In vitro catenation and decatenation of DNA and a novel eucaryotic ATP-dependent topoisomerase

Extracts from X. laevis germinal vesicles interlock duplex DNA circles to form catenanes. The catenation activity requires Mg++ and ATP. Negatively supercoiled or relaxed DNA can be used as substrates for the catenation reaction. Homology between donor and acceptor DNA is not required, since catenanes are formed between DNA molecules with unrelated sequences. In the course of the isolation of the activity responsible for the catenation reaction, we discovered a new ATP-dependent topoisomerase. The fractions containing the novel topoisomerase catenate and decatenate DNA, the ionic strength dictating which of the two opposing reactions will occur.     

Condensin aids sister chromatid decatenation by topoisomerase II

The condensin complex is a key determinant of mitotic chromosome architecture. In addition, condensin promotes resolution of sister chromatids during anaphase, a function that is conserved from prokaryotes to human. Anaphase bridges observed in cells lacking condensin are reminiscent of chromosome segregation failure after inactivation of topoisomerase II (topo II), the enzyme that removes catenanes persisting between sister chromatids following DNA replication. Circumstantial evidence has linked condensin to sister chromatid decatenation but, because of the difficulty of observing chromosome catenation, this link has remained indirect. Alternative models for how condensin facilitates chromosome resolution have been put forward. Here, we follow the catenation status of circular minichromosomes of three sizes during the Saccharomyeces cerevisiae cell cycle. Catenanes are produced during DNA replication and are for the most part swiftly resolved during and following S-phase, aided by sister chromatid separation. Complete resolution, however, requires the condensin complex, a dependency that becomes more pronounced with increasing chromosome size. Our results provide evidence that condensin prevents deleterious anaphase bridges during chromosome segregation by promoting sister chromatid decatenation.     

Mitochondrial topoisomerase II activity is essential for kinetoplast DNA minicircle segregation.

Etoposide, a nonintercalating antitumor drug, is a potent inhibitor of topoisomerase II activity. When Trypanosoma equiperdum is treated with etoposide, cleavable complexes are stabilized between topoisomerase II and kinetoplast DNA minicircles, a component of trypanosome mitochondrial DNA (T. A. Shapiro, V. A. Klein, and P. T. Englund, J. Biol. Chem. 264:4173-4178, 1989). Etoposide also promotes the time-dependent accumulation of small minicircle catenanes. These catenanes are radiolabeled in vivo with [3H]thymidine. Dimers are most abundant, but novel structures containing up to five noncovalently closed minicircles are detectable. Analysis by two-dimensional gel electrophoresis and electron microscopy indicates that dimers joined by up to six interlocks are late replication intermediates that accumulate when topoisomerase II activity is blocked. The requirement for topoisomerase II is particularly interesting because minicircles do not share the features postulated to make this enzyme essential in other systems: for minicircles, the replication fork is unidirectional, access to the DNA is not blocked by nucleosomes, and daughter circles are extensively nicked and (or) gapped.     

Identification of a unique domain essential for Escherichia coli DNA topoisomerase III-catalysed decatenation of replication intermediates

A 17-amino-acid residue domain has been identified in Escherichia coli DNA topoisomerase III (Topo III) that is essential for Topo III-mediated resolution of DNA replication intermediates in vitro. Deletion of this domain reduced Topo III-catalysed resolution of DNA replication intermediates and decatenation of multiply linked plasmid DNA dimers by four orders of magnitude, whereas reducing Topo III-catalysed relaxation of negatively supercoiled DNA substrates only 20-fold. The presence of this domain has been detected in multiple plasmid-encoded topoisomerases, raising the possibility that these enzymes may also be decatenases.     

