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At least 19 recordsLinked to original sources
PubMed2001

Mechanism of topology simplification by type II DNA topoisomerases.

Type II DNA topoisomerases actively reduce the fractions of knotted and catenated circular DNA below thermodynamic equilibrium values. To explain this surprising finding, we designed a model in which topoisomerases introduce a sharp bend in DNA. Because the enzymes have a specific orientation relative to the bend, they act like Maxwell's demon, providing unidirectional strand passage. Quantitative analysis of the model by computer simulations proved that it can explain much of the experimental data. The required sharp DNA bend was demonstrated by a greatly increased cyclization of short DNA fragments from topoisomerase binding and by direct visualization with electron microscopy.

Computer Simulation↗
PubMed2003

Phylogenomics of type II DNA topoisomerases.

Type II DNA topoisomerases (Topo II) are essential enzymes implicated in key nuclear processes. The recent discovery of a novel kind of Topo II (DNA topoisomerase VI) in Archaea led to a division of these enzymes into two non-homologous families, (Topo IIA and Topo IIB) and to the identification of the eukaryotic protein that initiates meiotic recombination, Spo11. In the present report, we have updated the distribution of all Topo II in the three domains of life by a phylogenomic approach. Both families exhibit an atypical distribution by comparison with other informational proteins, with predominance of Topo IIA in Bacteria, Eukarya and viruses, and Topo IIB in Archaea. However, plants and some Archaea contain Topo II from both families. We confront this atypical distribution with current hypotheses on the evolution of the three domains of life and origin of DNA genomes.

Animals↗
PubMed1998

Type II DNA topoisomerases.

Type II DNA topoisomerases are enzymes capable of passing one DNA duplex through another. A combination of structural and biochemical analyses is illuminating the mechanistic details of this transport reaction, revealing the sites of DNA and nucleotide binding and the existence of large-scale domain motions.

Archaeal Proteins↗
PubMed1996

Bacterial diversity based on type II DNA topoisomerase genes.

Type II DNA topoisomerases are essential and ubiquitous DNA metabolic enzymes that alter DNA topology. Eubacteria have two indispensable type II DNA topoisomerases, DNA gyrase encoded by gyrB and gyrA and topoisomerase IV encoded by parE and parC. These genes belong to a single family whose members span both eukaryotes and prokaryotes. The highly conserved motifs in these genes provide a rationale for the design of universal primers used in the polymerase chain reaction in order to systematically generate a data set suitable for bacterial diversity studies at the macro-diversity level, as well as at the micro-diversity level displaying individual species and isolates. This family of genes is the subject of intensive biochemical and genetic analyses, which provide an opportunity for comprehensive understanding of sequence conservation and variability and their relationship to function. These genes are ideally suited for microbial identification and biodiversity analyses.

Amino Acid Sequence↗
PubMed1980

Type II DNA topoisomerases: enzymes that can unknot a topologically knotted DNA molecule via a reversible double-strand break.

The T4 DNA topoisomerase is a recently discovered multisubunit protein that appears to have an essential role in the initiation of T4 bacteriophage DND replication. Treatment of double-stranded circular DNA with large amounts of this topoisomerase in the absence of ATP yields new DNA species which are knotted topological isomers of the double-stranded DNA circle. These knotted DNA circles, whether covalently closed or nicked, are converted to unknotted circles by treatment with trace amounts of the T4 topoisomerase in the presence of ATP. Very similar ATP-dependent enzyme activities capable of unknotting DNA are present in extracts of Drosophila eggs. Xenopus laevis eggs and mammalian tissue culture cells. The procaryotic enzyme, DNA gyrase, is also capable of unknotting DNA. We propose that these unknotting enzymes constitute a new general class of DNA topoisomerases (type II DNA topoisomerases). These enzymes must act via mechanisms that involve the concerted cleavage and rejoining of two opposite DNA strands, such that the DNA double helix is transiently broken. The passage of a second double-stranded DNA segment through this reversible double-strand break results in a variety of DNA topoisomerization reactions, including relaxation:super-coiling; knotting:unknotting and catenation:decatenation. In support of this type of mechanism, we demonstrate that the T4 DNA topoisomerase changes the linking number of a covalently closed double-stranded circular DNA molecule only by multiples of two. We discuss the possible roles of such enzymes in a variety of biological functions, along with their probable molecular mechanisms.

Animals↗
PubMed1984

Site-specific recognition of bacteriophage T4 DNA by T4 type II DNA topoisomerase and Escherichia coli DNA gyrase.

