Functional dissection of the phosphorylated termini of fission yeast DNA topoisomerase II

Fission Yeast DNA topoisomerase II (165 kD) consists of an enzymatically active 125-kD core, approximately 10-kD NH2-terminal and 30-kD COOH-terminal domains. The question addressed in the present study is what is the role of the topo II termini. Although deletion of either the NH2 or the COOH terminus is viable, deletion of both termini is lethal; the termini share an essential role for viability. We show here that topo II phosphorylation sites are localized in the terminal domains, but dephosphorylated topo II is still active. The topo II terminal sequences are required for nuclear localization; topo II double terminal deletion mutants are deficient for nuclear targeting, whereas wild-type and single deletion mutant topo IIs are transported into the nucleus with different efficiencies. Functional subdomains in the NH2 terminus are further dissected; we identified a 15 amino acid nuclear localization sequence (NLS) which is essential for viability and nuclear localization when the COOH terminus is deleted. This NLS could be substituted with SV-40 large T-antigen NLS. Two other functional subdomains were found; a non-essential acidic stretch which is phosphorylated and apparently enhances the nuclear localization and an essential hydrophilic stretch of unknown function. Motifs similar to these three NH2-terminal subdomains are also found in the COOH terminus. Our results support the possibility that phosphorylation of topo II does not play an essential role in fission yeast.     

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.     

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.     

Expression of the 170-kDa and 180-kDa isoforms of DNA topoisomerase II in resting and proliferating human lymphocytes

Abstract. The expression of the 170-kDa (α) and the 180-kDa ( β ) isoforms of DNA topoisomerase II (topo II) was investigated with two specific monoclonal antibodies (MoAbs) in human peripheral blood lymphocytes (PBL), before and after phyto-haemoagglutinin (PHA) stimulation. Binding of each MoAb was detected by indirect immunofluorescence labelling and quantified with flow cytometry. In resting PBL, the intensity of immunostaining was very low for both isozymes; however, topo II β -associated immunofluorescence was about 2.5 times significantly higher ( P< 0.001) than that associated with the a isoform. Between 48 and 72 h of PHA stimulation, when the highest percentage of cells in S and G₂+M phases was found, the levels of topo IIα and β increased up to about 30 and 10 times the value measured in resting PBL, respectively. Thus, the two isoforms reached comparable immunofluorescence values. At longer stimulation periods (96–120 h), topo IIα immunofluorescence was not significantly changed, while that relative to topo II β declined to about 50% of the peak value (P<0.02). At this time however, topo IIα-associated immunofluorescence was not significantly different from that related to the β isozyme. These results suggest that in resting PBL topo IIα is required at levels lower than topo II β , while in proliferating lymphocytes both isoforms are expressed to significantly higher levels.     

In vivo phosphorylation of the 170-kDa form of eukaryotic DNA topoisomerase II. Cell cycle analysis

We have examined the level of incorporation of 32P into DNA topoisomerase II in vivo in chicken lymphoblastoid cells that were fractionated into the various cell cycle phases by centrifugal elutriation. We find that topoisomerase II is phosphorylated in vivo, with the level of incorporation being approximately 3.5-fold higher in the G2 + M fraction than earlier in the cell cycle. Our antibody studies have revealed that topoisomerase II antigen exists as a number of discrete polypeptide species in these cells. Of these, the 170-kDa intact polypeptide is phosphorylated approximately 4.5-fold more than several antigenic fragments that actually comprise the bulk of the topoisomerase II antigen in these cells at mitosis. Phosphorylation of the 170-kDa form of the enzyme may be involved in activation of the enzyme for its role in the disjunction of sister chromatids at anaphase.     

Monoclonal antibodies to human DNA topoisomerase I and the two isoforms of DNA topoisomerase II: 170- and 180-kDa isozymes

Several monoclonal antibodies of different isotypes specific to human DNA topoisomerase I, to 170- and 180-kDa DNA topoisomerase II isozymes, were produced and characterized. The specificity of monoclonal antibodies was confirmed by comparison with polyclonal antibodies by Western blot, by immunoprecipitation of enzyme activity, and by immunoprecipitation of DNA topoisomerases with characterized polyclonal antisera. Morphological studies performed by immunofluorescence indicate that the three groups of monoclonal antibodies (MoAbs) stain the nucleus with characteristic patterns, which can be compared with those obtained with polyclonal antibodies. In particular the MoAbs to the 100-kDa DNA topoisomerase I stain the nucleolus and the nucleoplasm; the MoAbs to 170- and 180-kDa DNA topoisomerase II give completely distinct intranuclear patterns: those to the 170-kDa protein stain mainly the nucleoplasm, whereas those to the 180-kDa protein stain only the nucleolus. The two DNA topoisomerase II isozymes clearly exhibit fluctuations in their expression during cell growth: the 170-kDa isozyme is more abundant during the logarithmic phase of growth, while the 180-kDa isozyme is mainly present during the plateau phase of growth.     

