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PubMed1986

The nucleotide sequence of the fission yeast DNA topoisomerase II gene: structural and functional relationships to other DNA topoisomerases.

We have determined the complete nucleotide sequence of a 5.3-kb long genomic DNA fragment of the fission yeast Schizosaccharomyces pombe that encodes DNA topoisomerase II. It contains a 4293 bp long single open reading frame. The predicted polypeptide has 1431 residues (mol. wt 162,000) and shows three characteristic domains; the large C-terminal region, which consists of alternating acidic-basic stretches and might be a chromatin-binding domain, the NH2 half domain homologous to the ATP-binding gyrB subunit of bacterial gyrase and the central-to-latter part which is homologous to the NH2 domain of the catalytic gyrA subunit, suggesting a possible evolutionary consequence of the gene fusion of the bacterial gyrase subunits into the eucaryotic DNA topoisomerase II gene. We have found that the cloned fission yeast TOP2 gene can complement the budding yeast top2 mutation, although the fission yeast TOP2 protein sequence is only 50% homologous to the recently determined sequence of budding yeast (J.C. Wang, personal communication). Conversely, the budding yeast TOP2 gene can complement the fission yeast top2 mutations, indicating that their DNA topoisomerase II genes are functionally exchangeable.

Amino Acid Sequence↗
PubMed2001

Developmental regulation of DNA-topoisomerases during Drosophila embryogenesis.

Type I and type II DNA-topoisomerases are essential enzymes that mediate replication, transcription, recombination, and mitosis in multicellular eukaryotes but the extent of their interchange for specific reactions in vivo is controversial. Expression patterns for topoisomerase I and topoisomerase II during the embryogenesis of Drosophila melanogaster were compared with patterns of DNA replication and expression of the histone genes. In late oogenesis the maternally supplied top2 mRNA was evenly distributed throughout the egg with elevated levels at the posterior tip, a pattern that is maintained in syncytial blastoderm embryos. During gastrulation, top2 mRNA became differentially localized only to regions of DNA replication, including new expression in the gonads preceding mitosis/meiosis. Significantly higher levels of top2 mRNA were found in mitotic compared to endoreplicating tissues. The total histone mRNA was exclusively associated with DNA replication but, in contrast to top2 mRNA, mitotic and endoreplicating cells contained similar expression levels with no expression in the gonads. Striking differences exist between the distribution of the top2 mRNA and topoisomerase II protein. The protein localizes to all evolving nuclei where it persists throughout embryogenesis. A high level of top1 mRNA transcript was present without differential tissue distribution throughout embryogenesis.

Animals↗
PubMed1992

Dissociation of cytotoxicity and DNA cleavage activity induced by topoisomerase II-reactive intercalating agents in hamster-human somatic cell hybrids.

Previous studies using the mutant Chinese hamster ovary cell line VpmR-5 indicate that its resistance to epipodophyllotoxins and intercalating agents is likely to be mediated through a qualitative change in type II topoisomerase that confers resistance to drug-stimulated DNA cleavage activity. In a further investigation of the genetic basis of drug resistance in VpmR-5 cells, we fused a hypoxanthine-guanine phosphoribosyl transferase-deficient subline of VpmR-5 (Vtgm-6) with normal human lymphocytes and analyzed the resultant hybrid lines (HL) for altered drug sensitivity. In all, 3 of 16 hybrid clones exhibited partial reconstitution of sensitivity to etoposide, mitoxantrone, doxorubicin, and 5-iminodaunorubicin while retaining complete resistance to m-AMSA. However, enhanced sensitivity to drug-induced DNA cleavage activity was observed only for etoposide. Biochemical and molecular-marker analysis of the hybrids failed to identify human chromosome 17 (the provisional location of TOP2) or any other human chromosome that is consistently and uniquely associated with drug sensitivity. We therefore sought to verify the chromosomal assignment of TOP2 by Southern blot hybridization of TOP2 cDNA on a human hybrid mapping panel and confirmed its location on chromosome 17. However, no hybridizing sequence to the TOP2 cDNA was found in any of the 16 Vtgm-6 hybrid lines. Efforts to select more directly for human chromosome 17 VpmR-5 hybrids using microcell fusion of mouse A9 cells carrying human 17 linked to pSV2neo were unsuccessful. None of the five hybrid clones thus obtained had 17q markers, including the gene for TOP2. Although the mechanism underlying partial reversion to a drug-sensitive phenotype in the original Vtgm-6 hybrid lines has yet to be defined, the data obtained in these lines indicate that anthracycline- and anthracenedione-induced cytotoxic effects can be dissociated from DNA cleavage activity. This suggests that pathways distal to cleavable-complex formation or, alternatively, independent of interactions with topoisomerase II that involve other intracellular targets are important in mediating the cytotoxicity produced by these drugs.

