Conditional growth of Escherichia coli caused by expression of vaccinia virus DNA topoisomerase I.

Active vaccinia virus topoisomerase I is expressed in Escherichia coli containing plasmid p1940 (S. Shuman, M. Golder, and B. Moss, J. Biol. Chem. 263:16401-16407, 1988). Growth curves showed a decline of 2 to 3 logs in the number of viable cells at 42 degrees C after shift from 30 degrees C because of increased vaccinia virus topoisomerase I level. Mutations in the gyrA and gyrB genes allowed cells to grow equally well at 42 and 30 degrees C. The presence of gyrase inhibitor also improved growth at 42 degrees C.     

On the molecular basis of the thermal sensitivity of an Escherichia coli topA mutant.

Studies of two temperature-sensitive Escherichia coli topA strains AS17 and BR83, both of which were supposed to carry a topA amber mutation and a temperature-sensitive supD43,74 amber-suppressor, led to conflicting results regarding the essentiality of DNA topoisomerase I in cells grown in media of low osmolarity. We have therefore reexamined the molecular basis of the temperature sensitivity of strain AS17. We find that the supD allele in this strain had lost its temperature sensitivity. The temperature sensitivity of the strain, in media of all osmolarity, results from the synthesis of a mutant DNA topoisomerase I that is itself temperature-sensitive. Nucleotide sequencing of the AS17 topA allele and studies of its expected cellular product show that the mutant enzyme is not as active as its wild-type parent even at 30 degrees C, a permissive temperature for the strain, and its activity relative to the wild-type enzyme is further reduced at 42 degrees C, a nonpermissive temperature. Our results thus implicate an indispensable role of DNA topoisomerase I in E. coli cells grown in media of any osmolarity.     

Characterization of a potent catenation activity of HeLa cell nuclei

Using an assay which measures catenation of a supercoiled DNA template, we have characterized and quantitated a potent activity identified in crude fractions of HeLa cell nuclei. Catenation requires Mg-ATP and a DNA-condensing agent, polyvinyl alcohol. A filter-binding or agarose gel assay can be used to quantitate activity. In this reaction, DNA topoisomerase I relaxes the input supercoiled DNA to provide DNA topoisomerase II, a strongly favored template for catenation. DNA topoisomerase II preferentially catenates relaxed DNA over supercoiled DNA by a factor of 100. One molecule of DNA topoisomerase II is able to catenate about 20 circles of relaxed DNA/min at 30 degrees C but only 0.16 circle of supercoiled DNA/min at 30 degrees C. The purified HeLa topoisomerase I can also catenate DNA under these assay conditions, yet in an ATP-independent fashion. It is much less efficient than topoisomerase II; one molecule of topoisomerase I catenates only about 3.8 X 10(-3) molecules of supercoiled DNA/min at 30 degrees C with a DNA template containing 5% nicked circles. This remarkable difference between the two enzymes allows quantitation of DNA topoisomerase II activity seen in the presence of excess topoisomerase I. Unlike Escherichia coli topoisomerase I (omega), catenation by the HeLa topoisomerase I is not stimulated by gapped circles.     

DNA topoisomerase III from extremely thermophilic archaebacteria. ATP-independent type I topoisomerase from Desulfurococcus amylolyticus drives extensive unwinding of closed circular DNA at high temperature.

A second type I topoisomerase was purified from the extremely thermophilic archaebacterium Desulfurococcus amylolyticus. In contrast to the previously described reverse gyrase from this organism, the novel enzyme designated as Dam topoisomerase III is an ATP-independent relaxing topoisomerase. It is a monomer with Mr 108,000, as determined by electrophoresis under denaturing conditions and by size exclusion chromatography. Dam topoisomerase III, like other bacterial type I topoisomerases, absolutely requires Mg2+ for activity and is specific for single-stranded DNA. At 60-80 degrees C, it relaxes negatively but not positively supercoiled DNA and is inhibited by single-stranded M13 DNA. At 95 degrees C, the enzyme unwinds both positively and negatively supercoiled substrates and produces extensively unwound form I* and I** DNA. The peculiarities of DNA topoisomerization at high temperatures are discussed.     

