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PubMed1996

Pat1: a topoisomerase II-associated protein required for faithful chromosome transmission in Saccharomyces cerevisiae.

Saccharomyces cerevisiae top2 mutants deficient in topoisomerase II activity are defective in chromosome segregation during both mitotic and meiotic cell divisions. To identify proteins that act in concert with topoisomerase II during chromosome segregation in S.cerevisiae, we have used a two-hybrid cloning approach. We report the isolation of the PAT1 gene (for protein associated with topoisomerase II), which encodes a novel 90 kDa proline- and glutamine-rich protein that interacts with a highly conserved, leucine-rich region of topoisomerase II in vivo. Strains lacking Pat1p exhibit a slow growth rate and a phenotype reminiscent of conditional top2 mutants grown at the semi-permissive temperature; most notably, a reduced fidelity of chromosome segregation during both mitosis and meiosis. These findings indicate that the PAT1 gene is necessary for accurate chromosome transmission during cell division in eukaryotic cells and suggest that the interaction of Pat1p and topoisomerase II is an important component of this function.

Binding Sites↗
PubMed1997

Cellular distribution of mammalian DNA topoisomerase II is determined by its catalytically dispensable C-terminal domain.

Mammalian cells express two genetically distinct isoforms of DNA topoisomerase II, designated topoisomerase IIalphaand topoisomerase IIbeta. We have recently shown that mouse topoisomerase IIalpha can substitute for the yeast topoisomerase II enzyme and complement yeast top2 mutations. This functional complementation allowed functional analysis of the C-terminal domain (CTD) of mammalian topoisomerase II, where the amino acid sequences are divergent and species-specific, in contrast to the highly conserved N-terminal and central domains. Several C-terminal deletion mutants of mouse topoisomerase IIalpha were constructed and expressed in yeast top2 cells. We found that the CTD of topoisomerase IIalphais dispensable for enzymatic activity in vitro but is required for nuclear localization in vivo. Interestingly, the CTD of topoisomerase IIbetawas also able to function as a signal for nuclear targeting. We therefore examined whether the CTD alone is sufficient for nuclear localization in vivo . The C-terminal region was fused to GFP (green fluorescent protein) and expressed under the GAL1 promoter in yeast cells. As expected, GFP signal was exclusively detected in the nucleus, irrespective of the CTD derived from either topoisomerase IIalphaor IIbeta. Surprisingly, when the upstream sequence of each CTD was added nuclear localization of the GFP signal was found to be cell cycle dependent: topoisomerase IIalpha-GFP was seen in the mitotic nucleus but was absent from the interphase nucleus, while topoisomerase IIbeta-GFP was detected predominantly in the interphase nucleus and less in the mitotic nucleus. Our results suggest that the catalytically dispensable CTD of topoisomerase II is sufficient as a signal for nuclear localization and that yeast cells can distinguish between the two isoforms of mammalian topoisomerase II, localizing each protein properly.

Amino Acid Sequence↗
PubMed1997

Cell cycle regulation of RPA1 transcript levels in the trypanosomatid Crithidia fasciculata.

Transcripts of both mitochondrial and nuclear DNA replication genes accumulate periodically during the cell cycle in Crithidia fasciculata. An octameric consensus sequence with a conserved hexameric core was found previously to be required for cycling of the TOP2 transcript, encoding the mitochondrial DNA topoisomerase. We show here that the rate of synthesis of the p51 protein, the large subunit of nuclear replication protein-A encoded by the RPA1 gene, varies during the cell cycle in parallel with RPA1 mRNA level. Plasmids expressing a truncated form of RPA1 (Delta RPA1 ) were used to identify cis elements required for cycling of the Delta RPA1 transcript. Sequences within the RPA1 5'-untranslated region (UTR) were found to be necessary for cycling of the Delta RPA1 transcript. These sequences also function when transposed 3'of the Delta RPA1 coding sequence. A 121 bp fragment of this sequence can confer cycling on a heterologous transcript, but is inactivated when two consensus octamers within the sequence are mutated. Mutation of these two octamers in the full-length 5'-UTR ofDelta RPA1 is insufficient to abolish cycling of the mRNA unless three additional octamers having single base changes within the hexameric core are also mutated. Thus, common octameric sequence elements are involved in periodic accumulation of both the TOP2 and RPA1 transcripts.

