Comparative analysis of nuclear and mitochondrial DNA topoisomerase I from <Emphasis Type="Italic">Zea mays</Emphasis>

The properties were compared for maize nuclear and mitochondrial DNA topoisomerases I (topo I). Some differences in their ability to bind to single-stranded DNA were revealed. Mitochondrial topo I was active only in the presence of Mg²⁺, whereas the activity of the nuclear enzyme did not completely depend on Mg²⁺, although being essentially stimulated in the presence of Mg²⁺. The mitochondrial enzyme covalently bound to the 5′ DNA end, as unique to prokaryotic topo I. The nuclear enzyme, like all eukaryotic topo I, covalently bound to the 3′ DNA end. A search for homologous sequences in several databases revealed genes probably encoding mitochondrial topo I in other higher plants. Using cDNA sequencing and in silico analysis, an orthologous gene was revealed in the maize genome. The gene was strongly homologous to the genes encoding prokaryotic topo I, which could explain the differences in properties between mitochondrial and nuclear topo I from maize. The presence of prokaryotic topo I in mitochondria of higher plants is interesting and important for studying the evolution of these plant organelles and the mechanisms of mitochondrial genome expression.     

Thirteen-exon-motif signature for vertebrate nuclear and mitochondrial type IB topoisomerases

DNA topoisomerases contribute to various cellular activities that involve DNA. We previously identified a human nuclear gene that encodes a mitochondrial DNA topoisomerase. Here we show that genes for mitochondrial DNA topoisomerases (type IB) exist only in vertebrates. A 13-exon topoisomerase motif was identified as a characteristic of genes for both nuclear and mitochondrial type IB topoisomerases. The presence of this signature motif is thus an indicator of the coexistence of nuclear and mitochondrial type IB DNA topoisomerases. We hypothesize that the prototype topoisomerase IB with the 13-exon structure formed first, and then duplicated. One topoisomerase specialized for nuclear DNA and the other for mitochondrial DNA.     

Purification and characterization of a type I DNA topoisomerase from calf thymus mitochondria

A type I topoisomerase has been purified more than 4000-fold from calf thymus mitochondria. The enzyme is membrane associated and is effectively solubilized by 1% Triton X-100 treatment of purified mitochondrial inner membranes. This ATP-independent enzyme relaxes positively and negatively supercoiled DNA with delta LK = 1. At low ionic strength, the native enzyme appears to be a monomer (sedimentation coefficient of 4.3 S and Stokes radius of 34 A), but it can form a weakly associated dimer at higher salt concentrations (sedimentation coefficient of 7.0 S and Stokes radius of 47.5 A). The mitochondrial type I topoisomerase is distinguishable from the nuclear enzyme by its (1) pH profile, (2) thermal stability, (3) response to dimethyl sulfoxide and Berenil, and (4) molecular weight. The mitochondrial enzyme is inhibited by elevated concentrations of the bacterial DNA gyrase inhibitor novobiocin, but not nalidixic or oxolinic acids. Sensitivity to N-ethylmaleimide indicates the importance of cysteine for catalytic activity. It is estimated that there are at least five copies of topoisomerase I per mammalian mitochondrion or a minimum of one to two per mitochondrial genome. In a manner similar to that observed with leukemia (nuclear and mitochondrial), calf thymus (nuclear), and HeLa (nuclear) cell type I topoisomerase, the calf thymus mitochondrial enzyme is inhibited by physiological concentrations of ATP.     

