tagged variant appears largely cytosolic. Scale bar, 10 μm.