Subject(s)
DNA Topoisomerases, Type II/metabolism , DNA Topoisomerases, Type I/metabolism , DNA-Binding Proteins/metabolism , DNA/metabolism , Centrifugation, Density Gradient , Cesium/metabolism , Chlorides/metabolism , DNA Topoisomerases, Type I/analysis , DNA Topoisomerases, Type II/analysis , DNA-Binding Proteins/analysis , DNA-Binding Proteins/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Humans , Immunoblotting , Sarcosine/analogs & derivatives , Sarcosine/pharmacology , Teniposide/pharmacology , Topoisomerase I Inhibitors , Topoisomerase II Inhibitors , Topotecan/pharmacology , Tumor Cells, CulturedABSTRACT
The entire human topoisomerase II alpha (hTopoII alpha) dimer was expressed in the yeast Saccaromyces cerevisiae, purified to homogeneity, and subjected to atomic force microscopy (AFM) under a tapping mode. Molecular images obtained exhibited a 'heart or donut-like' structure with a large axial hole. The main benefit of the application of AFM to study the hTopoII alpha is that clear images of the internal 'pore' have been achieved without crystallization, staining, or fixation of the sample. These images are consistent with the model in which topoisomerase II has a large internal gate for DNA strand passage.
Subject(s)
DNA Topoisomerases, Type II , DNA Topoisomerases, Type II/chemistry , Isoenzymes/chemistry , Microscopy, Atomic Force/methods , Antigens, Neoplasm , DNA Topoisomerases, Type II/isolation & purification , DNA Topoisomerases, Type II/ultrastructure , DNA-Binding Proteins , Humans , Isoenzymes/isolation & purification , Isoenzymes/ultrastructure , Models, Molecular , Protein Conformation , Saccharomyces cerevisiae/enzymologyABSTRACT
A study of the distribution of Topoisomerase II alpha (Topo II) in cells of six tissue culture cell lines, human (HeLa), mouse (L929), rat, Indian muntjac, rat kangaroo (PTK-2), and wallaby revealed the following features: (1) There is a cell cycle association of a specific population of Topo II with the centromere. (2) The centromere is distinguished from the remainder of the chromosome by the intensity of its Topo II reactivity. (3) The first appearance of a detectable population of Topo II at the centromere varies between species but is correlated with the onset of centromeric heterochromatin condensation. (4) Detectable centromeric Topo II declines at the completion of cell division. (5) The distribution pattern of Topo II within the centromere is species- and stage-specific and is conserved only within the kinetochore domain. In addition, we report that the Topo II inhibitor ICRF-193 can prevent the normal accumulation of Topo II at the centromere. This results in the disruption of chromatin condensation sub-adjacent to the kinetochore as well as the perturbation of kinetochore structure. Taken together, our studies indicate that the distribution of Topo II at the centromere is unlike that reported for the remainder of the chromosome and is essential for proper formation of centromere/kinetochore structure.