Studies on mitochondrial type I topoisomerase and on its function

We have reported previously that rat liver mitochondria contain a topoisomerase and have shown it to be distinct from the nuclear enzyme by its sensitivity to Berenil and ethidium bromide. We report here some additional characterization. The enzyme differs further from its nuclear counterpart in its failure to bind to ssDNA cellulose and its chromatographic behavior on Sephadex; the latter procedure yields an Mr of 44 000 for the mitochondrial and 70 000 for the nuclear enzyme. The topoisomerase is strongly associated with mitochondrial membranes; only 10% of the activity could be extracted. The pH optimum of the enzyme falls between 6.0 and 8.5, with an NaCl optimum of 0.13 M in 0.1 M Tris (pH 8.3). Dithiothreitol is required, while N-ethylmaleimide is inhibitory. Tosylphenylalanine chloromethyl ketone, a serine proteinase inhibitor, abolishes activity; another, phenylmethanesulfonyl fluoride, has no effect. Berenil, a non-intercalating drug, and four of its analogues all inhibit with up to 100-fold differences in potency. No dependence on ATP, Mg2+, or both together could be shown. Neither novobiocin nor oxolinic acid shows any inhibitory effect. Nicked circles are generated in the presence of DMSO. These three observations are consistent with the topoisomerase being of the Type I class. Positively supercoiled pBR322 DNA, whose 6-8 positive turns were generated by altering solution conditions, is relaxed by the enzyme, indicating a lack of requirement for a negatively supercoiled substrate. We have also examined a partially purified preparation of the corresponding mitochondrial enzyme from mouse L cells. This enzyme is largely similar in properties to the rat liver enzyme. In isolated mitochondria, Berenil causes biphasic alterations in [3H]dATP incorporation into DNA, 10(-4) mM stimulating 2-fold, while higher concentrations inhibit. [3H]UTP incorporation into mitochondrial RNA also follows this pattern.     

Sequence specific interaction of Mycobacterium smegmatis topoisomerase I with duplex DNA.

We have identified strong topoisomerase sites (STS) for Mycobacteruim smegmatis topoisomerase I in double-stranded DNA context using electrophoretic mobility shift assay of enzyme-DNA covalent complexes. Mg2+, an essential component for DNA relaxation activity of the enzyme, is not required for binding to DNA. The enzyme makes single-stranded nicks, with transient covalent interaction at the 5'-end of the broken DNA strand, a characteristic akin to prokaryotic topoisomerases. More importantly, the enzyme binds to duplex DNA having a preferred site with high affinity, a property similar to the eukaryotic type I topoisomerases. The preferred cleavage site is mapped on a 65 bp duplex DNA and found to be CG/TCTT. Thus, the enzyme resembles other prokaryotic type I topoisomerases in mechanistics of the reaction, but is similar to eukaryotic enzymes in DNA recognition properties.     

Higher order chromatin structures in maize and Arabidopsis.

We are investigating the nature of plant genome domain organization by using DNase I- and topoisomerase II-mediated cleavage to produce domains reflecting higher order chromatin structures. Limited digestion of nuclei with DNase I results in the conversion of the >800 kb genomic DNA to an accumulation of fragments that represents a collection of individual domains of the genome created by preferential cleavage at super-hypersensitive regions. The median size of these fragments is approximately 45 kb in maize and approximately 25 kb in Arabidopsis. Hybridization analyses with specific gene probes revealed that individual genes occupy discrete domains within the distribution created by DNase I. The maize alcohol dehydrogenase Adh1 gene occupies a domain of 90 kb, and the maize general regulatory factor GRF1 gene occupies a domain of 100 kb in length. Arabidopsis Adh was found within two distinct domains of 8.3 and 6.1 kb, whereas an Arabidopsis GRF gene occupies a single domain of 27 kb. The domains created by topoisomerase II-mediated cleavage are identical in size to those created by DNase I. These results imply that the genome is not packaged by means of a random gathering of the genome into domains of indiscriminate length but rather that the genome is gathered into specific domains and that a gene consistently occupies a discrete physical section of the genome. Our proposed model is that these large organizational domains represent the fundamental structural loop domains created by attachment of chromatin to the nuclear matrix at loop basements. These loop domains may be distinct from the domains created by the matrix attachment regions that typically flank smaller, often functionally distinct sections of the genome.     