Animals↗
PubMed1989

Function of DNA topoisomerases as replication swivels in Saccharomyces cerevisiae.

We have examined the roles of eukaryotic DNA topoisomerases I and II in DNA replication by the use of a set of four isogenic strains of Saccharomyces cerevisiae that are TOP1+ TOP2+, TOP1+ top2 ts, delta top1 TOP2+, and delta top1 top2 ts. Cells synchronized by treatment with the alpha-mating factor, or by cycles of feeding and starvation, were released from cell-cycle arrest, and the size distribution of DNA chains that were synthesized after the cells reentered the S-phase was determined as a function of time. The results indicate that synthesis of short DNA chains several thousand nucleotides in length can initiate in the absence of both topoisomerases, but their further elongation requires at least one of the two topoisomerases. Inactivation of DNA topoisomerase II does not alter significantly the time dependence of the patterns of nascent DNA chain synthesis, which is consistent with the notion that the requirement of this enzyme for viability is due to its essential role during mitosis, when pairs of intertwined newly replicated chromosomes are being segregated. The absence of DNA topoisomerase I leads to a temporary delay in the extension of the short DNA chains; this delay in chain elongation is also reflected in the rate of total DNA synthesis in the delta top1 mutant during the early S-phase. Thus, in wild-type cells, DNA topoisomerase I is probably the major replication swivel. The patterns of DNA synthesis in asynchronously grown delta top1 top2 ts cells at permissive and non-permissive temperatures are also consistent with the above conclusions.

DNA Replication↗
PubMed2025

DNA topoisomerase II promotes N6-adenosine mRNA methylation.

DNA topoisomerase II (TOP2) is an enzyme that regulates DNA topology, primarily by removing DNA supercoiling. This function is crucial during transcription, as the movement of RNA polymerase II (RNAPII) generates torsional stress. However, the specific role of TOP2 in the regulation of gene expression remains to be fully elucidated, as both TOP2 inhibitors and poisons have been shown to upregulate specific genes. In this study, we show that TOP2 poisoning negatively affects transcription elongation of genes repressed at the level of promoter-proximal pausing. Importantly, this effect is counteracted by defective mRNA N6-adenosine methylation (m6A), which results in altered RNA turnover and pre-mRNA splicing. We propose that TOP2 serves a dual function, supporting the maintenance of basal transcription elongation while simultaneously promoting m6A modification in pre-mRNAs to reduce the overall gene expression output.

RNA Methylation↗
PubMed2003

Function of the loop residue Thr792 in human DNA topoisomerase II alpha.

We studied the mutation effect of one of the putative loop residues Thr792 in human DNA topoisomerase II alpha (TOP2 alpha). Thr792 mutants were expressed from high or low copy plasmids in a temperature sensitive yeast strain deficient in TOP2 (top2-1). When expressed from a high copy plasmid, mutants with small side chains complemented the yeast defect; however, from a low copy plasmid, only wild-type, Ser, and Cys substitution mutants complemented the yeast defect. Interestingly, at the permissive temperature other mutants (e.g., Val, Gly, and Glu substitutions) showed the dominant negative effect to the top2-1 allele, which was not observed by the control alpha 4-helix mutants. T792E mutant was 10-fold less active than wild-type and the T792P had no decatenation activity in vitro. These results suggest that Thr792 in human TOP2 alpha is involved in enzyme catalysis.

