Rapid exchange of mammalian topoisomerase II alpha at kinetochores and chromosome arms in mitosis

A stable cell line (GT2-LPk) derived from LLC-Pk was created in which endogenous DNA topoisomerase II alpha (topoII alpha) protein was downregulated and replaced by the expression of topoII alpha fused with enhanced green fluorescent protein (EGFP-topoII alpha). The EGFP-topoII alpha faithfully mimicked the distribution of the endogenous protein in both interphase and mitosis. In early stages of mitosis, EGFP-topoII alpha accumulated at kinetochores and in axial lines extending along the chromosome arms. During anaphase, EGFP-topoII alpha diminished at kinetochores and increased in the cytoplasm with a portion accumulating into large circular foci that were mobile and appeared to fuse with the reforming nuclei. These cytoplasmic foci appearing at anaphase were coincident with precursor organelles of the reforming nucleolus called nucleolus-derived foci (NDF). Photobleaching of EGFP-topoII alpha associated with kinetochores and chromosome arms showed that the majority of the protein rapidly exchanges (t1/2 of 16 s). Catalytic activity of topoII alpha was essential for rapid dynamics, as ICRF-187, an inhibitor of topoII alpha, blocked recovery after photobleaching. Although some topoII alpha may be stably associated with chromosomes, these studies indicate that the majority undergoes rapid dynamic exchange. Rapid mobility of topoII alpha in chromosomes may be essential to resolve strain imparted during chromosome condensation and segregation.     

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.     

Cleavage of DNA by mammalian DNA topoisomerase II

Using the P4 unknotting assay, DNA topoisomerase II has been purified from several mammalian cells. Similar to prokaryotic DNA gyrase, mammalian DNA topoisomerase II can cleave double-stranded DNA and be trapped as a covalent protein-DNA complex. This cleavage reaction requires protein denaturant treatment of the topoisomerase II-DNA complex and is reversible with respect to salt and temperature. The product after reversal of the cleavage reaction remains supertwisted, suggesting that the two ends of the putatively broken DNA are held tightly by the topoisomerase. Alternatively, the enzyme-DNA interaction is noncovalent, and the covalent linking of topoisomerase to DNA is induced by the protein denaturant. Detailed characterization of the cleavage products has revealed that topoisomerase II cuts DNA with a four-base stagger and is covalently linked to the protruding 5'-phosphoryl ends of each broken DNA strand. Calf thymus DNA topoisomerase II cuts SV40 DNA at multiple and specific sites. However, no sequence homology has been found among the cleavage sites as determined by direct nucleotide-sequencing studies.     

Mitotic spindle pulls but fails to separate chromosomes in type II DNA topoisomerase mutants: uncoordinated mitosis

The fission yeast top2 locus is defined by five temperature-sensitive mutations that cause heat-labile activity of type II DNA topoisomerase in the cell extracts. We show that the top2 locus is a structural gene for type II topoisomerase by cloning a genomic DNA fragment that complements top2. The top2 mutants at restrictive temperature produce abnormal chromosomes at the time of mitosis; these are transiently extended into filamentous structures along with the elongating mitotic spindle but are not separated. A primary defect in top2 appears to be the formation of aberrant mitotic chromosomes inseparable by the force generated by the spindle apparatus. Consistently, the top2 cells that become lethal during mitosis contain a catenated dimer of an ARS plasmid. DNA and RNA continue to be synthesized if cytokinesis is blocked. Uncoordinated mitosis, that is the occurrence of spindle dynamics without chromosome separation, is revealed in top2, and is discussed in relation to mitotic regulation. Different phenotypes between top2 and top1-top2 described in the present paper can be explained by a previously proposed hypothesis that type II topoisomerase has dual in vivo functions: one that decatenates and unknots duplex DNAs is essential in mitosis, whereas the other which relaxes supercoils is required throughout the cell cycle if type I topoisomerase is absent.     

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.     