Animals↗
PubMed2003

Cloning, functional analysis and post-transcriptional regulation of a type II DNA topoisomerase from Leishmania infantum. A new potential target for anti-parasite drugs.

We identified a type II topoisomerase enzyme from Leishmania infantum, a parasite protozoon causing disease in humans. This protein, named Li topo II, which displays a variable C-terminal end, is located in the kinetoplast. The cloned gene encoding Li-TOP2 compensates for the slow growth of topo II-deficient mutants of Saccharomyces cerevisiae, resulting in a catalytically active DNA topoisomerase in yeast. Analysis of the specific mRNA levels of the Li-TOP2 gene showed variations throughout the parasite cell cycle in synchronized cells as well as between the distinct forms of the parasite. Thus, the enzyme had higher levels of mRNA expression in the highly infective intracellular form of the parasite, the amastigote, than in the extracellular promastigote form, suggesting a relation with the distinct developmental and infectious phases of the protozoon. In addition, western blot analysis showed differences in protein expression between the proliferative and non-proliferative forms of L.infantum promastigotes, which displayed similar levels of mRNA. This indicated possible post-transcriptional regulation mechanisms. The data suggest that Li topo II has a part in DNA decatenation and probably at the initial stages of proliferation in the intracellular form of L.infantum, a parasite that has to proliferate into the host macrophage to survive its hostile environment in its first moments of intracellular infection.

Amino Acid Sequence↗
PubMed1998

A controllable gene-expression system for the pathogenic fungus Candida glabrata.

A system for controlling gene expression was established in the pathogenic fungus Candida glabrata to elucidate the physiological functions of genes. To control the expression of the gene of interest, the C. glabrata cells were first transformed with the plasmid carrying the tetracycline repressor-transactivator fusion tetR::GAL4, then with the DNA fragment containing the controllable cassette, the tetracycline operator chimeric promoter (tetO::ScHOP1). The peptide elongation factor 3 (CgTEF3) and DNA topoisomerase II (CgTOP2) genes from C. glabrata were cloned and their expression assessed using this system. When the promoter of CgTEF3 or CgTOP2 was replaced with tetO::ScHOP1, doxycycline almost completely repressed the expression of both mRNAs, and impaired growth. Repression of the TOP2 or TEF3 gene by doxycycline also hampered the survival of C. glabrata cells in mice; in mouse kidneys the number of C. glabrata cells, in which the TOP2 or TEF3 promoter was replaced with the tetO::ScHOP1 controllable cassette, did not increase when the mice were given doxycycline. Thus, it appears that the gene repression mediated by doxycycline occurred not only in culture media but also in animals; therefore, this system can be used to elucidate the function of the gene in fungal infections and pathogenesis.

Animals↗
PubMed1999

Activation of topoisomerase II-mediated excision of chromosomal DNA loops during oxidative stress.

Hydrogen peroxide (H2O2), a reactive oxygen species (ROS), is known to induce oxidative stress and apoptosis. U937 cells treated with H2O2 were shown to produce high molecular weight (HMW) DNA fragments approximately 50 to 100 kb in size in <1 min. The formation of these HMW DNA fragments is reversible and shown to be mediated by DNA topoisomerase II (TOP2). Following this initial event, formation of irreversible HMW DNA fragments and nucleosomal ladders occurs. Our results thus demonstrate a potential role of TOP2 in oxidative damage of DNA and apoptotic cell death.

DNA Damage↗
PubMed1992

Topoisomerases and yeast rRNA transcription: negative supercoiling stimulates initiation and topoisomerase activity is required for elongation.

Previous work has shown that rRNA synthesis is strongly inhibited in yeast top1-top2 double mutants. Here, we show that inactivation of yeast topoisomerases can have paradoxical effects on transcription by RNA polymerase I. For example, transcription of ribosomal minigenes on extrachromosomal plasmids is greatly stimulated in top1-top2 cells while accumulation of full-length endogenous rRNA is strongly inhibited. We present evidence for a mechanism that can partly account for these opposing effects on transcription. On the one hand, transcription initiation can be stimulated owing to an accumulation of negative superhelicity because polymerase I prefers to initiate on negatively supercoiled templates. Conversely, synthesis of full-length rRNA is inhibited owing to the fact that chain elongation requires a DNA relaxing activity.