The site specificity of bacteriophage T4-induced type II DNA topoisomerase action on double-stranded DNA has been explored by studying the sites where DNA cleavages are induced by the enzyme. Oxolinic acid addition increases the frequency at which phi X174 duplex DNA is cut by the enzyme by about 100-fold, to the point where nearly every topoisomerase molecule causes a double-stranded DNA cleavage event. The effect of oxolinic acid on the enzyme is very similar to its effect on another type II DNA topoisomerase, the Escherichia coli DNA gyrase. A filter-binding method was developed that allows efficient purification of topoisomerase-cleaved DNA fragments by selecting for the covalent attachment of this DNA to the enzyme. Using this method, T4 topoisomerase recognition of mutant cytosine-containing T4 DNA was found to be relatively nonspecific, whereas quite specific recognition sites were observed on native T4 DNA, which contains glucosylated hydroxymethylcytosine residues. The increased specificity of native T4 DNA recognition seems to be due entirely to the glucose modification. In contrast, E. coli DNA gyrase shows a high level of specificity for both the mutant cytosine-containing DNA and native T4 DNA, recognizing about five strong cleavage sites on both substrates. An unexpected feature of DNA recognition by the T4 topoisomerase is that the addition of the cofactor ATP strongly stimulates the topoisomerase-induced cleavage of native T4 DNA, but has only a slight effect on cleavage of cytosine-containing T4 DNA.

DNA Restriction Enzymes↗
PubMed1997

Effects of nucleosomes and anti-tumor drugs on the catalytic activity of type II DNA topoisomerase from rat testis.

To gain insight into the relative catalytic efficiencies of mammalian type I and type II DNA topoisomerases, in the cellular context, we have used naked DNA and DNA incorporated into nucleosomes as substrates. We observed that the relaxation activity of both the enzymes declined with DNA containing increasing densities of nucleosomes; however, kinetic analysis revealed that topoisomerase I seemed less affected than topoisomerase II. The addition of histone H1, in stoichiometric amounts, to naked DNA or minichromosomes lessened the activity of topoisomerase II, and required 7-fold less for complete inhibition when the latter was used as the substrate. To ascertain if the observed differences are specific to topoisomerase II from testis, we examined the effect of nucleosomes on the catalytic efficiency of its isoform from liver. Interestingly, the suppression of relaxation activity of liver topoisomerase II required substrates containing higher mass ratios of histone octamer/DNA. Studies on the effect of nucleosomes on the action of teniposide displayed significant differences in the kinetics of the reaction, in its IC50 values, and have provided biochemical evidence for the first time that nucleosomes increased inhibition caused by teniposide. Further, this feature appears to be specific for topoisomerase II-directed drugs and is not shared by the generic class of either DNA-intercalating or non-DNA-intercalating ligands.

Animals↗
PubMed1988

Localization of a type II DNA topoisomerase to two sites at the periphery of the kinetoplast DNA of Crithidia fasciculata.

A type II DNA topoisomerase (topollmt), purified to near homogeneity from the trypanosomatid C. fasciculata has been shown to be localized to the single mitochondrion of these kinetoplastid protozoa. Immunoblots show at least a 10-fold higher level of topollmt (per milligram of protein) in preparations of partially purified mitochondria as compared with those from whole cells. Analyses of type I and type II topoisomerase activities in both mitochondrial and whole cell extracts show a 4- to 5-fold higher specific activity of topollmt in mitochondrial extracts while a nuclear type I topoisomerase has a 4- to 5-fold lower specific activity in the same extract. Immunolocalizations using anti-topollmt antibodies show the enzyme to be present in close association with the mitochondrial DNA networks (kinetoplast DNA or kDNA). This association appears at two distinct locations on opposite sides of the kDNA network.

Animals↗
PubMed1998

Expression and cellular localization of Trypanosoma cruzi type II DNA topoisomerase.

Topoisomerases are enzymes that participate in many cellular functions involving topological manipulation of DNA strands. There are two types of topoisomerases in the cell: (a) type I topoisomerases; and (b) type II topoisomerases (topo II). Previously we have cloned and sequenced the gene encoding Trypanosoma cruzi topo II (TcTOP2). This study group has raised an antiserum against recombinant type II DNA topoisomerase (TctopoII) to study the expression of this gene during T. cruzi differentiation and to determine the cellular location of the enzyme. Western blot analysis showed that T. cruzi TctopoII is expressed in the replicative epimastigotes but not in the infective and non-replicative trypomastigotes. However, slot blot analysis of RNAs extracted from epimastigotes and metacyclic trypomastigotes showed that the mRNA encoding the enzyme is present in both developmental stages of the parasite. Confocal laser microscopy using the antiserum raised against recombinant TctopoII showed that the enzyme is located exclusively in the nucleus of the parasite. Similar results were obtained by immunofluorescence analysis of Crithidia fasciculata. However, monoclonal antisera against the corresponding enzyme extracted from C. fasciculata recognizes a kinetoplast protein in both T. cruzi and Crithidia.

