A distinct subnuclear localization of mammalian DNA topoisomerase IIbeta in yeast

Mammalian topoisomerase II isoforms alpha and beta are diverged in their C-terminal domain (CTD), but both isoforms complement the yeast top2 mutation. In this study, mammalian topoisomerase IIalpha-CTD and IIbeta-CTD were tagged with yellow fluorescent protein (YFP), expressed in yeast cells, and their localization was examined. YFP tagged-topoisomerase IIalpha-CTD was distributed evenly throughout the nucleus, while YFP tagged-topoisomerase IIbeta-CTD was sequestered into a subnuclear compartment. Deletion analysis revealed that two regions (amino acids 1207-1234 and 1513-1573) of the topoisomerase IIbeta-CTD are essential for specific localization of the beta isoform: if either of the two regions is removed, the mutant topoisomerase IIbeta-CTD distributes evenly throughout the nucleus. The data suggest that yeast cells distinguish the nuclear and subnuclear localization signals associated with these two mammalian topoisomerase II isoforms.     

Human DNA topoisomerase IIbeta binds and cleaves four-way junction DNA in vitro.

We have used gel retardation analysis to show that human DNA topoisomerase IIbeta can bind a 40 bp linear duplex containing a single DNA topoisomerase IIbeta cleavage site. Furthermore, we demonstrate for the first time that human DNA topoisomerase IIbeta binds to four-way junction DNA. This supports previous suggestions that topoisomerase II may be targeted to supercoiled DNA through the recognition of DNA cruciforms, helix-helix crossovers and hairpins. DNA topoisomerase IIbeta had a 4-fold higher affinity for the four-way junction than for the linear duplex, as demonstrated by protein titration and competition analysis. Furthermore, the DNA topoisomerase IIbeta:four-way junction complex was significantly more salt stable than the complex with linear DNA. The four-way junction contained potential topoisomerase IIbeta cleavage sites straddling the points of strand exchange, and indeed, topoisomerase IIbeta was able to cleave three of these four predicted sites. This indicates that topoiso-merase IIbeta can bind to the centre of the junction. Topoisomerase II has to bind both the transported and the gated DNA helices prior to strand passage, and it is possible that both helices are provided by the four-way junction in this case. The stable complex of DNA topoisomerase IIbeta with four-way junction DNA may provide an ideal substrate for further studies into the mechanism of substrate recognition and binding by DNA topoisomerase II.     

A truncated form of DNA topoisomerase IIbeta associates with the mtDNA genome in mammalian mitochondria.

Despite the likely requirement for a DNA topoisomerase II activity during synthesis of mitochondrial DNA in mammals, this activity has been very difficult to identify convincingly. The only DNA topoisomerase II activity conclusively demonstrated to be mitochondrial in origin is that of a type II activity found associated with the mitochondrial, kinetoplast DNA network in trypanosomatid protozoa [Melendy, T., Sheline, C., and Ray, D.S. (1988) Cell 55, 1083-1088; Shapiro, T.A., Klein, V.A., and Englund, P.A. (1989) J. Biol. Chem.264, 4173-4178]. In the present study, we report the discovery of a type DNA topoisomerase II activity in bovine mitochondria. Identified among mtDNA replicative proteins recovered from complexes of mtDNA and protein, the DNA topoisomerase relaxes a negatively, supercoiled DNA template in vitro, in a reaction that requires Mg2+ and ATP. The relaxation activity is inhibited by etoposide and other inhibitors of eucaryotic type II enzymes. The DNA topoisomerase II copurifies with mitochondria and directly associates with mtDNA, as indicated by sensitivity of some mtDNA circles in the isolated complex of mtDNA and protein to cleavage by etoposide. The purified activity can be assigned to a approximately 150-kDa protein, which is recognized by a polyclonal antibody made against the trypanosomal mitochondrial topo II enzyme. Mass spectrometry performed on peptides prepared from the approximately 150-kDa protein demonstrate that this bovine mitochondrial activity is a truncated version of DNA topoisomerase IIbeta, one of two DNA topoisomerase II activities known to exist in mammalian nuclei.     

