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.     

Sequence determinants of nuclear localization in the alpha and beta isoforms of human topoisomerase II.

The alpha and beta isoforms of DNA topoisomerase II (topo II) are targets for several widely used chemotherapeutic agents, and resistance to some of these drugs may be associated with reduced nuclear localization of the alpha isoform. Human topo IIalpha contains a strong bipartite nuclear localization signal (NLS) sequence between amino acids 1454 and 1497 (alphaNLS(1454-1497)). In the present study, we show that human topo IIalpha tagged with green fluorescence protein is still detectable in the nucleus when alphaNLS(1454-1497) has been disrupted. Seven additional regions in topo IIalpha containing overlapping potential bipartite NLSs were evaluated for their nuclear targeting abilities using a beta-galactosidase reporter system. A moderately functional NLS was identified between amino acids 1259 and 1296. When human topo IIbeta was examined in a similar fashion, it was found to contain two strongly functional sequences betaNLS(1522-1548) and betaNLS(1538-1573) in the region of topo IIbeta comparable to the region in topo IIalpha that contains the strongly functional alphaNLS(1454-1497). The third, betaNLS(1294-1332), although weaker than the other two beta sequences, is significantly stronger than the analogous alphaNLS(1259-1296). Differences in the NLS sequences of human topo II isoforms may contribute to their differences in subnuclear localization.     

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.     

Topoisomerase II binds importin alpha isoforms and exportin/CRM1 but does not shuttle between the nucleus and cytoplasm in proliferating cells

Resistance to anticancer drugs that target DNA topoisomerase II (topo II) isoforms alpha and/or beta is associated with decreased nuclear and increased cytoplasmic topo IIalpha. Earlier studies have confirmed that functional nuclear localization and export signal sequences (NLS and NES) are present in both isoforms. In this study, we show that topo II alpha and beta bind and are imported into the nucleus by importin alpha1, alpha3, and alpha5 in conjunction with importin beta. Topo IIalpha also binds exportin/CRM1 in vitro. However, wild-type topo IIalpha has only been observed in the cytoplasm of cells that are entering plateau phase growth. This suggests that topo IIalpha may shuttle between the nucleus and the cytoplasm with the equilibrium towards the nucleus in proliferating cells but towards the cytoplasm in plateau phase cells. The CRM1 inhibitor Leptomycin B increases the nuclear localization of GFP-tagged topo IIalpha with a mutant NLS, suggesting that its export is being inhibited. However, homokaryon shuttling experiments indicate that fluorescence-tagged wild-type topo II alpha and beta proteins do not shuttle in proliferating Cos-1 or HeLa cells. We conclude that topo II alpha and beta nuclear export is inhibited in proliferating cells so that these proteins do not shuttle.     

Mitotic specific phosphorylation of serine-1212 in human DNA topoisomerase IIalpha.

It is known that topoisomerase IIalpha is phosphorylated by several kinases. To elucidate the role of phosphorylation of topoisomerase IIalpha in the cell cycle, we have examined the cell cycle behavior of phosphorylated topoisomerase IIalpha in HeLa cells using antibodies against several phospho-oligopeptides of this enzyme. Here we demonstrate that serine1212 in topoisomerase IIalpha is phosphorylated only in the mitotic phase. Using an antibody against an oligopeptide containing phosphoserine-1212 in topoisomerase IIalpha (PS1212), subcellular localization of topoisomerase IIalpha phosphorylated at serine1212 was examined by indirect immunofluorescence staining, and compared with that of overall topoisomerase IIalpha. Serine1212-phosphorylated topoisomerase IIalpha was localized specifically on mitotic chromosomes, but not on interphase chromosomes; this result contrasts with overall topoisomerase IIalpha which was observed on chomosomes in both interphase and mitosis. Serine1212-phosphorylated topoisomerase lIalpha first appeared on chromosome arms in prophase, became concentrated on the centromeres in metaphase, and disappeared in early telophase. In addition, ICRF-193, a catalytic inhibitor of topoisomerase II, prevented accumulation of serine1212-phosphorylated topoisomerase IIalpha at the centromeres. These results indicate that serine1212 of topoisomerase IIalpha is phosphorylated specifically during mitosis, and suggest that the serine1212-phosphorylated topoisomerase IIalpha acts on resolving topological constraint progressively from the chromosome arm to the centromere during metaphase chromosome condensation.     

Analysis of functional domain organization in DNA topoisomerase II from humans and Saccharomyces cerevisiae.

The functional domain structure of human DNA topoisomerase IIalpha 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 IIalpha 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 IIalpha is dispensable for catalytic activity but contains elements that specifically locate the protein to the nucleus.     