Antigens, Neoplasm↗
PubMed2002

The SUMO-1 isopeptidase Smt4 is linked to centromeric cohesion through SUMO-1 modification of DNA topoisomerase II.

In S. cerevisiae, posttranslational modification by the ubiquitin-like Smt3/SUMO-1 protein is essential for survival, but functions and cellular targets for this modification are largely unknown. We find that one function associated with the Smt3/SUMO-1 isopeptidase Smt4 is to control chromosome cohesion at centromeric regions and that a key Smt3/SUMO-1 substrate underlying this function is Top2, DNA Topoisomerase II. Top2 modification by Smt3/SUMO-1 is misregulated in smt4 strains, and top2 mutants resistant to Smt3/SUMO-1 modification suppress the smt4 cohesion defect. top2 mutants display aberrant chromatid stretching at the centromere in response to mitotic spindle tension and altered chromatid reassociation following microtubule depolymerization. These results suggest Top2 modification by Smt3/SUMO-1 regulates a component of chromatin structure or topology required for centromeric cohesion.

Cell Cycle Proteins↗
PubMed2004

Interaction model for anthracycline activity against DNA topoisomerase II.

DNA topoisomerase II (Top2) is an essential nuclear enzyme and a target of very effective anticancer drugs including anthracycline antibiotics. Even though several aspects of drug activity against Top2 are understood, the drug receptor site is not yet known. Several Top2 mutants have altered drug sensitivity and have provided information of structural features determining drug action. Here, we have investigated the sensitivity to three closely related anthracycline derivatives of yeast Top2 bearing mutations in the CAP-like domain and integrated the findings with computer models of ternary drug-enzyme-DNA complexes. The results suggest a model for the anthracycline receptor wherein a drug molecule has specific interactions with the cleaved DNA as well as amino acid residues of the CAP-like domain of an enzyme monomer. The drug molecule is intercalated into DNA at the site of cleavage, and interestingly, drug-enzyme contacts involve one side of the four-ring chromophore and the side chain of the anthracycline molecule. The findings may explain several established structure-activity relationships of antitumor anthracyclines and may thus provide a framework for further developments of effective Top2 poisons.

Anthracyclines↗
PubMed1991

Genetic analysis of the gyrase A-like domain of DNA topoisomerase II of Saccharomyces cerevisiae.

We have undertaken a genetic analysis of heat-sensitive and cold-sensitive mutations in TOP2, the gene encoding yeast DNA topoisomerase II. Deletion mapping was used to localize 14 heat-sensitive and four cold-sensitive top2 mutations created by a method biased toward mutations in the 3' two-thirds of the gene. The mutations all appear to be located in the region of DNA topoisomerase II that shows homology to the "A" subunit of bacterial DNA gyrase. The heat-sensitive mutations and one cold-sensitive mutation lie in the center of the gene near the sequence that encodes the active site tyrosine. The three other cold-sensitive mutations map farther toward the 3' end of the gene. The cold-sensitive mutations exhibit intragenic complementation, and the complementation groups correspond to the physical map. We sequenced nine top2 mutations and found that the mutations are usually single missense mutations, frequently involve proline, and affect conserved regions of the protein. Suppressor analysis yielded two intragenic suppressors and seven independent isolates of an allele-specific extragenic suppressor we named tos1; tos1 is not allelic to any genes predicted to encode type I topoisomerase-related proteins. The two intragenic suppressors were tested for allele-specificity; the results revealed a complex pattern of suppression between heat-sensitive and cold-sensitive top2 alleles. These top2 mutations may have compensatory effects on the general stability of the protein.

Base Sequence↗
PubMed1994

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.

Amino Acid Sequence↗
PubMed1991

A functional 125-kDa core polypeptide of fission yeast DNA topoisomerase II.