Identification of a potent decatenating enzyme from Escherichia coli

A topoisomerase has been purified from extracts of a topoisomerase I-deficient strain of Escherichia coli based solely on its ability to segregate pBR322 DNA replication intermediates in vitro. This enzyme rapidly decatenated multiply linked form II:form II DNA dimers to form II DNA, provided that the DNA substrate contained single-stranded regions. Efficient relaxation of negatively supercoiled DNA was observed when reaction mixtures were incubated at 52 degrees C, but not at 30 degrees C (the temperature at which decatenation was readily observed). This topoisomerase was insensitive to the DNA gyrase inhibitor norfloxacin and unaffected by antibody directed against topoisomerase I. Relaxation of a unique plasmid topoisomer revealed that this decatenase changed the linking number of the DNA in steps of one and was therefore a type 1 topoisomerase. The cleavage pattern of a fragment of single-stranded phi X174 DNA generated by this decatenase was virtually identical to that reported for topoisomerase III, the least characterized topoisomerase present in E. coli.     

Leakage of periplasmic enzymes from lipopolysaccharide-defective mutants of Salmonella typhimurium.

Mutants of Salmonella typhimurium with defects in the heptose region of the lipopolysaccharide (LPS) molecule (heptose-deficient, chemotype Re) leak periplasmic enzymes (acid phosphatase (EC 3.1.3.2), cyclic phosphodiesterase, ribonuclease I (EC 3.1.4.22), and phosphoglucose isomerase (EC 5.3.1.9) (PGI is at least partially periplasmic in E. coli and S. typhimurium; see below)) and do not leak an internal enzyme (glucose-6-phosphate dehydrogenase) into the growth medium. The extent of this leakage is markedly increased at higher temperature (42 degrees C). Leakage of periplasmic enzymes from the strains lacking units distal to heptose I in the LPS molecule (chemotype Rd2) occurs only at 42 degrees C, and not at 30 or 37 degrees C. The extent of leakage of these enzymes from smooth strain and mutants of other LPS chemotypes (Rc, Rd1) is not significant, and is not influenced by growth temperatures. The kinetics of leakage of periplasmic enzymes after shift to 42 degrees C in nutrient broth reveal an accelerated release into the medium from heptose-deficient strains of cyclic phosphodiesterase and ribonuclease I after 30 min at 42 degrees C, and phosphoglucose isomerase after 60 min at 42 degrees C; at 30 degrees C the rate of release of cyclic phosphodiesterase and ribonuclease I is relatively slower. After 60 min at 42 degrees C in nutrient broth, growth of these strains has either slowed down or stopped. In L-broth, which permits the growth of the heptose-deficient strain (SA1377) at 42 degrees C, leakage of cyclic phosphodiesterase and phosphoglucose isomerase occurs, whereas there is no detectable leakage of these enzymes from the isogenic smooth strain (SA1355). Thus, leakage of the periplasmic enzymes from the heptose-deficient strain occurs with or without growth. Mg2+ (0.75 mM), sodium chloride (50 mM), and sucrose (100 mM) in nutrient broth at 42 degrees C prevent the leakage of these enzymes. The shedding of LPS from the heptose-deficient as well as the smooth strains is enhanced by high temperature (42 degrees C), whereas considerable leakage of protein occurs only in the heptose-deficient strain at 42 degrees C and not in the smooth strain. The smooth and heptose-deficient strains are equally sensitive to osmotic shock although a significant proportion of acid phosphatase and cyclic phosphodiesterase activities from the heptose-deficient cells grown at 42 degrees C comes off in the Tris-NaCl wash step suggesting a rather loose attachment of these enzymes onto the cell surface.     

Induction of mammalian DNA topoisomerase I mediated DNA cleavage by antitumor indolocarbazole derivatives.