Cloning, Molecular↗
PubMed2006

Bisindenoisoquinoline bis-1,3-{(5,6-dihydro-5,11-diketo-11H-indeno[1,2-c]isoquinoline)-6-propylamino}propane bis(trifluoroacetate) (NSC 727357), a DNA intercalator and topoisomerase inhibitor with antitumor activity.

Indenoisoquinolines are topoisomerase (Top) I inhibitors developed to overcome some of the limitations of camptothecins and expand their anticancer spectrum. Bis-1,3-{(5,6-dihydro-5,11-diketo-11H-indeno[1,2-c]isoquinoline)-6-propylamino}-propane bis(trifluoroacetate) (NSC 727357) is a novel dimeric indenoisoquinoline derivative with potent antiproliferative activity in the NCI-60 cell line panel, promising hollow fiber activity (score of 32) and activity against xenografts. Submicromolar concentrations of the bisindenoisoquinoline NSC 727357 induce Top1 cleavage complexes at specific sites in biochemical assays. At higher concentrations, inhibition of Top1 catalytic activity and DNA intercalation is observed. NSC 727357 also induces a limited number of Top2-DNA cleavage complexes. In contrast to the effect of other Top1 inhibitors, cells treated with the bisindenoisoquinoline NSC 727357 show an arrest of cell cycle progression in G(1) with no significant inhibition of DNA synthesis after a short exposure to the drug. Moreover, unlike camptothecin and the indenoisoquinoline MJ-III-65 (NSC 706744, 6-[3-(2-hydroxyethyl)aminopropyl]-5,6-dihydro-5,11-diketo-2,3-dimethoxy-(methylenedioxy)-11H-indeno[1,2-c]isoquinoline hydrochloride), the cytotoxicity of bisindenoisoquinoline NSC 727357 is only partially dependent on Top1 and p53, indicating that this drug has additional targets besides Top1 and Top2.

Animals↗
PubMed1987

MKT1, a nonessential Saccharomyces cerevisiae gene with a temperature-dependent effect on replication of M2 double-stranded RNA.

The MKT1 gene was defined by recessive alleles present in many laboratory strains of Saccharomyces cerevisiae that result in loss of M2 double-stranded RNA at temperatures above 30 degrees C if L-A-HN double-stranded RNA is present but not if L-A-H is present. I mapped MKT1 near TOP2 and isolated the gene by chromosome walking from TOP2. The gene location was defined by deletions, and a 2.8-kilobase transcript corresponding to the gene was detected. The recessive natural-variant mutations are not deletions as judged by Southern blots, but deletions of the MKT1 gene constructed in vitro and used to replace the normal gene surprisingly resulted in the same phenotype as that of the mkt1 natural variants, namely, a temperature-dependent maintenance of M2 double-stranded RNA. Thus the MKT1 gene product is only needed for M2 replication or maintenance at temperatures above 30 degrees C and if L-A-HN is present. The temperature dependence does not reflect the thermolability of a mutant gene product, as had previously been thought, nor does L-A double-stranded RNA need MKT1, as previously hypothesized. MKT1 may be involved in the process of packaging M2 double-stranded RNA. MKT1 is dispensable for host cell growth, mating, meiosis, and spore germination.

DNA Restriction Enzymes↗
PubMed1994

The C-terminal domain of Saccharomyces cerevisiae DNA topoisomerase II.