Yeast DNA topoisomerase II. An ATP-dependent type II topoisomerase that catalyzes the catenation, decatenation, unknotting, and relaxation of double-stranded DNA rings

An activity from the yeast Saccharomyces cerevisiae, initially noted for its catalysis of aggregation of covalently closed double-stranded DNA rings in the presence of ATP, has been identified as a type II DNA topoisomerase and is designated yeast DNA topoisomerase II. The formation of the DNA aggregate, which has been shown to be a network of DNA rings that are topologically interlocked, requires the presence of a yeast DNA-binding protein in addition to the topoisomerase. In the absence of the binding protein, yeast DNA topoisomerase II catalyzes decatenation and unknotting of duplex DNA rings and the relaxation of negatively or positively supercoiled DNA. All reactions are ATP-dependent and require Mg(II). Similar to other eukaryotic and phage T4-type II DNA topoisomerases, the yeast enzyme does not catalyze DNA supercoiling under the assay conditions employed. The activity is not sensitive to the gyrase inhibitor nalidixic acid, oxolinic acid, or novobiocin. Coumermycin inhibits the activity, however, at a concentration as low as 5 microgram/ml.     

In vivo inhibition of trypanosome mitochondrial topoisomerase II: effects on kinetoplast DNA maxicircles.

Kinetoplast DNA, the mitochondrial DNA of trypanosomes, is a topologically complex structure composed of interlocked minicircles and maxicircles. We previously reported that etoposide, a potent inhibitor of topoisomerase II, promotes the cleavage of about 20% of network minicircle DNA (T. A. Shapiro, V. A. Klein, and P. T. Englund, J. Biol. Chem. 264:4173-4178, 1989). We now find that virtually all maxicircles are released from kinetoplast DNA networks after trypanosomes are treated with etoposide. As expected for a topoisomerase II cleavage product, the linearized maxicircles have protein bound to both 5' ends. After etoposide treatment, the residual minicircle catenanes have a sedimentation coefficient which is only 70% that of controls, and by electron microscopy the networks are less compact. Double-size networks, the characteristic dumbbell-shape forms that normally arise in the final stages of network replication, are replaced by aberrant unit-size forms.     

Step-wise DNA relaxation and decatenation by NaeI-43K.

Nae I protein was originally isolated for its restriction endonuclease properties. Nae I was later discovered to either relax or cleave supercoiled DNA, depending upon whether Nae I position 43 contains a lysine (43K) or leucine (43L) respectively. Nae I-43K DNA relaxation activity appears to be the product of coupling separate endonuclease and ligase domains within the same polypeptide. Whereas Nae I relaxes supercoiled DNA like a topoisomerase, even forming a transient covalent intermediate with the substrate DNA, Nae I shows no obvious sequence similarity to the topoisomerases. To further characterize the topoisomerase activity of Nae I, we report here that Nae I-43K changes the linking number of a single negatively supercoiled topoisomer of pBR322 by units of one and therefore is a type I topoisomerase. Positively supercoiled pBR322 was resistant to Nae I-43K. At low salt concentration Nae I-43K was processive; non-saturating amounts of enzyme relaxed a fraction of the DNA. At high salt concentration the same non-saturating amounts of Nae I-43K partially relaxed all the DNA in a step-wise fashion to give a Gaussian distribution of topoisomers, demonstrating a switch from a processive to a distributive mode of action. Nae I-43K decatenated kinetoplast DNA containing nicked circles, implying that Nae I-43K can cleave opposite a nick. The products of the reaction are decatenated nicked circles under both processive and distributive conditions. The behavior of Nae I-43K is consistent with that of a prokaryotic type I topoisomerase.     

Minicircular kinetoplast DNA from Trypanosomatidae

Analysis of primary structure and organization of mitochondrial (kinetoplast) DNA of flagellates occupies a prominent place in the studies of eukaryote mitochondrial genomes, owing to its unusual organization and functioning as well as to the epidemiological role of the Trypanosomatidae family. According to contemporary notions, living zooflagellates are direct descendants of the ancestral forms that gave rise to all eukaryotic kingdoms. Hence, comparative mtDNA studies of recent Trypanosomatidae open broad prospects for phylogenetic reconstructions and analysis of presumable routes of eukaryote evolution. The structure, characteristics, and functions of Trypanosomatidae minicircular kinetoplast DNA are discussed here.     