Nuclear interactions of topoisomerase II alpha and beta with phospholipid scramblase 1

DNA topoisomerase (topo) II modulates DNA topology and is essential for cell division. There are two isoforms of topo II (α and β) that have limited functional redundancy, although their catalytic mechanisms appear the same. Using their COOH-terminal domains (CTDs) in yeast two-hybrid analysis, we have identified phospholipid scramblase 1 (PLSCR1) as a binding partner of both topo II α and β. Although predominantly a plasma membrane protein involved in phosphatidylserine externalization, PLSCR1 can also be imported into the nucleus where it may have a tumour suppressor function. The interactions of PLSCR1 and topo II were confirmed by pull-down assays with topo II α and β CTD fusion proteins and endogenous PLSCR1, and by co-immunoprecipitation of endogenous PLSCR1 and topo II α and β from HeLa cell nuclear extracts. PLSCR1 also increased the decatenation activity of human topo IIα. A conserved basic sequence in the CTD of topo IIα was identified as being essential for binding to PLSCR1 and binding of the two proteins could be inhibited by a synthetic peptide corresponding to topo IIα amino acids 1430-1441. These studies reveal for the first time a physical and functional interaction between topo II and PLSCR1.     

Mutant isolation of mouse DNA topoisomerase II alpha in yeast.

For characterizing in vivo functions of a mammalian protein, it is informative to obtain conditional mutations and apply them to the mouse genetic system. However, the isolation of conditional mutations has been quite difficult in cultured cells. We report here that functional expression of a heterologous mammalian gene in the yeast Saccharomyces cerevisiae provides a system for isolating mutated genes. We found that the cloned mouse TOP2 alpha cDNA, which encodes mouse DNA topoisomerase II (topo II) alpha, could rescue the lethal phenotype caused by yeast top2 null mutation. In order to generate and select temperature-sensitive mouse topo II alpha, an expression plasmid was mutagenized in vitro and was transformed, using the plasmid shuffling method, into the yeast strain, in which the endogenous TOP2 gene had been disrupted. We observed that one of such clone of yeast cells harboring a mutagenized mouse TOP2 alpha showed temperature-sensitive growth. Enzymatic assays and sequencing analysis revealed that this phenotype was caused by the thermosensitive nature of the mutant mouse protein, which has isoleucine at amino acid 961 instead of threonine. Therefore we have isolated the first conditional mutation in the mouse TOP2 alpha.     

Pattern of recognition of DNA by mammalian DNA topoisomerase II

The antitumor drug VP-16 stabilizes the topoisomerase II-DNA covalent complexes formed in an intermediate step of the isomerization reaction. The location of the sites of formation of these complexes and their relative strength were studied in vitro using pBR322. Sequences alignment of the regions containing the 24 detectable sites allows to identify GCGCGC-(N) alpha-TGAC with 9 less than or equal to alpha less than or equal to 25 as the DNA sequence recognized by topoisomerase II to form a cleavable complex. Changes in the last two nucleotides of the sequence determine weaker complexes.     

DNA topoisomerase II must act at mitosis to prevent nondisjunction and chromosome breakage.

The hypothesis that DNA topoisomerase II facilitates the separation of replicated sister chromatids was tested by examining the consequences of chromosome segregation in the absence of topoisomerase II activity. We observed a substantial elevation in the rate of nondisjunction in top2/top2 cells incubated at the restrictive temperature for one generation time. In contrast, only a minor increase in the amount of chromosome breakage was observed by either physical or genetic assays. These results suggest that aneuploidy is a major cause of the nonviability observed when top2 cells undergo mitosis at the restrictive temperature. In related experiments, we determined that topoisomerase II must act specifically during mitosis. This latter observation is consistent with the hypothesis that the mitotic spindle is necessary to allow topoisomerase II to complete the untangling of sister chromatids.     

DNA topoisomerase II is required at the time of mitosis in yeast

We have constructed five new temperature-sensitive DNA topoisomerase II mutations and have analyzed their physiological consequences in yeast. Several lines of evidence suggest that the activity of topoisomerase II is required specifically at the time of miosis. First, top2 mutations cause dramatic lethality at the restrictive temperature, but only if the mutant cells are actively traversing the cell cycle. Second, temperature-shift experiments with synchronized cultures show that the onset of inviability coincides with the time of mitosis. Third, fluorescence microscopy reveals that the normal progression of mitosis is disturbed in mutant cells at the restrictive temperature. Finally, inviability at the restrictive temperature is prevented by nocodazole, an inhibitor of tubulin polymerization that prevents formation of the mitotic spindle. These results are consistent with the hypothesis that the essential function of topoisomerase II is to allow the separation of intertwined chromosomal DNA molecules during mitosis.     

