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* Medizinische Poliklinik and 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 (
Institute of Pathology, University of Würzburg, Germany; § Department of Human Genetics and
Department of Molecular and Structural Biology, University of Aarhus, Denmark; ¶ Institute of Molecular Medicine, John
Radcliffe Hospital, University of Oxford, United Kingdom; and ** Laboratory of Medical Mycology, Institute for Disease
Mechanism and Control, Nagoya University, Japan
) or reticular (
)
nuclear patterns throughout interphase. In contrast to
topoisomerase II
, topoisomerase II
was completely
excluded from nucleoli. In mitosis, topoisomerase II
diffused completely into the cytosol, whereas topoisomerases I and II
remained chromosome bound.
Chromosomal staining of topoisomerase I was homogeneous, whereas topoisomerase II
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
escaped poisoning. Refractoriness to
etoposide could be assigned to the salt-stable scaffold
fraction of topoisomerase II
, which increased from
<2% in G1 phase to 48% in mitosis. Topoisomerases I
and II
remained completely extractable throughout
the cell cycle. In summary, expression of topoisomerases increases towards mitosis, but specific activities decrease. Topoisomerase II
is released from the
heterochromatin, whereas topoisomerase I and II
remain chromosome bound. Scaffold-associated topoisomerase II
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.
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