M phase-specific association of human topoisomerase IIIbeta with chromosomes

Two isoforms, 1 and 2, of human DNA topoisomerase IIIbeta were expressed in HeLa cells as a fusion protein to the C-terminus of green fluorescent protein (GFP). The fusion protein of the isoform 1 was found to be localized to the nucleus, and to be associated with chromosomes during metaphase and anaphase. As yeast top3 mutants are known to exhibit phenotypes indicative of defective chromosome segregation, the result suggests that the isoform 1 of the human enzyme may also be involved in chromosome segregation. Two-hybrid screening for interaction partners of the isoform identified three candidate genes: CENP-F, a gene encoding a centromere protein and two genes of no known function, one of which was novel. The GFP fusion of the isoform 2 was found in the cytoplasm, indicating the nuclear localization signal sequence in the isoform 1 is in the C-terminal part that is different between the two isoforms.     

Enhanced processing of UVA-irradiated DNA by human topoisomerase II in living cells

Solar UV light induces a variety of DNA lesions in the genome. Enhanced cleavage of such base modifications by topoisomerase II has been demonstrated in vitro, but it is unclear what will arise from an interplay of these mechanisms in the genome of a living cell exposed to UV light. To address this question, we have subjected cells expressing biofluorescent topoisomerase IIalpha or IIbeta to DNA base modifications inflicted by a UVA laser at 364 nm through a confocal microscope in a locally confined manner. At DNA sites thus irradiated, we observed rapid, long term (>90 min) accumulation of topoisomerase IIalpha and IIbeta, which was accompanied by a decrease in mobility but not immobilization of the enzyme. The catalytic topoisomerase II inhibitor ICRF-187 prevented the effect when added to the cell culture before the UVA pulse but promoted it when added thereafter. Self-primed in situ extension with rhodamine-dUTP revealed massive DNA breakage at the UVA-exposed spot. Culturing the cells with ICRF-187 before UVA-exposure prevented such breaks. In conclusion, we show in a living cell nucleus that UVA-modified DNA is preferentially targeted and processed by topoisomerase IIalpha and IIbeta. This results in increased levels of topoisomerase II-mediated DNA breaks, but formation of immobile, stable topoisomerase II.DNA intermediates is not notably promoted. Inhibition of topoisomerase II activity by ICRF-187 greatly diminishes UVA-induced DNA breakage, implying topoisomerase IIalpha and IIbeta as endogenous co-factors modulating and possibly aggravating the impact of UVA light on the genome.     

Preparation and characterization of nuclear matrix/chromosome scaffold in situ

A method of nuclear matrix and chromosomal scaffold preparation from cultured animal cells was developed. After the high-salt extraction, interphase and mitotic cells were not detached from the coverslips that enabled us to analyse the nuclear matrix and chromosomal scaffold in cells at all mitotic phases. Morphological methods (phase contrast microscopy and electron microscopy of ultrathin sections) did not reveal any structures that could be identified as a chromosomal scaffold. However, after staining with antibodies to XCAP-E and topoisomerase IIalpha some structures were revealed in metaphase cells having both localization and morphology of a chromosomal scaffold. The cell residuals were not stained with antibodies to XCAP-E and topoisomerase IIalpha, if the nuclear matrix and chromosomal scaffold were destabilized by addition of beta-mercaptoethanol.     

Stimulation of human DNA topoisomerase II activity by its direct association with the beta subunit of protein kinase CKII

DNA topoisomerase II copurifies with and is phosphorylated by protein kinase CKII. In this study, a yeast two-hybrid system was used to investigate the interaction between human topoisomerase II isozymes and CKII subunits. The two-hybrid test clearly showed that both topoisomerase IIalpha and IIbeta interact with the CKIIbeta, but not the CKIIalpha subunit. The two-hybrid test also demonstrated that topoisomerase IIbeta residues 1099-1263 and topoisomerase IIalpha residues 1078-1182 mediate the interaction with the CKIIbeta subunit, providing evidence that the leucine zipper motif and the major CKII-dependent phosphorylation sites of topoisomerase II are unnecessary for its physical binding to CKIIbeta. Furthermore, a DNA relaxation assay demonstrated that the CKII subunit enhances topoisomerase II activity by physical interaction with topoisomerase II.     