A set of carboxy-terminal deletion mutants of Saccharomyces cerevisiae DNA topoisomerase II were constructed for studying the functions of the carboxyl domain in vitro and in vivo. The wild-type yeast enzyme is a homodimer with 1,429 amino acid residues in each of the two polypeptides; truncation of the C terminus to Ile-1220 has little effect on the function of the enzyme in vitro or in vivo, whereas truncations extending beyond Gln-1138 yield completely inactive proteins. Several mutant enzymes with C termini in between these two residues were found to be catalytically active but unable to complement a top2-4 temperature-sensitive mutation. Immunomicroscopy results suggest that the removal of a nuclear localization signal in the C-terminal domain is likely to contribute to the physiological dysfunction of these proteins; the ability of these mutant proteins to relax supercoiled DNA in vivo shows, however, that at least some of the mutant proteins are present in the nuclei in a catalytically active form. In contrast to the ability of the catalytically active mutant proteins to relax supercoiled intracellular DNA, all mutants that do not complement the temperature-dependent lethality and high frequency of chromosomal nondisjunction of top2-4 were found to lack decatenation activity in vivo. The plausible roles of the DNA topoisomerase II C-terminal domain, in addition to providing a signal for nuclear localization, are discussed in the light of these results.

Adenosine Triphosphatases↗
PubMed1993

Identification of seven new cut genes involved in Schizosaccharomyces pombe mitosis.

Fission yeast cut mutants cause cytokinesis in the absence of normal nuclear division. These mutants show abnormal uncoupled mitosis and are known to be the result of mutations in the genes encoding DNA topoisomerase II, proteins related to spindle pole duplication, and a kinesin-related mitotic motor. We have screened 717 temperature-sensitive (ts) mutants by individually observing their cytological phenotypes at the restrictive temperature, and have newly isolated 25 cut mutants. Genetic analyses indicate that 14 of them fall into five previously identified loci, namely, top2, cut1, cut5, cut7 and cut9, whereas nine have been mapped onto seven new loci, designated cut13 to cut19. The cytological phenotypes of the newly identified cut mutants can be classified into three groups. One group consists of mutants in which a portion of the nuclear chromatin is stretched by the elongated spindle but the entire nucleus is not separated, reminiscent of, but not identical to, the phenotypes of top2 and cut1; mutants cut14-208, cut15-85, cut16-267 and cut17-275 display such a phenotype. Another group exhibits non-disjunctioned and condensed chromosomes in the presence of the spindle; cut13-131 belongs to this group. The cut19-708 mutant has also been found to have condensed chromosomes. The remaining group has a mixed phenotype of the above two groups; namely, stretched chromatin and condensed chromosomes; cut18-447 exhibits such a phenotype. The isolation and characterization of the mutated genes will be the subjects of future investigations.

Cell Division↗
PubMed1992

Cytotoxicity of quinolones toward eukaryotic cells. Identification of topoisomerase II as the primary cellular target for the quinolone CP-115,953 in yeast.

The quinolone CP-115,953 (6,8-difluoro-7-(4-hydroxyphenyl)-1-cyclopropyl-4- quinolone-3-carboxylic acid) represents a novel mechanistic class of drugs with potent activity against eukaryotic topoisomerase II in vitro (Robinson, M. J., Martin, B. A., Gootz, T. D., McGuirk, P. R., Moynihan, M., Sutcliffe, J. A., and Osheroff, N. (1991) J. Biol. Chem. 266, 14585-14592). Although the quinolone is highly toxic to mammalian cells in culture, its mechanism of cytotoxic action is not known. Therefore, yeast was used as a model system to determine whether topoisomerase II is the primary target responsible for the in vivo effects of CP-115,953. The quinolone was equipotent to etoposide at enhancing DNA breakage mediated by the Saccharomyces cerevisiae type II enzyme. Moreover, at concentrations as low as 5 microM, CP-115,953 was cytotoxic to yeast cells that carried wild type topoisomerase II (TOP2+). By utilizing a yeast strain that expressed the top2-1 temperature-sensitive mutant, the effect of topoisomerase II activity on quinolone cytotoxicity was determined. At the permissive temperature of 25 degrees C, cells were highly sensitive to CP-115,953. However, at the semipermissive temperature of 30 degrees C (where in vivo enzyme activity is present but is greatly diminished), cells displayed only marginal sensitivity to the quinolone at concentrations as high as 50 microM. These results strongly suggest that topoisomerase II is the primary physiological target responsible for quinolone cytotoxicity and that CP-115,953 kills cells by converting the type II enzyme into a cellular poison.

Anti-Infective Agents↗
PubMed1987

DNA topoisomerase activity is required as a swivel for DNA replication and for ribosomal RNA transcription.