Animals↗
PubMed2001

Functional and regulatory characteristics of eukaryotic type II DNA topoisomerase.

DNA topoisomerases are ubiquitous nuclear enzymes that govern the topological interconversions of DNA by transiently breaking/rejoining the phosphodiester backbone of one (type I) or both (type II) strands of the double helix. Consistent with these functions, topoisomerases play key roles in many aspects of DNA metabolism. Type II DNA topoisomerase (topo II) is vital for various nuclear processes, including DNA replication, chromosome segregation, and maintenance of chromosome structure. Topo II expression is regulated at multiple stages, including transcriptional, posttranscriptional, and posttranslational levels, by a multitude of signaling factors. Topo II is also the cellular target for a variety of clinically relevant anti-tumor drugs. Despite significant progress in our understanding of the role of topo II in diverse nuclear processes, several important aspects of topo II function, expression, and regulation are poorly understood. We have focused this review specifically on eukaryotic DNA topoisomerase II, with an emphasis on functional and regulatory characteristics.

Adenosine Triphosphate↗
PubMed1983

Knotting of the circular duplex DNA by type II DNA topoisomerase from Drosophila melanogaster.

DNA molecules with topological knots can be detected in a reaction mixture of circular DNA and type II DNA topoisomerase purified from the embryos of Drosophila melanogaster. Similar to the DNA knotting reaction by bacteriophage T4-induced topoisomerase (Liu, L. F., Liu, C. C., and Alberts, B. M. (1980) Cell 19, 697-707), the knotting reaction by the Drosophila topoisomerase II requires a high ratio of enzyme/DNA. However, they differ in the following aspects: 1) the knotting reaction by the Drosophila enzyme is greatly stimulated by the presence of ATP; 2) the knotted DNA is a stable product in the reaction mixture over prolonged incubation; and 3) both the covalently closed circular DNA and its nicked form, which is devoid of any superhelical structure, can be knotted. The knotted DNA generated by the Drosophila topoisomerase II can be efficiently unknotted by a catalytic amount of Drosophila topoisomerase II, but not by topoisomerase I. The ionic strength optimum for the knotting reaction is close, but not identical, to the optimal condition for the unknotting and supercoil relaxation reactions. Aspects of DNA knotting are discussed.

Animals↗
PubMedDate not supplied

[The import of DNA topoisomerase type II from Drosophila melanogaster into the chloroplasts of transgenic Nicotiana tabacum plants].

The construct containing the structural gene of the type II DNA topoisomerase of Drosophila melanogaster fused to the sequence of the chloroplast transit peptide directed by the 35 S promoter was produced on the basis of the binary vector Bin 19. The construct was introduced into Nicotiana tabacum plants by Agrobacterium-mediated transformation. The integration of the construct into genomic DNA was demonstrated by PCR and Southern blot hybridization. Expression of the chimeric gene at the transcription level was studied by Northern blotting. The protein produce of the expression was identified in chloroplasts by Western blot hybridization with polyclonal anti-type II Drosophila topoisomerase antibodies.

Animals↗
PubMed1994

Localization of an aminoacridine antitumor agent in a type II topoisomerase-DNA complex.

Type II topoisomerases are the targets of several classes of chemotherapeutic agents that stabilize an intermediate of the catalytic cycle with the enzyme covalently linked to cleaved DNA. We have used 3-azido-AMSA [4'-(3-azido-9-acridinylamino)methanesulfon-m-anisidide], a photo-activatible analog of the inhibitor m-AMSA [4'-(9-acridinylamino)methanesulfon-m-anisidide], to localize the inhibitor binding site in a cleavage complex consisting of an oligonucleotide substrate and the bacteriophage T4 type II DNA topoisomerase. Upon photoactivation, the inhibitor covalently attached to the substrate only in the presence of topoisomerase. Sites of inhibitor attachment were detected by primer-extension analysis and by piperidine-induced cleavage of the covalently modified substrate. 3-Azido-AMSA reacted with bases immediately adjacent to the two phosphodiester bonds cleaved by the enzyme. Therefore, topoisomerase creates or stabilizes preferential binding sites for the inhibitor precisely at the two sites of DNA cleavage.