Drug-promoted cleavage of kinetoplast DNA minicircles. Evidence for type II topoisomerase activity in trypanosome mitochondria

Minicircle DNA, the major component of the mitochondrial DNA of trypanosomes (kinetoplast DNA), is linearized when living Trypanosoma equiperdum cells are treated with inhibitors of mammalian type II topoisomerases and then lysed with sodium dodecyl sulfate. A variety of intercalating and nonintercalating compounds (the epipodophyllotoxins, 4'-(9-acridinylamino)-methanesulfon-m-anisidine, 2-methyl-9-hydroxyellipticine, and acriflavine) are active, but novobiocin and specific gyrase inhibitors (the quinolones) are not. The linearized minicircles are in a DNA-protein complex, as their electrophoretic mobility is increased by Proteinase K treatment. They are digested by exonuclease III but not by lambda exonuclease, indicating that the protein must be linked to both 5' ends. Drug-induced cleavage sites vary with different compounds and are found throughout the minicircle sequence. These results indicate that trypanosome mitochondria contain a type II topoisomerase with some properties similar to those of type II topoisomerases in the nucleus of higher eukaryotes. A maximum of 12% of all minicircles is cleaved in the presence of VP16-213, indicating there are at least 600 molecules of mitochondrial type II topoisomerase/cell or about one enzyme/8 kilobases of minicircle DNA.     

Decatenation of kinetoplast DNA by topoisomerases

Kinetoplast DNA is the mitochondrial DNA of trypanosomatids such as Crithidia fasciculata. This DNA is in the form of networks containing thousands of DNA circles which are apparently catenated (interlocked). Some topoisomerases, such as T4 phage topoisomerase and DNA gyrase, catalyze a decatenation of the networks to form individual covalently closed circles.     

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.     

In vivo catenation and decatenation of DNA.

We have noted previously that when circular, but not linear, DNA or chromatin was injected into Xenopus laevis oocytes, much of it went through an intermediate form in which it did not readily enter an agarose gel; after a few hours, it reappeared as monomer DNA that had acquired its full complement of nucleosomes (T. J. Miller and J. E. Mertz, Mol. Cell. Biol. 2:1581-1593, 1982). We determined, using electron microscopy and a variety of biochemical techniques, the structure of this aggregated material. Most of it was oligomeric and multimeric catenanes of the injected sample. In addition, injection of DNA that had been catenated in vitro with DNA gyrase resulted in the conversion of most of it back to monomer circles. These findings demonstrate directly that both catenation and decatenation of DNA occur in vivo under physiological conditions. Whether these reactions play a crucial role in nucleosome formation, as well as in DNA replication and recombination, remains to be determined.     

DNA gyrase-catalyzed decatenation of multiply linked DNA dimers

One possible intermediate during the terminal stages of the replication of a closed circular DNA is a catenated DNA dimer of the two completed daughter molecules. The two monomer DNA rings in these DNA dimers can be linked as many as 20-30 times. In Escherichia coli, DNA gyrase could act on these catenated dimers to eliminate the linkages between the daughter duplexes, yielding the final monomer product. In this report, this reaction has been studied biochemically. The in vitro pBR322 DNA replication system (Minden, J., and Marians, K. J. (1985) J. Biol. Chem. 260, 9316-9325) was used to manufacture large amounts of multiply linked catenated DNA dimers for use as a substrate for DNA gyrase-catalyzed decatenation. Studies presented here demonstrate that this decatenation reaction is more efficient with supercoiled as opposed to relaxed DNA dimers, proceeds in a distributive fashion, is inhibited by moderate amounts of salt (80 mM KCl), and is stimulated by the E. coli protein HU.