Cell Cycle–coupled Relocation of Types I and II Topoisomerases and Modulation of Catalytic Enzyme Activities

Abstract

We visualized DNA topoisomerases in A431 cells and isolated chromosomes by isoenzyme-selective immunofluorescence microscopy. In interphase, topoisomerase I mainly had a homogeneous nuclear distribution. 10–15% of the cells exhibited granular patterns, 30% showed bright intranucleolar patches. Topoisomerase II isoenzymes showed spotted (α) or reticular (β) nuclear patterns throughout interphase. In contrast to topoisomerase IIα, topoisomerase IIβ was completely excluded from nucleoli. In mitosis, topoisomerase IIβ diffused completely into the cytosol, whereas topoisomerases I and IIα remained chromosome bound. Chromosomal staining of topoisomerase I was homogeneous, whereas topoisomerase IIα accumulated in the long axes of the chromosome arms and in the centriols. Topoisomerase antigens were 2–3-fold higher in mitosis than in interphase, but specific activities of topoisomerase I and II were reduced 5- and 2.4-fold, respectively. These changes were associated with mitotic enzyme hyperphosphorylation. In interphase, topoisomerases could be completely linked to DNA by etoposide or camptothecin, whereas in mitosis, 50% of topoisomerase IIα escaped poisoning. Refractoriness to etoposide could be assigned to the salt-stable scaffold fraction of topoisomerase IIα, which increased from <2% in G1 phase to 48% in mitosis. Topoisomerases I and IIβ remained completely extractable throughout the cell cycle. In summary, expression of topoisomerases increases towards mitosis, but specific activities decrease. Topoisomerase IIβ is released from the heterochromatin, whereas topoisomerase I and IIα remain chromosome bound. Scaffold-associated topoisomerase IIα appears not to be involved in catalytic DNA turnover, though it may play a role in the replicational cycle of centriols, where it accumulates during M phase.

The N-terminal Domain Anchors Human Topoisomerase I at Fibrillar Centers of Nucleoli and Nucleolar Organizer Regions of Mitotic Chromosomes

“…Immunohistochemical studies have firmly established topoisomerase I as a mostly nucleolar protein (5,(21)(22)(23), but it is unclear what actually directs the enzyme to the nucleolus and for what purpose. The most likely candidate responsible for targeting topoisomerase I to defined locations within the cell nucleus is the non-conserved N-terminal domain spanning amino acid residues 1-210 of the human enzyme.…”

Mitotic Phosphorylation of DNA Topoisomerase II α by Protein Kinase CK2 Creates the MPM-2 Phosphoepitope on Ser-1469

“…Interestingly, while the MPM-2 motif is highly conserved among topoisomerase II␣ from different mammalian species, it is not present on topoisomerase II␤. The two different topoisomerase II isoforms show different cellular localization during mitosis, since topoisomerase II␣ remains tightly associated with the mitotic chromosomes whereas the ␤ isoform dissociates from the chromatin during this step of the cell cycle (73).…”

Micromechanical studies of mitotic chromosomes

“…Metazoan cells contain topo IIa and b isoforms; during mitosis topo IIa is mainly resident on chromosomes, while topo IIb is mainly cytoplasmic (Meyer et al 1997). Observations of GFP-topo IIa fusion proteins in vivo show it to rapidly exchange on and off chromosomes (Christensen et al 2002, Tavormina et al 2002.…”