A novel active DNA topoisomerase I in Leishmania donovani

A common feature shared by type I DNA topoisomerases is the presence of a "serine, lysine, X, X, tyrosine" motif as conventional enzyme active site. Preliminary data have shown that Leishmania donovani DNA topoisomerase I gene (LdTOP1A) lacked this conserved motif, giving rise to different theories about the reconstitution of an active DNA topoisomerase I in this parasite. We, herein, describe the molecular cloning of a new DNA topoisomerase I gene from L. donovani (LdTOP1B) containing the highly conserved serine, lysine, X, X, tyrosine motif. DNA topoisomerase I activity was detected only when both genes (LdTOP1A and LdTOP1B) were co-expressed in a yeast expression system, suggesting the existence of a dimeric DNA topoisomerase I in Leishmania parasites.     

Human NADH:Ubiquinone Oxidoreductase

NADH:ubiquinone oxidoreductase consists of at least 43 proteins; seven are encoded by the mitochondrial genome, while the remainder are encoded by the nuclear genome. A deficient activity of this enzyme complex is frequently observed in the clinical heterogeneous group of mitochondrial disorders, with Leigh (-like) disease as the main contributor. Enzyme complex activity measurement in skeletal muscle is the mainstay of the diagnostic process. Fibroblast studies are a prerequisite whenever prenatal enzyme diagnosis is considered. Mitochondrial DNA mutations are found in approximately 5–10% of all complex I deficiencies. Recently, all structural nuclear complex I genes have been determined at the cDNA level and several at the gDNA level. A comprehensive mutational analysis study of all complex I nuclear genes in a group of 20 patients exhibiting this deficiency revealed mutations in about 40%. Here, we describe the enzymic methods we use and the recent progress made in genomics and cell biology of human complex I.     

Localization of a DNA topoisomerase II to mitochondria inDictyostelium discoideum: Deletion mutant analysis and mitochondrial targeting signal presequence

DNA topoisomerase II ofDictyostelium discoideum (TopA), the gene (topA) encoding which we cloned, was shown to have an additional N-terminal region which contains a putative mitochondrial targeting signal presequence. We constructed overexpression mutants which expressed the wild-type or the N-terminally deleted enzyme, and examined its localization by immunofluorescence microscopy and proteinase K digestion experiment. These experiments revealed that the enzyme is located in the mitochondria by virtue of the additional N-terminal region. Furthermore, in the cell extract depleted the enzyme by immunoprecipitation, nuclear DNA topoisomerase II activity was not decreased. These results confirmed that TopA is located in the mitochondria, even through its amino acid sequence is highly similar to those of nuclear type topoisomerase II of other organisms. Thus, this report is the first to establish the location of the mitochondrial targeting signal presequence in DNA topoisomerase II and in proteins ofD. discoideum directly by analyzing deletion mutants. Tsukuba Advanced Research Alliance (TARA researcher for the Sakabe project)     

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.     

Normal Mitochondrial Genome in Brain from Patients with Parkinson''s Disease and Complex I Defect

The mitochondrial genome codes for 13 proteins which are located in the respiratory chain. In postmortem brain of patients with Parkinson's disease, decreased activity of complex I of the respiratory chain could be demonstrated. Because seven subunits of complex I are coded by the mitochondrial genome, we analyzed the mitochondrial DNA of human postmortem substantia nigra, putamen, and frontal cortex by the Southern blot technique. No deletions of the mitochondrial genome could be demonstrated, thus indicating that either subunits which are encoded by the nuclear genome are decreased or enzyme activity is diminished by metabolites, toxins, or increase of Fe3+.     