Bioflavonoids as poisons of human topoisomerase II alpha and II beta

Bioflavonoids are human dietary components that have been linked to the prevention of cancer in adults and the generation of specific types of leukemia in infants. While these compounds have a broad range of cellular activities, many of their genotoxic effects have been attributed to their actions as topoisomerase II poisons. However, the activities of bioflavonoids against the individual isoforms of human topoisomerase II have not been analyzed. Therefore, we characterized the activity and mechanism of action of three major classes of bioflavonoids, flavones, flavonols, and isoflavones, against human topoisomerase IIalpha and IIbeta. Genistein was the most active bioflavonoid tested and stimulated enzyme-mediated DNA cleavage approximately 10-fold. Generally, compounds were more active against topoisomerase IIbeta. DNA cleavage with both enzyme isoforms required a 5-OH and a 4'-OH and was enhanced by the presence of additional hydroxyl groups on the pendant ring. Competition DNA cleavage and topoisomerase II binding studies indicate that the 5-OH group plays an important role in mediating genistein binding, while the 4'-OH moiety contributes primarily to bioflavonoid function. Bioflavonoids do not require redox cycling for activity and function primarily by inhibiting enzyme-mediated DNA ligation. Mutagenesis studies suggest that the TOPRIM region of topoisomerase II plays a role in genistein binding. Finally, flavones, flavonols, and isoflavones with activity against purified topoisomerase IIalpha and IIbeta enhanced DNA cleavage by both isoforms in human CEM leukemia cells. These data support the hypothesis that bioflavonoids function as topoisomerase II poisons in humans and provide a framework for further analysis of these important dietary components.     

Vaccinia virus DNA ligase recruits cellular topoisomerase II to sites of viral replication and assembly

Vaccinia virus replication is inhibited by etoposide and mitoxantrone even though poxviruses do not encode the type II topoisomerases that are the specific targets of these drugs. Furthermore, one can isolate drug-resistant virus carrying mutations in the viral DNA ligase and yet the ligase is not known to exhibit sensitivity to these drugs. A yeast two-hybrid screen was used to search for proteins binding to vaccinia ligase, and one of the nine proteins identified comprised a portion (residue 901 to end) of human topoisomerase IIbeta. One can prevent the interaction by introducing a C(11)-to-Y substitution mutation into the N terminus of the ligase bait protein, which is one of the mutations conferring etoposide and mitoxantrone resistance. Coimmunoprecipitation methods showed that the native ligase and a Flag-tagged recombinant protein form complexes with human topoisomerase IIalpha/beta in infected cells and that this interaction can also be disrupted by mutations in the A50R (ligase) gene. Immunofluorescence microscopy showed that both topoisomerase IIalpha and IIbeta antigens are recruited to cytoplasmic sites of virus replication and that less topoisomerase was recruited to these sites in cells infected with mutant virus than in cells infected with wild-type virus. Immunoelectron microscopy confirmed the presence of topoisomerases IIalpha/beta in virosomes, but the enzyme could not be detected in mature virus particles. We propose that the genetics of etoposide and mitoxantrone resistance can be explained by vaccinia ligase binding to cellular topoisomerase II and recruiting this nuclear enzyme to sites of virus biogenesis. Although other nuclear DNA binding proteins have been detected in virosomes, this appears to be the first demonstration of an enzyme being selectively recruited to sites of poxvirus DNA synthesis and assembly.     

Association of human DNA topoisomerase IIalpha with mitotic chromosomes in mammalian cells is independent of its catalytic activity.

DNA topoisomerase (topo) II is an essential nuclear enzyme that plays an important role in DNA metabolism and chromosome organization. In the present study, we expressed human topo IIalpha in mammalian cells by fusion to an enhanced green fluorescent protein (EGFP). Decatenation assays indicated that the EGFP-topo IIalpha is catalytically active in vitro. Assays for band depletion, growth inhibition, and cytotoxicity by topo II inhibitors suggested that the fusion protein is also functional in vivo. By following its subcellular localization throughout the cell cycle in living cells, we found that the fusion protein is localized to the nucleus and nucleolus at interphase, and it is bound to chromosomal DNA at every stage of mitosis. Of importance, a mutant EGFP-topo IIalpha, in which the active Tyr 805 is replaced by Phe (Y805F) and is catalytically inactive, still binds to chromosomal DNA throughout the cell cycle like the wild-type enzyme. Together, our results suggest that the ability of topo IIalpha to bind to chromosomal DNA in the cell, a presumed requirement for its structural role, can be separated from its catalytic activity.     

The cytochrome b5 tail anchors and stabilizes subdomains of human DNA topoisomerase II alpha in the cytoplasm of retrovirally infected mammalian cells.