DNA topoisomerases have been shown to be important therapeutic targets in cancer chemotherapy. We found that KT6006 and KT6528, synthetic antitumor derivatives of indolocarbazole antibiotic K252a, were potent inducers of a cleavable complex with topoisomerase I. In DNA cleavage assay using purified calf thymus DNA topoisomerase I and supercoiled pBR322 DNA, KT6006 induced topoisomerase I mediated DNA cleavage in a dose-dependent manner at drug concentrations up to 50 microM, while DNA cleavage induced by KT6528 was saturated at 5 microM. The maximal amount of nicked DNA produced by KT6006 was more than 50% of substrate DNA, which was comparable to that of camptothecin. Heat treatment (65 degrees C) of the reaction mixture containing these compounds and topoisomerase I resulted in a substantial reduction in DNA cleavage, suggesting that topoisomerase I mediated DNA cleavage induced by KT6006 and KT6528 is through the mechanism of stabilizing the reversible enzyme-DNA "cleavable complex". Both KT6006 and KT6528 did not induce topoisomerase II mediated DNA cleavage in vitro. KT6006 and KT6528 were found to induce nearly identical topoisomerase I mediated DNA cleavage patterns, which was distinctly different from that with camptothecin. In contrast to the similarity between KT6006 and KT6528 in their structures and the nature of their cleavable complex with topoisomerase I, these drugs have different properties with respect to their interaction with DNA: KT6006 is a very weak intercalator whereas KT6528 is a strong intercalator with potentials comparable to that of adriamycin. These results indicate that KT6006 and KT6528 represent a new distinct class of mammalian DNA topoisomerase I active antitumor drugs.     

Induction of thermotolerance in T cells protects nuclear DNA topoisomerase I from heat stress.

In this study, we have demonstrated that topoisomerase I DNA relaxing activity is protected against a severe heat shock in T cells made thermotolerant by a prior modest heat treatment. However, following a severe heat-shock challenge and incubation at 37 degrees C, topoisomerase activity in the control population eventually returned to levels similar to those detected in thermotolerant cells. This recovery of topoisomerase activity appears to result from the renaturation of heat-inactivated enzyme rather than from synthesis of new protein because the rate of recovery of catalytic activity was not inhibited by the presence of the protein synthesis inhibitor, cycloheximide.     

Comparison of responses of DNA topoisomerase I from Candida albicans and human cells to four new agents which stimulate topoisomerase-dependent DNA nicking

Abstract DNA topoisomerase I is a potential target for therapeutic antifungal agents predicted to have a fungicidal mode of action. This report describes four agents with varying degrees of selectivity for the fungal topoisomerase I compared to the human enzyme: 5-hydroxy-1H-indole-3-acetic acid (5-HIAA), quinizarin, dibenzo- p -dioxin-2-carboxylic acid and 7-amino-4-hydroxy-2-naphthalenesulfonic acid. Taken together with the response of topoisomerase to camptothecin and aminocatechol, these data suggest that there are sufficient structural differences between the topoisomerase I from Candida albicans and human cells to allow selective targeting of the fungal topoisomerase I over its human counterpart.     

Hyperthermophilic topoisomerase I from Thermotoga maritima. A very efficient enzyme that functions independently of zinc binding