Segregation of recombined chromosomes in meiosis I requires DNA topoisomerase II

To understand better the similarities and differences between meiosis and mitosis, we examined the meiotic role of DNA topoisomerase II, an enzyme that is required mitotically to disentangle sister chromatids at the time of chromosome segregation. In meiosis, we found that topoisomerase II is required only at the time of nuclear division. When cold-sensitive top2 mutants are induced to sporulate at the restrictive temperature, they undergo premeiotic DNA synthesis and commitment to meiotic levels of recombination but fail to complete the first meiotic nuclear division. The introduction of a mutation blocking recombination relieves the requirement for topoisomerase II in meiosis I. These results suggest that topoisomerase II is required at the time of chromosome segregation in meiosis I for the resolution of recombined chromosomes.     

DNA ligation catalyzed by human topoisomerase II alpha

The DNA ligation reaction of topoisomerase II is essential for genomic integrity. However, it has been impossible to examine many fundamental aspects of this reaction because ligation assays historically required the enzyme to cleave a DNA substrate before sealing the nucleic acid break. Recently, a cleavage-independent DNA ligation assay was developed for human topoisomerase IIalpha [Bromberg, K. D., Hendricks, C., Burgin, A. B., and Osheroff, N. (2002) J. Biol. Chem. 277, 31201-31206]. This assay overcomes the requirement for DNA cleavage by monitoring the ability of the enzyme to ligate a nicked oligonucleotide in which the 5'-terminal phosphate at the nick has been activated by covalent attachment to the tyrosine mimic, p-nitrophenol. The cleavage-independent ligation assay was used to more fully characterize the DNA ligation activity of human topoisomerase IIalpha. Results suggest that the active site tyrosine contributes little to the catalysis of DNA ligation beyond its primary role as an activating/leaving group. Although arginine 804 (the residue immediately N-terminal to the active site tyrosine) has been proposed to help anchor the 5'-DNA terminus during cleavage, conversion of this residue to alanine had only a modest effect on DNA ligation. Thus, it appears that arginine 804 does not play an essential role in DNA strand joining. In contrast, disruption of base pairing at the 5'-DNA terminus abrogated DNA ligation in the absence of a covalent enzyme-DNA bond. Therefore, it is proposed that base pairing represents a secondary mechanism for aligning the 5'-DNA termini for ligation. Finally, the human enzyme appears to ligate the two scissile bonds of a cleavage site in a nonconcerted fashion.     

Ciprofloxacin: mammalian DNA topoisomerase type II poison in vivo

Ciprofloxacin (CF), a fluoroquinolone widely used as a potent antimicrobial drug, was evaluated in vivo in mouse bone marrow cells for its ability to induce clastogenicity and DNA damage in terms of increased sister-chromatid exchange (SCE) frequencies. Doses of 0.6, 6 and 20 mg/kg body weight of CF given intraperitoneally induced a positive dose-dependent significant clastogenicity (trend test α ⩽ 0.05), though the effects were not specific for specific phases of the cell cycle.The DNA-damaging effect observed as increased SCE frequencies using doses of 0.15, 0.30, 0.60, 1.2 and 6 mg/kg body weight showed a significant dose-dependent increase (trend test α ⩽ 0.05; lowest effective concentration 1.2 mg/kg of body weight).Compared to a potent eukaryotic DNA topoisomerase type II poison, etoposide (VP-16, 0.5, 1 and 5 mg/kg body weight, given intraperitoneally), ciprofloxacin produced comparable dose-dependent SCE frequency increases. Ciprofloxacin was postulated to be specific for the target DNA gyrase, the prokaryotic homologue of DNA topoisomerase type II enzyme. The present paper along with the existing earlier data strongly suggest that topoisomerase type II and DNA gyrase are physiological targets for the drug action. In view of the present significant in vivo mammalian DNA topoisomerase type II-mediated genotoxicity and clastogenicity data, ciprofloxacin should be administered with caution.     