Pea chloroplast topoisomerase I: purification,characterization, and role in replication

A DNA-relaxing enzyme was purified 5 000-fold to homogeneity from isolated chloroplasts of Pisum sativum. The enzyme consists of a single polypeptide of 112 kDa. The enzyme was able to relax negatively supercoiled DNA in the absence of ATP. It is resistant to nalidixic acid and novobiocin, and causes a unit change in the linkage number of supercoiled DNA. The enzyme shows optimum activity at 37°C with 50 mM KCl and 10 mM MgCl₂. From these properties, the enzyme can be classified as a prokaryotic type I topoisomerase.Using a partiall purified pea chloroplast DNA polymerase fraction devoid of topoisomerase I activity for in vitro replication on clones containing the pea chloroplast DNA origins of replication, a 2–6-fold stimulation of replication activity was obtained when the purified topoisomerase I was added to the reaction at 70–100 mM KCl. However, when the same reaction was carried out at 125 mM KCl, which does not affect DNA polymerase activity on calf thymus DNA but is completely inhibitory for topoisomerase I activity, a 4-fold drop in activity resulted. Novobiocin, an inhibitor of topoisomerase II, was not found to inhibit the in vitro replication of chloroplast DNA.     

Characterization of vaccinia virus DNA topoisomerase I expressed in Escherichia coli

The putative structural gene encoding the vaccinia virus type I DNA topoisomerase (EC 5.99.1.2) was expressed in Escherichia coli under the control of a bacteriophage T7 promoter. Provision of T7 RNA polymerase resulted in the accumulation to high level of a Mr = 33,000 type I topoisomerase with the properties of the vaccinia enzyme. A simple purification scheme yielded approximately 8 mg of recombinant vaccinia topoisomerase from 400 ml of bacteria. DNA unwinding by the enzyme was stimulated by magnesium, manganese, calcium, cobalt, and spermidine, but inhibited by copper and zinc. Like eukaryotic cellular type I topoisomerases, but unlike the prokaryotic counterpart, the recombinant topoisomerase relaxed positively and negatively supercoiled DNA. The viral topoisomerase I was, however, resistant to the effects of camptothecin, a drug that specifically inhibits cellular type I topoisomerases.     

N-terminal region of the large subunit of Leishmania donovani bisubunit topoisomerase I is involved in DNA relaxation and interaction with the smaller subunit

Leishmania donovani topoisomerase I is an unusual bisubunit enzyme. We have demonstrated earlier that the large and small subunit could be reconstituted in vitro to show topoisomerase I activity. We extend our biochemical study to evaluate the role of the large subunit in topoisomerase activity. The large subunit (LdTOP1L) shows a substantial degree of homology with the core DNA binding domain of the topoisomerase IB family. Two N-terminal truncation constructs, LdTOP1Delta39L (lacking amino acids 1-39) and LdTOP1Delta99L (lacking amino acids 1-99) of the large subunit were generated and mixed with intact small subunit (LdTOP1S). Our observations reveal that residues within amino acids 1-39 of the large subunit have significant roles in modulating topoisomerase I activity (i.e. in vitro DNA relaxation, camptothecin sensitivity, cleavage activity, and DNA binding affinity). Interestingly, the mutant LdTOP1Delta99LS was unable to show topoisomerase I activity. Investigation of the loss of activity indicates that LdTOP1Delta99L was unable to pull down glutathione S-transferase-LdTOP1S in an Ni(2+)-nitrilotriacetic acid co-immobilization experiment. For further analysis, we co-expressed LdTOP1L and LdTOP1S in Escherichia coli BL21(DE3)pLysS cells. The lysate shows topoisomerase I activity. Immunoprecipitation revealed that LdTOP1L could interact with LdTOP1S, indicating the subunit interaction in bacterial cells, whereas immunoprecipitation of bacterial lysate co-expressing LdTOP1Delta99L and LdTOP1S reveals that LdTOP1Delta99L was significantly deficient at interacting with LdTOP1S to reconstitute topoisomerase I activity. This study demonstrates that heterodimerization between the large and small subunits of the bisubunit enzyme appears to be an absolute requirement for topoisomerase activity. The residue within amino acids 1-39 from the N-terminal end of the large subunit regulates DNA topology during relaxation by controlling noncovalent DNA binding or by coordinating DNA contacts by other parts of the enzyme.