DNA topoisomerase (topo) II catalyses topological genomic changes essential for many DNA metabolic processes. It is also regarded as a structural component of the nuclear matrix in interphase and the mitotic chromosome scaffold. Mammals have two isoforms (α and β) with similar properties in vitro. Here, we investigated their properties in living and proliferating cells, stably expressing biofluorescent chimera of the human isozymes. Topo IIα and IIβ behaved similarly in interphase but differently in mitosis, where only topo IIα was chromosome associated to a major part. During interphase, both isozymes joined in nucleolar reassembly and accumulated in nucleoli, which seemed not to involve catalytic DNA turnover because treatment with teniposide (stabilizing covalent catalytic DNA intermediates of topo II) relocated the bulk of the enzymes from the nucleoli to nucleoplasmic granules. Photobleaching revealed that the entire complement of both isozymes was completely mobile and free to exchange between nuclear subcompartments in interphase. In chromosomes, topo IIα was also completely mobile and had a uniform distribution. However, hypotonic cell lysis triggered an axial pattern. These observations suggest that topo II is not an immobile, structural component of the chromosomal scaffold or the interphase karyoskeleton, but rather a dynamic interaction partner of such structures.
The DNA cleavage reaction of eukaryotic topoisomerase II produces nicked DNA along with linear nucleic acid products. Therefore, relationships between the enzyme's DNA nicking and double-stranded cleavage reactions were determined. This was accomplished by altering the pH at which assays were performed. At pH 5.0 Drosophila melanogaster topoisomerase II generated predominantly (greater than 90%) single-stranded breaks in duplex DNA. With increasing pH, less single-stranded and more double-stranded cleavage was observed, regardless of the buffer or the divalent cation employed. As has been shown for double-stranded DNA cleavage, topoisomerase II was covalently bound to nicked DNA products, and enzyme-mediated single-stranded cleavage was salt reversible. Moreover, sites of single-stranded DNA breaks were identical with those mapped for double-stranded breaks. To further characterize the enzyme's cleavage mechanism, electron microscopy studies were performed. These experiments revealed that separate polypeptide chains were complexed with both ends of linear DNA molecules generated during cleavage reactions. Finally, by use of a novel religation assay [Osheroff, N., & Zechiedrich, E. L. (1987) Biochemistry 26, 4303-4309], it was shown that nicked DNA is an obligatory kinetic intermediate in the topoisomerase II mediated reunion of double-stranded breaks. Under the conditions employed, the apparent first-order rate constant for the religation of the first break was approximately 6-fold faster than that for the religation of the second break. The above results indicate that topoisomerase II carries out double-stranded DNA cleavage/religation by making two sequential single-stranded breaks in the nucleic acid backbone, each of which is mediated by a separate subunit of the homodimeric enzyme.
The functional domain structure of human DNA topoisomerase II␣ and Saccharomyces cerevisiae DNA topoisomerase II was studied by investigating the abilities of insertion and deletion mutant enzymes to support mitotic growth and catalyze transitions in DNA topology in vitro. Alignment of the human topoisomerase II␣ and S. cerevisiae topoisomerase II sequences defined 13 conserved regions separated by less conserved or differently spaced sequences. The spatial tolerance of the spacer regions was addressed by insertion of linkers. The importance of the conserved regions was assessed through deletion of individual domains. We found that the exact spacing between most of the conserved domains is noncritical, as insertions in the spacer regions were tolerated with no influence on complementation ability. All conserved domains, however, are essential for sustained mitotic growth of S. cerevisiae and for enzymatic activity in vitro. A series of topoisomerase II carboxy-terminal truncations were investigated with respect to the ability to support viability, cellular localization, and enzymatic properties. The analysis showed that the divergent carboxy-terminal region of human topoisomerase II␣ is dispensable for catalytic activity but contains elements that specifically locate the protein to the nucleus.Eukaryotic DNA topoisomerase II is an abundant nuclear enzyme involved in regulating the conversion of DNA between different topological isoforms (42, 60). It fulfills essential functions in DNA replication (41, 53) and chromosome segregation during both mitosis (26, 27) and meiosis (46), and it is thought to play a key role in certain types of DNA recombination events (8,10,22,30,48). The enzyme has furthermore been suggested to constitute a component of nuclear scaffold structures (3, 23), where it may be involved in chromosome condensation (4, 58) and decondensation (45).Topoisomerase II binds to DNA as a dimer and cleaves its DNA substrate with a 4-bp stagger (40, 60) at sites with loosely defined sequences (54). Upon DNA cleavage, each subunit of topoisomerase II becomes transiently ligated to the 5Ј ends of the respective DNA strands through an O 4 -phosphotyrosine bond involving a tyrosine residue in the active site of the protein (7, 36). Catalytic activity is ATP dependent, and it is performed by passing one intact DNA helix through the enzyme-mediated double-stranded DNA break, followed by rejoining of the DNA strands (42).Although the mechanics of topoisomerase II function have been characterized in great detail, little information has emerged about the functional organization of the enzyme. Proteolysis studies on purified topoisomerase II from Saccharomyces cerevisiae (35), Schizosaccharomyces pombe (50), Drosophila melanogaster (34), and humans (9) suggest that the enzyme is composed of three major structural domains, of which two are constituted by the conserved N-terminal and central parts of the protein and a third is constituted by the highly divergent C-terminal region.The fragment originating from the N-...
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