Cytotoxic mechanism of XK469: resistance of topoisomerase IIbeta knockout cells and inhibition of topoisomerase I

Topoisomerase IIbeta knockout mouse cells (beta-/-) were found to have only slight resistance to m-AMSA, a dual topoisomerase IIalpha-IIbeta poison, as compared to wild-type cells (beta+/+) during 1 h or 3 day exposures to the drug. In contrast, the beta-/- cells were greater than threefold resistant to XK469, a selective topoisomerase IIbeta poison during three day drug exposures (beta+/+ IC(50) = 175 microM, beta-/- IC(50) = 581 microM). Short term (1 h) exposure to XK469 was not cytotoxic to either beta-/- or beta+/+ cells, suggesting that anticancer therapy with XK469 may be more efficacious if systemic levels can be prolonged. During studies on topoisomerase activity in nuclear extracts of the beta+/+ and beta-/- cells, we found evidence that XK469 is a weak topoisomerase I catalytic inhibitor. The high IC(50) for topoisomerase I inhibition (2 mM) suggests that topoisomerase I is not a significant target for XK469 cytotoxicity.     

Human topoisomerase IIalpha and IIbeta interact with the C-terminal region of p53

The p53 tumor suppressor protein is a critical regulator of cell cycle progression and apoptosis following exposure of cells to DNA damaging agents such as ionizing radiation or anticancer drugs. An important group of anticancer drugs, including compounds such as etoposide and doxorubicin (Adriamycin), interacts with DNA topoisomerase II (topo II), causing the accumulation of enzyme-DNA adducts that ultimately lead to double-strand breaks and cell death via apoptosis. Human topo IIbeta has previously been shown to interact with p53, and we have extended this analysis to show that both topo IIalpha and IIbeta interact with p53 in vivo and in vitro. Furthermore, we show that the regulatory C-terminal basic region of p53 (residues 364-393) is necessary and sufficient for interaction with DNA topo II.     

Involvement of topoisomerase III in telomere-telomere recombination

Telomere maintenance is required for chromosome stability, and telomeres are typically replicated by the action of telomerase. In both mammalian tumor and yeast cells that lack telomerase, telomeres are maintained by an alternative (ALT) recombination mechanism. In yeast, Sgs1p and its associated type IA topoisomerase, Top3p, may work coordinately in removing Holliday junction intermediates from a crossover-producing recombination pathway. Previous studies have also indicated that Sgs1 helicase acts in a telomere recombination pathway. Here we show that topoisomerase III is involved in telomere-telomere recombination. The recovery of telomere recombination-dependent survivors in a telomerase-minus yeast strain was dependent on Top3p catalytic activity. Moreover, the RIF1 and RIF2 genes are required for the establishment of TOP3/SGS1-dependent telomere-telomere recombination. In human Saos-2 ALT cells, human topoisomerase IIIalpha (hTOP3alpha) also contributes to telomere recombination. Strikingly, the telomerase activity is clearly enhanced in surviving si-hTOP3alpha Saos-2 ALT cells. Altogether, the present results suggest a potential role for hTOP3alpha in dissociating telomeric structures in telomerase-deficient cells, providing therapeutic implications in human tumors.     

The SUMO pathway is required for selective degradation of DNA topoisomerase IIbeta induced by a catalytic inhibitor ICRF-193(1)

DNA topoisomerase I and II have been shown to be modified with a ubiquitin-like protein SUMO in response to their specific inhibitors called 'poisons'. These drugs also damage DNA by stabilizing the enzyme-DNA cleavable complex and induce a degradation of the enzymes through the 26S proteasome system. A plausible link between sumoylation and degradation has not yet been elucidated. We demonstrate here that topoisomerase IIbeta, but not its isoform IIalpha, is selectively degraded through proteasome by exposure to the catalytic inhibitor ICRF-193 which does not damage DNA. The beta isoform immunoprecipitated from ICRF-treated cells was modified by multiple modifiers, SUMO-2/3, SUMO-1, and polyubiquitin. When the SUMO conjugating enzyme Ubc9 was conditionally knocked out, the ICRF-induced degradation of topoisomerase IIbeta did not occur, suggesting that the SUMO modification pathway is essential for the degradation.     