DNA topoisomerase II (topo II) is the target of many anticancer drugs and is often altered in drug-resistant cell lines. In some tumor cell lines truncated isoforms of topo IIalpha are localized to the cytoplasm. To study the localization and function of individual enzyme domains, we have epitope-tagged several fragments of human topo IIalpha and expressed them by retroviral infection of rodent and human cells. We find that fusion of the topo II fragments to the hydrophobic tail of human liver cytochrome b5 anchors the fusion protein to the outer face of cytoplasmic membranes, as determined by colocalization with calnexin and selective detergent permeabilization. Moreover, whereas the minimal ATPase domain (aa 1-266) is weakly and diffusely expressed, addition of the cytb5 anchor (1-266-b5) increases its steady-state level 16-fold with no apparent toxicity. Similar results are obtained with the complete ATPase domain (aa 1-426). A C-terminal domain (aa 1030-1504) of human topo IIalpha containing an intact dimerization motif is stably expressed and accumulates in the nucleus. Fusion to the cytb5 anchor counteracts the nuclear localization signal and relocalizes the protein to cytoplasmic membranes. In conclusion, we describe a technique that stabilizes and targets retrovirally expressed proteins such that they are exposed on the cytoplasmic surface of cellular membranes. This approach may be of general use for regulating the nuclear accumulation of drugs or proteins in living cells.     

Localisation of DNA topoisomerase IIalpha in mouse erythroleukemia cells.

The presence of DNA topoisomerase IIalpha was investigated in interphase and metaphase mouse erythroleukemia (MEL) Friend-S cells, and in extracted with 25 mM lithium diiodosalicylate buffer (Lis) nuclei using indirect immunofluorescence. The results showed that DNA topoisomerase IIalpha is localised in the nuclei. In the metaphase cells, we found high concentrations of this enzyme in the mitotic chromosomes. Our results support the idea of the accumulation of DNA topoisomerase IIalpha at the end of the cell cycle. The extractions of nuclei with 25 mM Lis led to the complete depletion of DNA topoisomerase IIalpha from the residual nuclear matrix. Using a high dilution of the first antibody, we established that the high level of heterochromatin compactisation in the interphase nuclei is caused by the high concentration of DNA topoisomerase IIalpha.     

Effects of wild-type p53 expression on the quantity and activity of topoisomerase IIalpha and beta in various human cancer cell lines.

The p53 null HL-60 cell line was transfected with plasmids coding for either the wild-type p53 or mutant p53 gene. The stable expression of wild-type p53 resulted in a significant increase in sensitivity to the topoisomerase II poisons etoposide and doxorubicin, but not to the topoisomerase II inhibitors razoxane and ADR-529. HL-60 cells expressing wild-type p53 demonstrated 8- to 10-fold more VP-16 induced DNA breaks by the alkaline elution assay. The effect of inducible expression of wild-type p53 was also studied in the p53 null erythroblastoid cell line K562 and in the human squamous carcinoma cell line SqCC. The inducible expression of wild-type p53 in the K562 cell line resulted in a 3-fold increase in sensitivity to VP-16. The quantity of topoisomerase IIalpha was not altered by the transfection as determined by immunoblotting, while the amount of the beta isoform was increased 2.5-fold in HL-60 cells. The topo II catalytic activity present in nuclear extracts was measured as the decatenation of kinetoplast DNA, and found to be unaltered by p53 expression. Immunostaining for topoisomerase IIalpha was substantially diminished in both stable and inducible wild-type p53 expressing cells when three different antibodies were used (two polyclonal and one monoclonal). However, the addition of VP-16 resulted in a rapid appearance of nuclear fluorescence for topoisomerase IIalpha. No changes in topoisomerase IIbeta immunostaining were observed. These results suggest that an epitope for topoisomerase IIalpha is concealed in cells expressing wild-type p53 and that a complex between topoisomerase IIalpha and p53 may be disrupted by the addition of antitumor drugs.     

SUMO-1 conjugation to human DNA topoisomerase II isozymes

Topoisomerase I-mediated DNA damage induced by camptothecin has been shown to induce rapid small ubiquitin-related modifier (SUMO)-1 conjugation to topoisomerase I. In the current study, we show that topoisomerase II-mediated DNA damage induced by teniposide (VM-26) results in the formation of high molecular weight conjugates of both topoisomerase IIalpha and IIbeta isozymes in HeLa cells. Immunological characterization of these conjugates suggests that both topoisomerase IIalpha and IIbeta isozymes are conjugated to SUMO-1. The involvement of SUMO-1/UBC9 in the modification of topoisomerase II isozymes is also supported by the demonstration of physical interaction between topoisomerase II and SUMO-1/UBC9. Surprisingly, ICRF-193, which does not induce topoisomerase II-mediated DNA damage but traps topoisomerase II into a circular clamp conformation, is also shown to induce similar SUMO-1 conjugation to topoisomerase II isozymes. In addition, we show that both oxidative and heat shock stresses, which can cause protein damage, rapidly increase nuclear SUMO-1 conjugates. These studies raise the question on whether SUMO-1 conjugation to topoisomerases is an indirect result of a DNA damage response or a direct result because of protein conformational changes.