Meyer K N, Kjeldsen E, Straub T, Knudsen B R, Hickson I D, Kikuchi A, Kreipe H, Boege F
Medizinische Poliklinik, University of Wurzburg, Germany.
J Cell Biol. 1997 Feb 24;136(4):775-88. doi: 10.1083/jcb.136.4.775.
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 IIalpha, topoisomerase IIbeta was completely excluded from nucleoli. In mitosis, topoisomerase IIbeta diffused completely into the cytosol, whereas topoisomerases I and IIalpha remained chromosome bound. Chromosomal staining of topoisomerase I was homogeneous, whereas topoisomerase IIalpha 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 IIalpha escaped poisoning. Refractoriness to etoposide could be assigned to the salt-stable scaffold fraction of topoisomerase IIalpha, which increased from <2% in G1 phase to 48% in mitosis. Topoisomerases I and IIbeta remained completely extractable throughout the cell cycle. In summary, expression of topoisomerases increases towards mitosis, but specific activities decrease. Topoisomerase IIbeta is released from the heterochromatin, whereas topoisomerase I and IIalpha remain chromosome bound. Scaffold-associated topoisomerase IIalpha 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.
我们通过同工酶选择性免疫荧光显微镜观察了A431细胞和分离染色体中的DNA拓扑异构酶。在间期，拓扑异构酶I主要呈均匀的核分布。10%-15%的细胞呈现颗粒状模式，30%显示明亮的核仁内斑块。拓扑异构酶II同工酶在整个间期呈现斑点状（α）或网状（β）核模式。与拓扑异构酶IIα不同，拓扑异构酶IIβ完全排除在核仁之外。在有丝分裂中，拓扑异构酶IIβ完全扩散到细胞质中，而拓扑异构酶I和IIα仍与染色体结合。拓扑异构酶I的染色体染色是均匀的，而拓扑异构酶IIα聚集在染色体臂的长轴和中心粒中。拓扑异构酶抗原在有丝分裂期比间期高2-3倍，但拓扑异构酶I和II的比活性分别降低了5倍和2.4倍。这些变化与有丝分裂酶的过度磷酸化有关。在间期，拓扑异构酶可被依托泊苷或喜树碱完全与DNA连接，而在有丝分裂期，50%的拓扑异构酶IIα逃脱中毒。对依托泊苷的耐药性可归因于拓扑异构酶IIα的盐稳定支架部分，其从G1期的<2%增加到有丝分裂期的48%。拓扑异构酶I和IIβ在整个细胞周期中仍可完全提取。总之，拓扑异构酶的表达在有丝分裂期增加，但比活性降低。拓扑异构酶IIβ从异染色质中释放，而拓扑异构酶I和IIα仍与染色体结合。与支架相关的拓扑异构酶IIα似乎不参与催化DNA周转，尽管它可能在中心粒的复制周期中起作用，在M期它在中心粒中积累。