The DNA topoisomerase IIbeta binding protein 1 (TopBP1) interacts with poly (ADP-ribose) polymerase (PARP-1)

We investigated the physical association of the DNA topoisomerase IIbeta binding protein 1 (TopBP1), involved in DNA replication and repair but also in regulation of apoptosis, with poly(ADP-ribose) polymerase-1 (PARP-1). This enzyme plays a crucial role in DNA repair and interacts with many DNA replication/repair factors. It was shown that the sixth BRCA1 C-terminal (BRCT) domain of TopBP1 interacts with a protein fragment of PARP-1 in vitro containing the DNA-binding and the automodification domains. More significantly, the in vivo interaction of endogenous TopBP1 and PARP-1 proteins could be shown in HeLa-S3 cells by co-immunoprecipitation. TopBP1 and PARP-1 are localized within overlapping regions in the nucleus of HeLa-S3 cells as shown by immunofluorescence. Exposure to UVB light slightly enhanced the interaction between both proteins. Furthermore, TopBP1 was detected in nuclear regions where poly(ADP-ribose) (PAR) synthesis takes place and is ADP-ribosylated by PARP-1. Finally, cellular (ADP-ribosyl)ating activity impairs binding of TopBP1 to Myc-interacting zinc finger protein-1 (Miz-1). The results indicate an influence of post-translational modifications of TopBP1 on its function during DNA repair.     

Dynamics of human DNA topoisomerases IIalpha and IIbeta in living cells

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 (alpha and beta) with similar properties in vitro. Here, we investigated their properties in living and proliferating cells, stably expressing biofluorescent chimera of the human isozymes. Topo IIalpha and IIbeta behaved similarly in interphase but differently in mitosis, where only topo IIalpha 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 IIalpha 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.     

Metaphase chromosome structure. Involvement of topoisomerase II

SCI is a prominent, 170,000 Mr, non-histone protein of HeLa metaphase chromosomes. This protein binds DNA and was previously identified as one of the major structural components of the residual scaffold structure obtained by differential protein extraction from isolated chromosomes. The metaphase scaffold maintains chromosomal DNA in an organized, looped conformation. We have prepared a polyclonal antibody against the SC1 protein. Immunolocalization studies by both fluorescence and electron microscopy allowed identification of the scaffold structure in gently expanded chromosomes. The micrographs show an immunopositive reaction going through the kinetochore along a central, axial region that extends the length of each chromatid. Some micrographs of histone-depleted chromosomes provide evidence of the substructural organization of the scaffold; the scaffold appears to consist of an assembly of foci, which in places form a zig-zag or coiled arrangement. We present several lines of evidence that establish the identity of SC1 as topoisomerase II. Considering the enzymic nature of this protein, it is remarkable that it represents 1% to 2% of the total mitotic chromosomal protein. About 60% to 80% of topoisomerase II partitions into the scaffold structure as prepared from isolated chromosomes, and we find approximately three copies per average 70,000-base loop. This supports the proposed structural role of the scaffold in the organization of the mitotic chromosome. The dual enzymic and apparent structural function of topoisomerase II (SC1) and its location at or near the base of chromatin loops allows speculation as to its involvement in the long-range control of chromatin structure.     

Involvement of Oct3/4 in the enhancement of neuronal differentiation of ES cells in neurogenesis-inducing cultures

Oct3/4 plays a critical role in maintaining embryonic stem cell pluripotency. Regulatable transgene-mediated sustained Oct3/4 expression in ES cells cultured in serum-free LIF-deficient medium caused accelerated differentiation to neuroectoderm-like cells that expressed Sox2, Otx1 and Emx2 and subsequently differentiated into neurons. Neurogenesis of ES cells is promoted by SDIA (stromal cell-derived inducing activity), which accumulates on the PA6 stromal cell surface. Oct3/4 expression in ES cells was maintained by SDIA whereas without it expression was promptly downregulated. Suppression of Oct3/4 abolished neuronal differentiation even after stimulation by SDIA. In contrast, sustained upregulated Oct3/4 expression enhanced SDIA-mediated neurogenesis of ES cells. Therefore, Oct3/4 appears to promote neuroectoderm formation and subsequent neuronal differentiation from ES cells.     

Dna topoisomerase activities in plants: Involvement in cell proliferation

Abstract

The role of DNA topoisomerases in cell processes related to DNA metabolism, their involvement in the regulation of cell proliferation and their distribution in plant tissues are discussed.