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.     

DNA topoisomerase II is required for condensation and separation of mitotic chromosomes in S. pombe

We show that DNA topoisomerase II (topo II) is continuously required for mitotic chromosome changes in Schizosaccharomyces pombe. We constructed cold-sensitive (cs) or temperature-sensitive (ts) strains mutated in the genes coding for topo II (top2) and beta-tubulin (nda3). The ATP-dependent activity of the top2cs gene product is cs in vitro. The cloned top2cs gene sequence predicts an amino acid substitution. A cs top2-cs nda3 double mutant at 20 degrees C shows long, entangled chromosomes, which condense and separate upon the shift to permissive temperatures. If spindle formation is prevented at permissive temperatures, the chromosomes condense but do not separate. Thus topo II is required for final chromosome condensation; moreover, pulse-shift experiments show that topo II is required for chromatid disjuction. Experiments with ts top2-cs nda3 cells show that topo II is also required for chromosome separation in anaphase: inactivation of topo II and activation of beta-tubulin allow normal spindle formation but result in "streaked" chromosomes.     

Structural homology between DNA binding sites of DNA polymerase beta and DNA topoisomerase II

Unsaturated long-chain fatty acids selectively bind to the DNA binding sites of DNA polymerase beta and DNA topoisomerase II, and inhibit their activities, although the amino acid sequences of these enzymes are markedly different from each other. Computer modeling analysis revealed that the fatty acid interaction interface in both enzymes has a group of four amino acid residues in common, forming a pocket which binds to the fatty acid molecule. The four amino acid residues were Thr596, His735, Leu741 and Lys983 for yeast DNA topoisomerase II, corresponding to Thr79, His51, Leu11 and Lys35 for rat DNA polymerase beta. Using three-dimensional structure model analysis, we determined the spatial positioning of specific amino acid residues binding to the fatty acids in DNA topoisomerase II, and subsequently obtained supplementary information to build the structural model.     

DNA cleavage and religation by human topoisomerase II alpha at high temperature.

A common DNA religation assay for topoisomerase II takes advantage of the fact that the enzyme can rejoin cleaved nucleic acids but cannot mediate DNA scission at suboptimal temperatures (either high or low). Although temperature-induced DNA religation assays have provided valuable mechanistic information for several type II enzymes, high-temperature shifts have not been examined for human topoisomerase IIalpha. Therefore, the effects of temperature on the DNA cleavage/religation activity of the enzyme were characterized. Human topoisomerase IIalpha undergoes two distinct transitions at high temperatures. The first transition occurs between 45 and 55 degrees C and is accompanied by a 6-fold increase in the level of DNA cleavage at 60 degrees C. It also leads to a loss of DNA strand passage activity, due primarily to an inability of ATP to convert the enzyme to a protein clamp. The enzyme alterations that accompany the first transition appear to be stable and do not revert at lower temperature. The second transition in human topoisomerase IIalpha occurs between 65 and 70 degrees C and correlates with a precipitous drop in the level of DNA scission. At 75 degrees C, cleavage falls well below amounts seen at 37 degrees C. This loss of DNA scission appears to result from a decrease in the forward rate of DNA cleavage rather than an increase in the religation rate. Finally, similar high-temperature alterations were observed for yeast topoisomerase II and human topoisomerase IIbeta, suggesting that parallel heat-induced transitions may be widespread among type II topoisomerases.     

Localization of topoisomerase II in mitotic chromosomes

In the preceding article we described a polyclonal antibody that recognizes cSc-1, a major polypeptide component of the chicken mitotic chromosome scaffold. This polypeptide was shown to be chicken topoisomerase II. In the experiments described in the present article we use indirect immunofluorescence and immunoelectron microscopy to examine the distribution of topoisomerase II within intact chromosomes. We also describe a simple experimental protocol that differentiates antigens that are interspersed along the chromatin fiber from those that occupy restricted domains within the chromosome. These experiments indicate that the distribution of the enzyme appears to be independent of the bulk chromatin. Our data suggest that topoisomerase II is bound to the bases of the radial loop domains of mitotic chromosomes.