We purified fission yeast DNA topoisomerase II (topo II) to apparent homogeneity. It consists of a single 165-kDa polypeptide in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and, upon treatment with a bifunctional reagent, doubles its molecular weight. Limited proteolysis of intact topo II by papain produces a 125-kDa core, which lacks the N-terminal 75 and the C-terminal approximately 260 amino acids but still contains regions similar to those of bacterial or phage T4 topo II subunits. The core retains relaxing and unknotting activities. Further digestion inactivates the core, cleaving it at the middle of the GyrB-like domain and at the beginning of the GyrA-like domain. Therefore, papain appears to cleave spatially distinct subdomains of topo II. We made top2 mutant genes deleted of the C-terminal 286 or N-terminal 74 amino acids, which can substitute for the wild-type top2+ gene in mitosis and meiosis. However, a mutant containing deletions of both termini cannot rescue the top2 null mutant, despite the fact that the product is enzymatically active. Therefore, the top2 product of the doubly truncated gene may not fulfill all of the in vivo requirements for top2+ function.

Amino Acid Sequence↗
PubMed1994

Analysis of yeast DNA topoisomerase II mutants resistant to the antitumor drug amsacrine.

Site-directed mutagenesis of regions within a plasmid-borne yeast TOP2 gene encoding DNA topoisomerase II and hydroxylamine mutagenesis of the entire plasmid were carried out, and the mutagenized plasmid DNA pools were used separately to transform yeast with a temperature-sensitive top2-4 mutation in the chromosomal TOP2 locus. By selecting transformants that grow in the presence of the antitumor drug amsacrine at 35 degrees C, a nonpermissive temperature for the top2-4 allele, plasmid-borne top2 mutants expressing amsacrine-resistant and physiologically functional DNA topoisomerase II were readily obtained. The causality between amsacrine resistance and the presence of these mutations in yeast DNA topoisomerase II has been firmly established, and this causality in turn shows that, in yeast at least, DNA topoisomerase II is the only significant cellular target of amsacrine. Three classes of such mutants have been identified: one involves single or multiple changes in a sequence PLRGK-MLN located at positions 474-481 of yeast DNA topoisomerase II, a highly conserved motif in all type II DNA topoisomerases; a second involving a single mutation changing Ala642 to threonine or glycine; and a third involving deletions of portions of the carboxy-terminal domain of the yeast enzyme. The nature of drug resistance of these different classes of mutants is discussed. The approaches used in this work should be readily applicable to yeast cells expressing heterologous DNA topoisomerases such as human DNA topoisomerase II alpha. Other DNA topoisomerase II-targeting drugs can also be studied in such a system.

Alleles↗
PubMed1993

Novel selection and genetic characterisation of an etoposide-resistant human leukaemic CCRF-CEM cell line.

We have studied the genetic alterations acquired during selection of a cloned human leukaemic cell line (CEM/VP-1) that is 15-fold more resistant to the anticancer topoisomerase II-inhibitor etoposide than parental CCRF-CEM cells. CEM/VP-1 cells exhibit an 'atypical MDR' phenotype: cross resistance to other topo II inhibitors (but not Vinca alkaloids) and expression of a drug-resistant topo II activity. Cytogenetic and molecular studies revealed that the cell line carried multiple genetic changes affecting TOP2 genes encoding both topo II alpha and beta isoforms. CEM/VP-1 was diploid, 47,XX,+20, and appears to have been preferentially selected from a 1% diploid subpopulation present in the tetraploid parental cells. The same chromosomal abnormalities were present in resistant and sensitive cells except for an acquired 3p- change most likely deleting one TOP2 beta allele. PCR/DNA sequence analysis and allele-specific hybridisation showed that one of two TOP2 alpha alleles expressed in CEM/VP-1 cells had acquired a Lys-797-->Asn codon change. This mutation lies close to the catalytic Tyr-804 residue of the protein and may interfere with drug-induced trapping of the cleavable complex. Alternatively, it could exert a loss of function phenotype. CEM/VP-1 cells did not exhibit codon 449 or 486 TOP2 alpha mutations in the ATP binding domain reported in two other resistant cell lines. Diploid selection and multiple changes observed in CEM/VP-1 cells appear to be consequences of the recessive phenotype of at-MDR. These results may be useful in approaching the mechanisms of clinical resistance.