Amsacrine↗
PubMed1985

Purification and characterization of a type II DNA topoisomerase from bovine calf thymus.

We report here the large scale purification of DNA topoisomerase II from calf thymus glands, using the unknotting of naturally knotted P4 phage DNA as an assay for enzymatic activity. Topoisomerase II was purified more than 1300-fold as compared to the whole cell homogenate, with 22% yield. Analysis of the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two bands of apparent molecular masses of 125 and 140 kDa. Tryptic maps of the two bands indicated that they derive from the same protein. Using these fragments, specific polyclonal antisera to topoisomerase II were raised in rabbits. Immunoblotting of whole cell lysates from various species indicated that topoisomerase II is well conserved among mammals and has a native subunit molecular mass of 180 kDa. Analytical sedimentation and gel filtration were used to determine a sedimentation coefficient of 9.8 S and a Stokes radius of 68 A. The calculated solution molecular mass of 277 kDa implies a dimer structure in solution. The purified topoisomerase II unknots P4 DNA in an ATP-dependent manner and is highly stimulated in its relaxation activity by ATP. A DNA-stimulated ATPase activity, as has been found with other type II topoisomerases, is associated with the purified enzyme. Approximate kinetic parameters for the ATPase reaction were determined to be: a Vmax of 0.06 nmol of ATP/(micrograms of protein) (min) and Km of 0.2 mM in the absence of DNA, and a Vmax of 0.2 nmol of ATP/(micrograms of protein) (min) and Km of 0.4 mM ATP in the presence of supercoiled plasmid DNA.

Adenosine Triphosphate↗
PubMed1996

Differential behaviors of Staphylococcus aureus and Escherichia coli type II DNA topoisomerases.

Staphylococcus aureus gyrA and gyrB genes encoding DNA gyrase subunits were cloned and coexpressed in Escherichia coli under the control of the T7 promoter-T7 RNA polymerase system, leading to soluble gyrase which was purified to homogeneity. Purified gyrase was catalytically indistinguishable from the gyrase purified from S. aureus and did not contain detectable amounts of topoisomerases from the E. coli host. Topoisomerase IV subunits GrlA and GrlB from S. aureus were also expressed in E. coli and were separately purified to apparent homogeneity. Topoisomerase IV, which was reconstituted by mixing equimolar amounts of GrlA and GrlB, had both ATP-dependent decatenation and DNA relaxation activities in vitro. This enzyme was more sensitive than gyrase to inhibition by typical fluoroquinolone antimicrobial agents such as ciprofloxacin or sparfloxacin, adding strong support to genetic studies which indicate that topoisomerase IV is the primary target of fluoroquinolones in S. aureus. The results obtained with ofloxacin suggest that this fluoroquinolone could also primarily target gyrase. No cleavable complex could be detected with S. aureus gyrase upon incubation with ciprofloxacin or sparfloxacin at concentrations which fully inhibit DNA supercoiling. This suggests that these drugs do not stabilize the open DNA-gyrase complex, at least under standard in vitro incubation conditions, but are more likely to interfere primarily with the DNA breakage step, contrary to what has been reported with E. coli gyrase. Both S. aureus gyrase-catalyzed DNA supercoiling and S. aureus topoisomerase IV-catalyzed decatenation were dramatically stimulated by potassium glutamate or aspartate (500- and 50-fold by 700 and 350 mM glutamate, respectively), whereas topoisomerase IV-dependent DNA relaxation was inhibited 3-fold by 350 mM glutamate. The relevance of the effect of dicarboxylic amino acids on the activities of type II topoisomerases is discussed with regard to the intracellular osmolite composition of S. aureus.

Anti-Bacterial Agents↗
PubMed1998

Eukaryotic DNA topoisomerase II beta.

Type II DNA topoisomerase activity is required to change DNA topology. It is important in the relaxation of DNA supercoils generated by cellular processes, such as transcription and replication, and it is essential for the condensation of chromosomes and their segregation during mitosis. In mammals this activity is derived from at least two isoforms, termed DNA topoisomerase II alpha and beta. The alpha isoform is involved in chromosome condensation and segregation, whereas the role of the beta isoform is not yet clear. DNA topoisomerase II beta was first reported in 1987. Here we review the research on DNA topoisomerase II beta over the last 10 years.

Amino Acid Sequence↗