Christine J. Farr

Type II topoisomerase enzymes are essential for resolving DNA topology problems arising through various aspects of DNA metabolism. In vertebrates two isoforms are present, one of which (TOP2A) accumulates on chromatin during mitosis. Moreover, TOP2A targets the mitotic centromere during prophase, persisting there until anaphase onset. It is the catalytically-dispensable C-terminal domain of TOP2 that is crucial in determining this isoform-specific behaviour. In this study we show that, in addition to the recently identified chromatin tether domain, several other features of the alpha-C-Terminal Domain (CTD). influence the mitotic localisation of TOP2A. Lysine 1240 is a major SUMOylation target in cycling human cells and the efficiency of this modification appears to be influenced by T1244 and S1247 phosphorylation. Replacement of K1240 by arginine results in fewer cells displaying centromeric TOP2A accumulation during prometaphase-metaphase. The same phenotype is displayed by cells expressing TOP2A in which either of the mitotic phosphorylation sites S1213 or S1247 has been substituted by alanine. Conversely, constitutive modification of TOP2A by fusion to SUMO2 exerts the opposite effect. FRAP analysis of protein mobility indicates that post-translational modification of TOP2A can influence the enzyme’s residence time on mitotic chromatin, as well as its subcellular localisation.

In addition to its roles in transcription and replication, topoisomerase 2 (topo 2) is crucial in shaping mitotic chromosomes and in ensuring the orderly separation of sister chromatids. As well as its recruitment throughout the length of the mitotic chromosome, topo 2 accumulates at the primary constriction. Here, following cohesin release, the enzymatic activity of topo 2 acts to remove residual sister catenations. Intriguingly, topo 2 does not bind and cleave all sites in the genome equally; one preferred site of cleavage is within the core centromere. Discrete topo 2-centromeric cleavage sites have been identified in α-satellite DNA arrays of active human centromeres and in the centromere regions of some protozoans. In this study, we show that topo 2 cleavage sites are also a feature of the centromere in Schizosaccharomyces pombe, the metazoan Drosophila melanogaster and in another vertebrate species, Gallus gallus (chicken). In vertebrates, we show that this site-specific cleavage is diminished by depletion of CENP-I, an essential constitutive centromere protein. The presence, within the core centromere of a wide range of eukaryotes, of precise sites hypersensitive to topo 2 cleavage suggests that these mark a fundamental and conserved aspect of this functional domain, such as a non-canonical secondary structure.

As proliferating cells transit from interphase into M-phase, chromatin undergoes extensive reorganization, and topoisomerase (topo) IIα, the major isoform of this enzyme present in cycling vertebrate cells, plays a key role in this process. In this study, a human cell line conditional null mutant for topo IIα and a derivative expressing an auxin-inducible degron (AID)-tagged version of the protein have been used to distinguish real mitotic chromosome functions of topo IIα from its more general role in DNA metabolism and to investigate whether topo IIβ makes any contribution to mitotic chromosome formation. We show that topo IIβ does contribute, with endogenous levels being sufficient for the initial stages of axial shortening. However, a significant effect of topo IIα depletion, seen with or without the co-depletion of topo IIβ, is the failure of chromosomes to hypercompact when delayed in M-phase. This requires much higher levels of topo II protein and is impaired by drugs or mutations that affect enzyme activity. A prolonged delay at the G2/M border results in hyperefficient axial shortening, a process that is topo IIα-dependent. Rapid depletion of topo IIα has allowed us to show that its function during late G2 and M-phase is truly required for shaping mitotic chromosomes.