Catalytic function of DNA topoisomerase II.

Although the genetic code is defined by a linear array of nucleotides, it is the three-dimensional structure of the double helix that regulates most of its cellular functions. Over the past two decades, it has become increasingly clear that aspects of this three-dimensionality which reflect topological relationships within the double helix (i.e., superhelical twisting, knotting, or tangling) influence virtually every facet of nucleic acid physiology. In vivo, DNA topology is modulated by ubiquitous enzymes known as topoisomerases. The type II enzyme is essential to the eukaryotic cell and is required for unlinking daughter chromosomes and maintaining chromosome structure. Moreover, topoisomerase II also has been identified as the primary cellular target for several widely used antineoplastic drugs. Before the physiological functions of topoisomerase II can be effectively dissected or its drug interactions fully exploited, it is imperative to understand the mechanism by which this important enzyme carries out its catalytic cycle.     

Structural organization and functional analysis of centromeric DNA in the fission yeast Schizosaccharomyces pombe.

Centromeric DNA in the fission yeast Schizosaccharomyces pombe was isolated by chromosome walking and by field inversion gel electrophoretic fractionation of large genomic DNA restriction fragments. The centromere regions of the three chromosomes were contained on three SalI fragments (120 kilobases [kb], chromosome III; 90 kb, chromosome II; and 50 kb, chromosome I). Each fragment contained several repetitive DNA sequences, including repeat K (6.4 kb), repeat L (6.0 kb), and repeat B, that occurred only in the three centromere regions. On chromosome II, these repeats were organized into a 35-kb inverted repeat that included one copy of K and L in each arm of the repeat. Site-directed integration of a plasmid containing the yeast LEU2 gene into K repeats at each of the centromeres or integration of an intact K repeat into a chromosome arm had no effect on mitotic or meiotic centromere function. The centromeric repeat sequences were not transcribed and possessed many of the properties of constitutive heterochromatin. Thus, S. pombe is an excellent model system for studies on the role of repetitive sequence elements in centromere function.     

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.     

A mutation in yeast topoisomerase II that confers hypersensitivity to multiple classes of topoisomerase II poisons

A mutation was constructed in the CAP homology domain of yeast topoisomerase II that resulted in hypersensitivity to the intercalating agent N-[4-(9-acridinylamino)-3-methoxy-phenyl]methanesulfonamide and the fluoroquinolone 6, 8-difluoro-7-(4'-hydroxyphenyl)-1-cyclopropyl-4-quinolone-3-carboxyli c acid, but not to etoposide. This mutation, which changes threonine at position 744 to proline, also confers hypersensitivity to anti-bacterial fluoroquinolones. The purified T744P mutant protein had wild type enzymatic activity in the absence of drugs, and no alteration in drug-independent DNA cleavage. Enhanced DNA cleavage in the presence of N-[4-(9-acridinylamino)-3-methoxy-phenyl]methanesulfonamide and fluoroquinolones was observed, in agreement with the results observed in vivo. DNA cleavage was also seen in the presence of norfloxacin and oxolinic acid, two quinolones that are inactive against eukaryotic topoisomerase II. The hypersensitivity was not associated with heat-stable covalent complexes, as was seen in another drug-hypersensitive mutant. Molecular modeling suggests that the mutation in the CAP homology domain may displace amino acids that play important roles in catalysis by topoisomerase II and may explain the drug-hypersensitive phenotype.     

DNA structure checkpoints in fission yeast

A DNA structure checkpoint can be defined as any checkpoint which responds to changes in the structure of the DNA either through the cell cycle, or in response to outside events such as DNA damage. Genetic analysis of DNA structure checkpoints in fission yeast has identified several distinct pathways responding to different circumstances. Three checkpoints have been identified which inhibit the onset of mitosis. (1) A radiation checkpoint which prevents mitosis after DNA damage. (2) A checkpoint linking S phase and mitosis (the S-M checkpoint) that prevents mitosis when DNA synthesis is incomplete. (3) A checkpoint linking G1 to mitosis (the G1-M checkpoint) that prevents the onset of mitosis in cells which are arrested in the G1 period of the cycle. A large number of genetic loci that are required for these checkpoints have been identified through mutant analysis, and the involvement of the relevant genes with the individual checkpoint pathways has been investigated. The largest class of checkpoint genes, known as the ‘checkpoint rad’ genes, are required for all the DNA structure checkpoints and the evidence suggests that they may also be involved in regulating DNA synthesis following precursor deprivation (hydroxyurea treatment) or when the replication fork encounters DNA damage. In this review, the available genetic and physiological evidence has been interpreted to suggest a close association between the ‘checkpoint rad’ class of gene products and the DNA-protein complexes that regulate and perform DNA synthesis. Biochemical evidence will be required in order to prove or disprove this hypothesis.     