Base Sequence↗
PubMed1985

Cloning of yeast TOP1, the gene encoding DNA topoisomerase I, and construction of mutants defective in both DNA topoisomerase I and DNA topoisomerase II.

Rabbit antibodies specific to yeast DNA topoisomerase I were used in immunological screening of a Saccharomyces cerevisiae genomic DNA library in Escherichia coli. One of the clones identified by its expression of antigenic determinants of the yeast enzyme is shown to contain the coding sequence of the enzyme: no active DNA topoisomerase I is detectable in cell extracts when insertion or deletion mutations are introduced into a 2-kilobase-pair (kb) region of the sequence in a haploid yeast genome. Blot hybridizations show that there is a single copy of the cloned sequence per haploid and that the sequence is transcribed to give a 2.7-kb poly(A)+ message. Mutants in which 1.7 kb of the sequence is deleted are viable. Temperature-shift experiments using synchronously grown cells of a delta top1 top2 temperature-sensitive (ts) double mutant and its isogenic top2 ts strain show that, whereas mitotic blocks can prevent killing of the top2 ts mutant at a nonpermissive temperature, the same treatments are ineffective in preventing cell death of the delta top1 top2 ts double mutant. These experiments suggest that in yeast DNA topoisomerase I serves a role auxiliary to DNA topoisomerase II.

Base Sequence↗
PubMed1999

Catalysis of ATP hydrolysis by two NH(2)-terminal fragments of yeast DNA topoisomerase II.

Catalysis of ATP hydrolysis by two NH(2)-terminal fragments of yeast DNA topoisomerase II was studied in the absence and presence of DNA, and in the absence and presence of inhibitor ICRF-193. The results indicate that purified Top2-(1-409), a fragment containing the NH(2)-terminal 409 amino acids of the yeast enzyme, is predominantly monomeric, with a low level of ATPase owing to weak association of two monomers to form a catalytically active dimer. The ATPase activity of Top2-(1-409) is independent of DNA in a buffer containing 100 mM NaCl, in which intact yeast DNA topoisomerase II exhibits robust DNA-dependent ATPase and DNA transport activities. Purified Top2-(1-660), a fragment containing the NH(2)-terminal 660 amino acid of the yeast enzyme, appears to be dimeric in the absence or presence of DNA, and the ATPase activity of the protein is significantly stimulated by DNA. These results are consistent with a model in which binding of an intact DNA topoisomerase II to DNA places the various subfragments of the enzyme in a way that makes the intramolecular dimerization of the ATPase domains more favorable. We believe that this alignment of subfragments is mainly achieved through the binding of the enzyme to the DNA segment within which the enzyme makes transient breaks. The ATPase activity of Top2-(1-409) is inhibited by ICRF-193, suggesting that the bisdioxopiperazine class of DNA topoisomerase II inhibitors directly interacts with the paired ATPase domains of the enzyme.

Adenosine Triphosphatases↗
PubMed2002

Failure to relax negative supercoiling of DNA is a primary cause of mitotic hyper-recombination in topoisomerase-deficient yeast cells.

