The C-terminal 31 residues of Topo IIα are important for CTR chromosomal association and association with DNA in vitro. (A) M. muntjak cells transfected with mCherry–Topo IIα CTR (Topo IIα residues 1,321–1,531) or mCherry–Topo IIα CTRΔ31 (Topo IIα residues 1,321–1,500) imaged in live metaphase cells. mCherry–Topo IIα CTR localizes to mitotic chromosomes, whereas mCherry–Topo IIα CTRΔ31 is localized diffusely through the nucleoplasm. (A, right) A representative quantification (>3 experimental repeats) of mCherry signal across mitotic chromosomes (the broken lines in the images). Bars, 5 µm. (B) The CTR of Topo IIα binds to plasmid DNA in vitro. EMSA analysis of supercoiled, linear, and relaxed pUC19 DNA mixed with recombinant histone H1° or CTR fragments of Topo IIα (defined in A) and resolved on agarose gels. M = 1 kb ladder. The star indicates the lane where the H1°–DNA complex is assumed to be net positively charged and has reversed its migration direction. Brackets highlight the mobility shifts in the 12.5–25 pmol range for CTR and CTRΔ31. (C) Schematic showing recombinant Topo IIα fragments used in DNA–beads binding assays (D). (D, left) Anti-His tag immunoblot of Topo IIα fragment pull-downs using DNA-coated beads and purified HIS-tagged Topo IIα CTR fragments. (D, right) Quantification of pull-down efficiency when uncoated bead background is subtracted (percentage of input). Topo IIα CTRΔ31 and Topo IIα CTRΔ52 bind DNA with reduced efficiency. The CTR of Topo IIβ (βCTR, residues 1,359–1,621) has negligible DNA-binding activity. n = 3. Error bars indicate SD. n.s., not a statistically significant difference.