Premeiotic DNA synthesis in fission yeast

Sporulating and various non-sporulating strains of S. pombe, especially several mutants deficient in conjugation or meiosis, were compared with respect to DNA synthesis under sporulation conditions. Meiosis and sporulation were induced by a transfer to nitrogen-free medium. As synchronized mitotic division was observed in all the strains as a first response to the shift, reducing the DNA amount per cell from the replicated state in G2 to the unreplicated state in the G1 phase of the cell cycle. Cells of the heterothallic wild-type strains ($\text{h}{}^{\mathrm{+}}\text{h}{}^{\mathrm{+}}$ or $\text{h}{}^{\mathrm{?}}\text{h}{}^{\mathrm{?}}$) accumulated in G1 with respect to DNA synthesis when they were incubated separately. In a mixed culture of these strains a period of enhanced DNA synthesis was observed after the start of zygote formation. This period of synthesis was absent in mutant fus1, where only prezygotes accumulated. Hence we conclude that in zygotic meiosis the premeiotic DNA synthesis is confined to zygotes after conjugation has been completed. In the diploid sporulating wild-type strain ($\text{h}{}^{\mathrm{+}}\text{h}{}^{\mathrm{?}}$), capable of azygotic meiosis without prior conjugation, premeiotic DNA synthesis occurred between $\text{2}\text{1}\text{2}$ and 5 h after the shift to the sporulation medium. There was no significant premeiotic DNA synthesis observed in diploid cells of the meiosis-deficient mutants mei1 or mei3, whereas premeiotic DNA synthesis proceeded normally in mutant mei4, which is blocked at a stage after commitment to meiosis in opposition to both the other mutants.     

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.     

A mutation in human topoisomerase II alpha whose expression is lethal in DNA repair-deficient yeast cells

Type II DNA topoisomerases are ATP-dependent enzymes that catalyze alterations in DNA topology. These enzymes are important targets of a variety of anti-bacterial and anti-cancer agents. We identified a mutation in human topoisomerase II alpha, changing aspartic acid 48 to asparagine, that has the unique property of failing to transform yeast cells deficient in recombinational repair. In repair-proficient yeast strains, the Asp-48 --> Asn mutant can be expressed and complements a temperature-sensitive top2 mutation. Purified Asp-48 --> Asn Top2alpha has relaxation and decatenation activity similar to the wild type enzyme, but the purified protein exhibits several biochemical alterations compared with the wild type enzyme. The mutant enzyme binds both covalently closed and linear DNA with greater avidity than the wild type enzyme. hTop2alpha(Asp-48 --> Asn) also exhibited elevated levels of drug-independent cleavage compared with the wild type enzyme. The enzyme did not show altered sensitivity to bisdioxopiperazines nor did it form stable closed clamps in the absence of ATP, although the enzyme did form elevated levels of closed clamps in the presence of a non-hydrolyzable ATP analog compared with the wild type enzyme. We suggest that the lethality exhibited by the mutant is likely because of its enhanced drug-independent cleavage, and we propose that alterations in the ATP binding domain of the enzyme are capable of altering the interactions of the enzyme with DNA. This mutant enzyme also serves as a new model for understanding the action of drugs targeting topoisomerase II.     

A consensus sequence for cleavage by vertebrate DNA topoisomerase II.

Topoisomerase II, purified from chicken erythrocytes, was reacted with a large number of different DNA fragments and cleavages were catalogued in the presence and absence of drugs that stabilize the cleavage intermediate. Cleavages were sequenced to derive a consensus for topoisomerase II that predicts catalytic sites. The consensus is: (sequence; see text) where N is any base and cleavage occurs at the indicated mark between -1 and +1. The consensus accurately predicts topoisomerase II sites in vitro. This consensus is not closely related to the Drosophila consensus sequence, but the two enzymes show some similarities in site recognition. Topoisomerase II purified from human placenta cleaves DNA sites that are essentially identical to the chicken enzyme, suggesting that vertebrate type II enzymes share a common catalytic sequence. Both viral and tissue specific enhancers contain sites sharing strong homology to the consensus and endogenous topoisomerase II recognizes some of these sites in vivo.     

Isolation of type I and II DNA topoisomerase mutants from fission yeast: single and double mutants show different phenotypes in cell growth and chromatin organization.