Yeast strains with mutations in the genes for DNA topoisomerases I and II have been identified previously. The topoisomerase II mutants (top2) are conditional-lethal, temperature-sensitive mutants defective in the termination of DNA replication and the segregation of daughter chromosomes. The topoisomerase I mutants (top1), including strains with null mutations, are viable and exhibit no obvious growth defects, demonstrating that DNA topoisomerase I is not essential for viability in yeast. In contrast to the single mutants, top1 top2 double mutants grow poorly at the permissive temperature and stop DNA and ribosomal RNA synthesis at the restrictive temperature. Transfer RNA synthesis remains relatively normal. The rate of polyA+ RNA synthesis is down about 3-fold in the double mutant at the non-permissive temperature but the synthesis of three specific RNA polymerase II transcripts is unaffected. The results suggest that DNA replication and at least ribosomal RNA synthesis require an active topoisomerase, presumably to act as a swivel to relieve torsional stress, and that either topoisomerase can perform the required function (except for termination of DNA replication where topoisomerase II is required).

DNA Replication↗
PubMed1993

A temperature sensitive topoisomerase II allele confers temperature dependent drug resistance on amsacrine and etoposide: a genetic system for determining the targets of topoisomerase II inhibitors.

We have developed a simple system for determining the specific contribution of topoisomerase II targeting to the cytotoxic activity of a drug. We have constructed yeast strains that are permeable to anti-topoisomerase II drugs, carry a DNA repair mutation, rad52, and also have a temperature sensitive topoisomerase II mutation, top2-1. Strains carrying the top2-1 mutation have nearly normal topoisomerase II activity at 25 degrees C but less than 10% of wild type activity at 36 degrees C. We find that at a semi-permissive temperature (30 degrees C), there is sufficient topoisomerase II activity for viability; but since the topoisomerase II activity is greatly reduced, the cells are very resistant to anti-topoisomerase II drugs. Conversely, such cells are hypersensitive to the topoisomerase I inhibitor camptothecin. These results provide strong support for the model that drug stabilized DNA cleavage, rather than a lack of enzyme activity, is responsible for cell killing by eukaryotic anti-topoisomerase II agents. They also show that there is a minimum level of topoisomerase II activity in yeast that is consistent with viability but also allows a high degree of resistance to anti-topoisomerase II agents.

Alleles↗
PubMed1996

Functional expression of human topoisomerase II alpha in yeast: mutations at amino acids 450 or 803 of topoisomerase II alpha result in enzymes that can confer resistance to anti-topoisomerase II agents.

DNA topoisomerase II is the target of a variety of important antitumor agents, including etoposide, adriamycin, and amsacrine. We have constructed a system for analyzing the action of anti-topoisomerase II agents using the yeast Saccharomyces cerevisiae and have constructed vectors for expressing human topoisomerase II functionally in yeast. We have demonstrated that temperature-conditional yeast TOP2 mutants can be complemented by expression of wild-type human topoisomerase II alpha. Furthermore, expression of human topoisomerase II in yeast results in a quantitatively unique pattern of sensitivity to amsacrine. We also have constructed mutations in human TOP2 based on previously identified mutations from a human cell line selected for resistance to teniposide. Our experiments demonstrate that mutation of either arginine 450 or proline 803 of human topoisomerase II can result in an enzyme that has altered sensitivity to anti-topoisomerase II agents, and that a human enzyme carrying both mutations confers a higher level of drug resistance than enzymes carrying either single mutation.

Alleles↗
PubMed2026

Analysis of gene expression changes upon topobexin treatment and TOP2B-knockout in hiPSC-derived cardiomyocytes.

