Cell cycle-coupled relocation of types I and II topoisomerases and modulation of catalytic enzyme activities

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 (alpha) or reticular (beta) nuclear patterns throughout interphase. In contrast to topoisomerase II alpha, topoisomerase II beta was completely excluded from nucleoli, In mitosis, topoisomerase II beta diffused completely into the cytosol, whereas topoisomerases I and II alpha remained chromosome bound. Chromosomal staining of topoisomerase I was homogeneous, whereas topoisomerase II alpha 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 II alpha escaped poisoning. Refractoriness to etoposide could be assigned to the salt-stable scaffold fraction of topoisomerase II alpha, which increased from <2% in G(1) phase to 48% in mitosis. Topoisomerases I and II beta remained completely extractable throughout the cell cycle. In summary, expression of topoisomerases increases towards mitosis, but specific activities decrease. Topoisomerase II beta is released from the heterochromatin, whereas topoisomerase I and II alpha remain chromosome bound. Scaffold-associated topoisomerase II alpha 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.