We have isolated mutants defective in DNA topoisomerases and an endonuclease from the fission yeast Schizosaccharomyces pombe by screening individual extracts of mutagenized cells. Two type I topoisomerase mutants (top1) and three endonuclease mutants (end1) were all viable. The double mutant top1 end1 was also viable and, in its extract, Mg2+- and ATP- dependent type II activity could be detected. Three temperature-sensitive (ts-) mutants having heat-sensitive (hs-) type II enzymes were isolated, and the ts- marker cosegregated with the hs- type II activity. All the ts- mutations fell in one gene (top2) tightly linked to leul in chromosome II. The nuclear division of single top2 mutants was blocked at the restrictive temperature, but the formation of a septum was not inhibited so that the nucleus was cut across with the cell plate. In contrast, the double top1 top2 mutants were rapidly arrested at various stages of the cell cycle, showing a strikingly altered nuclear chromatin region. The type II topoisomerase may have an essential role in the compaction and/or segregation of chromosomes during the nuclear division but also complement the defect of the type I enzyme whose major function is the maintenance of chromatin organization throughout the cell cycle.     

DNA topoisomerase II from Drosophila melanogaster. Relaxation of supercoiled DNA

In order to study the double-strand DNA passage reaction of eukaryotic type II topoisomerases, a quantitative assay to monitor the enzymic conversion of supercoiled circular DNA to relaxed circular DNA was developed. Under conditions of maximal activity, relaxation catalyzed by the Drosophila melanogaster topoisomerase II was processive and the energy of activation was 14.3 kcal . mol-1. Removal of supercoils was accompanied by the hydrolysis of either ATP or dATP to inorganic phosphate and the corresponding nucleoside diphosphate. Apparent Km values were 200 microM for pBR322 plasmid DNA, 140 microM for SV40 viral DNA, 280 microM for ATP, and 630 microM for dATP. The turnover number for the Drosophila enzyme was at least 200 supercoils of DNA relaxed/min/molecule of topoisomerase II. The enzyme interacts preferentially with negatively supercoiled DNA over relaxed molecules, is capable of removing positive superhelical twists, and was found to be strongly inhibited by single-stranded DNA. Kinetic and inhibition studies indicated that the beta and gamma phosphate groups, the 2'-OH of the ribose sugar, and the C6-NH2 of the adenine ring are important for the interaction of ATP with the enzyme. While the binding of ATP to Drosophila topoisomerase II was sufficient to induce a DNA strand passage event, hydrolysis was required for enzyme turnover. The ATPase activity of the topoisomerase was stimulated 17-fold by the presence of negatively supercoiled DNA and approximately 4 molecules of ATP were hydrolyzed/supercoil removed. Finally, a kinetic model describing the switch from a processive to a distributive relaxation reaction is presented.     

The fission yeast UVDR DNA repair pathway is inducible.

In addition to nucleotide excision repair (NER), the fission yeast Schizosaccharomyces pombe possesses a UV damage endonuclease (UVDE) for the excision of cyclobutane pyrimidine dimers and 6-4 pyrimidine pyrimidones. We have previously described UVDE as part of an alternative excision repair pathway, UVDR, for UV damage repair. The existence of two excision repair processes has long been postulated to exist in S.pombe, as NER-deficient mutants are still proficient in the excision of UV photoproducts. UVDE recognizes the phosphodiester bond immediately 5'of the UV photoproducts as the initiating event in this process. We show here that UVDE activity is inducible at both the level of uve1+ mRNA and UVDE enzyme activity. Further, we show that UVDE activity is regulated by the product of the rad12 gene.     

A novel 53-kDa polypeptide from chicken embryo.

We have isolated from chicken embryos a novel 53-kDa protein possessing properties which are similar, but not identical to the 55-kDa PDI polypeptide from chicken embryos. The novel 53-kDa polypeptide copurifies with PDI, but is separated by ion-exchange chromatography. The novel 53-kDa polypeptide cross-reacts strongly with antibodies specific for bovine PDI and cross-reacts to varying degrees with six different preparations of antibodies specific for chicken PDI which is identical to the beta-subunit of chicken prolyl 4-hydroxylase. Anti-bovine PDI immunoglobulins selected by the purified 53-kDa polypeptide react with bovine PDI but not with the beta-subunit of prolyl 4-hydroxylase, suggesting that the 53-kDa polypeptide shares epitopes with bovine PDI but not with the chicken prolyl 4-hydroxylase beta-subunit. Amino acid compositional analysis of the purified polypeptide yielded unique data when compared to PDI and other PDI-like polypeptides. Edman degradation from the N terminus of the 53-kDa polypeptide yields a sequence very different from the N terminus of PDI. This sequence is unique when compared to all entries in available databases. A 20-residue sequence of an internal cyanogen bromide fragment of the 53-kDa polypeptide gives a nearly identical match with human beta-endorphin. The 53-kDa polypeptide is capable of cleaving the disulphides of insulin under conditions where PDI is active. The periodic acid-Schiff assay failed to detect bound carbohydrate. These observations support evidence for a family of PDI-like proteins in chicken embryo and suggest that PDI activity is not confined to only one protein.