The role of DNA topoisomerase II beta (TOP2B) in cardiomyocyte differentiation is poorly understood. To address this, human induced pluripotent stem cells (hiPSC) were differentiated into cardiomyocytes (CM) that were wild type (WT) or contained a genomic deletion of Topoisomerase 2B (BKO). Both WT and BKO hiPSC could be induced to differentiate into sheets of beating cardiomyocytes. BKO hiPSC take slightly longer to differentiate into sheets of beating CM than WT iPSC. RNA was prepared from both undifferentiated and differentiated WT and BKO hiPSC. RNA-seq was used to examine gene expression changes when the WT and BKO hiPSC were differentiated into CM. Gene expression changes following differentiation of BKO cells were largely similar to those in WT cells. In addition, the differentiated WT CM were treated with dexrazoxane (ICRF-187), a TOP2 catalytic inhibitor that targets both TOP2A and TOP2B, or topobexin, a new TOP2B selective catalytic inhibitor. Topobexin inhibition partially phenocopied a TOP2B deletion and thereby providing an alternative to TOP2B gene knockout in many cell lines. In future, hiPSC derived CM with and without TOP2B and inhibition by topobexin ex vivo CM could be used to study anthracycline-induced cardiotoxicity and to screen for cardioprotectants.

Myocytes, Cardiac↗
PubMed1988

Depletion of histone H4 and nucleosomes activates the PHO5 gene in Saccharomyces cerevisiae.

We have previously constructed a yeast strain (UKY403) whose sole histone H4 gene is under control of the GAL1 promoter. This yeast arrests in G2 upon glucose treatment as a result of histone H4 depletion. The yeast PHO5 gene contains phase nucleosomes covering promoter (UAS) sequences in the PHO5 repressed state and it has been suggested that nucleosomes prevent the binding of positively acting factors to these UAS sequences. Using UKY403 we examined the length of polynucleosomes and nucleosome phasing in the PHO5 upstream region by the use of micrococcal nuclease and indirect end-labeling. It was found that glucose arrest led to a severe disruption in PHO5 chromatin structure and that most nucleosomes had their position altered or were lost from the PHO5 promoter region. Cell undergoing nucleosome depletion synthesized large quantities of accurate PHO5 transcripts even under repressive, high inorganic phosphate conditions. Histone H4 depletion did not appear to affect the repression or activation of another inducible yeast gene, CUP1. Arrest with landmarks in early G1 (in the cell division cycle mutant cdc28) or in various stages of G2 (in cdc15, cdc17 and cdc20) does not activate PHO5; nor does arrest due to chromosome topology changes (in top2 or the top1top2 topoisomerase mutants). cdc14, which has its arrest landmark at a similar point in the cell cycle as cdc15, does derepress PHO5. However, since it also leads to derepression of CUP1 it is probably functioning through an independent mechanism. Therefore, our data suggest that nucleosomes regulate PHO5 transcription.

Gene Expression Regulation↗
PubMed2006

Spermatocyte responses in vitro to induced DNA damage.

Spermatocytes normally sustain many meiotically induced double-strand DNA breaks (DSBs) early in meiotic prophase; in autosomal chromatin, these are repaired by initiation of meiotic homologous-recombination processes. Little is known about how spermatocytes respond to environmentally induced DNA damage after recombination-related DSBs have been repaired. The experiments described here tested the hypothesis that, even though actively completing meiotic recombination, pachytene spermatocytes cultured in the absence of testicular somatic cells initiate appropriate chromatin remodeling and cell-cycle responses to environmentally induced DNA damage. Two DNA-damaging agents were employed for in vitro treatment of pachytene spermatocytes: gamma-irradiation and etoposide, a topoisomerase II (TOP2) inhibitor that results in persistent unligated DSBs. Chromatin modifications associated with DSBs were monitored after exposure by labeling surface-spread chromatin with antibodies against RAD51 (which recognizes DSBs) and the phosphorylated variant of histone H2AFX (herein designated by its commonly used symbol, H2AX), gammaH2AX (which modifies chromatin associated with DSBs). Both gammaH2AX and RAD51 were rapidly recruited to irradiation- or etoposide-damaged chromatin. These chromatin modifications imply that spermatocytes recruit active DNA damage responses, even after recombination is substantially completed. Furthermore, irradiation-induced DNA damage inhibited okadaic acid-induced progression of spermatocytes from meiotic prophase to metaphase I (MI), implying efficacy of DNA damage checkpoint mechanisms. Apoptotic responses of spermatocytes with DNA damage differed, with an increase in frequency of early apoptotic spermatocytes after etoposide treatment, but not following irradiation. Taken together, these results demonstrate modification of pachytene spermatocyte chromatin and inhibition of meiotic progress after DNA damage by mechanisms that may ensure gametic genetic integrity.

Animals↗