Contribution of Topoisomerase IIα chromatin-tether and C-terminal domain to mitotic chromosome association and structural organization

Mitotic functions of Topoisomerase IIα (TopoIIα) are dictated by its C-terminal domain (CTD), which is distinct from TopoIIβ. The chromatin-tether domain (ChT) within the αCTD recognizes methylated chromatin, an ability lacking for TopoIIβ and loss of αChT leads to ultrafine DNA bridges (UFBs) and chromosome bridges in anaphase. Because UFBs arise from incomplete decatenation of tangled genomic DNA, these observations suggest that αChT directs TopoIIα to sites of tangled DNA. Here, by chimeric swapping of ChT domains between the isoforms, TopoIIα-βChT and TopoIIβ-αChT, we reveal that αChT improves TopoIIβ's ability for UFB resolution but not for preventing chromosome bridges. This highlights the requirement for a complete αCTD for accurate chromosome segregation. Salt extraction assays show that the full αCTD is required for stable TopoIIα association with mitotic chromosomes, while the αChT enhances this association without affecting decatenation activity. Moreover, proteomic analysis showed altered levels of condensin and centromeric proteins in TopoII mutants. These results reveal the role of αChT in the αCTD-dependent chromosomal targeting mechanism of TopoIIα as well as an αChT-dependent requirement for robust structural organization of mitotic chromosomes.