SUMOylation of TOP2A CTD regulates Haspin binding and H3T3 phosphorylation on mitotic chromosomes. (A) Schematic representation of the primary structure of X. laevis TOP2A. Three lysines indicated in the CTD were mutated to arginine for a TOP2A mutant that could not be SUMOylated in the CTD (3KR). (B) Endogenous TOP2A in CSF XEEs was immunodepleted and replaced with either recombinant full-length T7-TOP2A WT or 3KR (left). β-Tubulin was used as a loading control of TOP2A levels in CSF XEEs. Mitotic chromosomes assembled in TOP2A-replaced CSF XEEs were analyzed by immunoblotting with indicated antibodies (right). Histone H4 was used as a loading control for mitotic chromosomes. (C) Quantification of Haspin and H3T3p levels on the mitotic chromosomes, as seen in B, relative to levels of TOP2A WT chromosomes from three independent experiments (n = 3) with levels normalized to histone H4 levels. Error bar represents SD. *, P < 0.05; **, P < 0.01 (Student’s t test).