In the yeast Saccharomyces cerevisiae, DNA topoisomerases I and II can functionally substitute for each other in removing positive and negative DNA supercoils. Yeast Delta top1 top2(ts) mutants grow slowly and present structural instability in the genome; over half of the rDNA repeats are excised in the form of extrachromosomal rings, and small circular minichromosomes strongly multimerize. Because these traits can be reverted by the extrachromosomal expression of either eukaryotic topoisomerase I or II, their origin is attributed to the persistence of unconstrained DNA supercoiling. Here, we examine whether the expression of the Escherichia coli topA gene, which encodes the bacterial topoisomerase I that removes only negative supercoils, compensates the phenotype of Delta top1 top2(ts) yeast cells. We found that Delta top1 top2(ts) mutants expressing E. coli topoisomerase I grow faster and do not manifest rDNA excision and minichromosome multimerization. Furthermore, the recombination frequency in repeated DNA sequences, which is increased by nearly two orders of magnitude in Delta top1 top2(ts) mutants relative to the parental TOP+ cells, is restored to normal levels when the bacterial topoisomerase is expressed. These results indicate that the suppression of mitotic hyper-recombination caused by eukaryotic topoisomerases I and II is effected mainly by the relaxation of negative rather than positive supercoils; they also highlight the potential of unconstrained negative supercoiling to promote homologous recombination.

DNA Topoisomerases, Type I↗
PubMed1999

Differential in vitro interactions of a series of clinically useful topoisomerase-interacting compounds with the cleavage/religation activity of the human topoisomerase IIalpha and IIbeta isoforms.

The topoisomerase II (TOP2)-associated DNA cleavage activity and the DNA sequence preference of 20 antitumor drugs, including 15 TOP2-interacting compounds, have been defined. Four major classes of drugs have been identified: (i) those which enhanced the stabilization of cleavable complexes at a single major site (e.g. amsacrine, doxorubicin), or (ii) at many sites (e.g. etoposide, azatoxin), with chemically related compounds having very similar, although not identical, cleavage patterns (e.g. etoposide, GL331 and Top-53); (iii) those which inhibited DNA breakage (e.g. aclarubicin, actinomycin D); and (iv) those which did not visibly interfere with TOP2-mediated cleavable complexes (e.g. ICRF-187, camptothecin). All drugs tested induced similar overall patterns of sites of preferred DNA cleavage, in the presence either of the two known isoforms, TOP2alpha or TOP2beta, although relative intensities of signals at each position varied. It has been further shown that etoposide and its derivatives blocked the religation step downstream of the DNA cleavage step, whereas amsacrine, ellipticine, azatoxin and genistein acted upstream through enhancement of DNA cleavage. The information provided by this mechanistically based comparison can now be exploited in designing or synthesizing novel TOP2-interacting agents.

Antineoplastic Agents↗
PubMed1997

Identification of yeast DNA topoisomerase II mutants resistant to the antitumor drug doxorubicin: implications for the mechanisms of doxorubicin action and cytotoxicity.

Doxorubicin is a therapeutically useful anticancer drug that exerts multiple biological effects. Its antitumor and cardiotoxic properties have been ascribed to anthracycline-mediated free radical damage to DNA and membranes. Evidence for this idea comes in part from the selection by doxorubicin from stationary phase yeast cells of mutants (petites) deficient in mitochondrial respiration and therefore defective in free radical generation. However, doxorubicin also binds to DNA topoisomerase II, converting the enzyme into a DNA damaging agent through the trapping of a covalent enzyme-DNA complex termed the 'cleavable complex.' We have used yeast to determine whether stabilization of cleavable complexes plays a role in doxorubicin action and cytotoxicity. A plasmid-borne yeast TOP2 gene was mutagenized with hydroxylamine and used to transform drug-permeable yeast strain JN394t2-4, which carries a temperature-sensitive top2-4 mutation in its chromosomal TOP2 gene. Selection in growth medium at the nonpermissive temperature of 35 degrees in the presence of doxorubicin resulted in the isolation of plasmid-borne top2 mutants specifying functional doxorubicin-resistant DNA topoisomerase II. Single-point changes of Gly748 to Glu or Ala642 to Ser in yeast topoisomerase II, which lie in and adjacent to the CAP-like DNA binding domain, respectively, were identified as responsible for resistance to doxorubicin, implicating these regions in drug action. None of the mutants selected in JN394t2-4, which has a rad52 defect in double-strand DNA break repair, was respiration-deficient. We conclude that topoisomerase II is an intracellular target for doxorubicin and that the genetic background and/or cell proliferation status can determine the relative importance of topoisomerase II- versus free radical-killing.

Alleles↗