Topoisomerases, by controlling DNA supercoiling state, are key enzymes for adaptation to high temperatures in thermophilic organisms. We focus here on the topoisomerase I from the hyperthermophilic bacterium Thermotoga maritima (optimal growth temperature, 80 degrees C). To determine the properties of the enzyme compared with those of its mesophilic homologs, we overexpressed T. maritima topoisomerase I in Escherichia coli and purified it to near homogeneity. We show that T. maritima topoisomerase I exhibits a very high DNA relaxing activity. Mapping of the cleavage sites on a variety of single-stranded oligonucleotides indicates a strong preference for a cytosine at position -4 of the cleavage, a property shared by E. coli topoisomerase I and archaeal reverse gyrases. As expected, the mutation of the putative active site Tyr 288 to Phe led to a totally inactive protein. To investigate the role of the unique zinc motif (Cys-X-Cys-X(16)-Cys-X-Cys) present in T. maritima topoisomerase I, experiments have been performed with the protein mutated on the tetracysteine motif. Strikingly, the results show that zinc binding is not required for DNA relaxation activity, contrary to the E. coli enzyme. Furthermore, neither thermostability nor cleavage specificity is altered in this mutant. This finding opens the question of the role of the zinc-binding motif in T. maritima topoisomerase I and suggests that this hyperthermophilic topoisomerase possesses a different mechanism from its mesophilic homolog.     

DNA cleavage and religation by human topoisomerase II alpha at high temperature.

A common DNA religation assay for topoisomerase II takes advantage of the fact that the enzyme can rejoin cleaved nucleic acids but cannot mediate DNA scission at suboptimal temperatures (either high or low). Although temperature-induced DNA religation assays have provided valuable mechanistic information for several type II enzymes, high-temperature shifts have not been examined for human topoisomerase IIalpha. Therefore, the effects of temperature on the DNA cleavage/religation activity of the enzyme were characterized. Human topoisomerase IIalpha undergoes two distinct transitions at high temperatures. The first transition occurs between 45 and 55 degrees C and is accompanied by a 6-fold increase in the level of DNA cleavage at 60 degrees C. It also leads to a loss of DNA strand passage activity, due primarily to an inability of ATP to convert the enzyme to a protein clamp. The enzyme alterations that accompany the first transition appear to be stable and do not revert at lower temperature. The second transition in human topoisomerase IIalpha occurs between 65 and 70 degrees C and correlates with a precipitous drop in the level of DNA scission. At 75 degrees C, cleavage falls well below amounts seen at 37 degrees C. This loss of DNA scission appears to result from a decrease in the forward rate of DNA cleavage rather than an increase in the religation rate. Finally, similar high-temperature alterations were observed for yeast topoisomerase II and human topoisomerase IIbeta, suggesting that parallel heat-induced transitions may be widespread among type II topoisomerases.     

DNA topoisomerase I phosphorylation in murine fibroblasts treated with 12-O-tetradecanoylphorbol-13-acetate and in vitro by protein kinase.

The phosphorylation of DNA topoisomerase I in quiescent murine 3T3-L1 fibroblasts treated with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) was characterized by in vivo labeling with [32P] orthophosphate and immunoprecipitation with a scleroderma anti-DNA topoisomerase I autoantibody. DNA topoisomerase I phosphorylation was stimulated 4-fold by 2 h of TPA treatment (TPA at 100 ng/ml maximally enhanced phosphorylation). Purified DNA topoisomerase I was phosphorylated in vitro in a Ca2+ and phospholipid-dependent fashion by types I, II, and III protein kinase C. The phosphorylation reaction was stimulated by TPA and had an apparent K(m) of 0.4 microM. DNA topoisomerase I was phosphorylated in vivo and in vitro predominantly at serine. The major tryptic phosphopeptides from DNA topoisomerase I in TPA-treated fibroblasts and phosphorylated by protein kinase C comigrated in thin-layer electrophoresis. The half-life of incorporated phosphate on DNA topoisomerase I was 40 min in both TPA-treated and control cells. These results suggest that phosphorylation is a mechanism for activating DNA topoisomerase I in fibroblasts treated with TPA and that protein kinase C functions in the phosphorylation.     

Induction of mammalian DNA topoisomerase I and II mediated DNA cleavage by saintopin, a new antitumor agent from fungus.

Saintopin is an antitumor antibiotic recently discovered in mechanistically oriented screening using purified calf thymus DNA topoisomerases. Saintopin induced topoisomerase I mediated DNA cleavage comparable to that of camptothecin, and topoisomerase II mediated DNA cleavage equipotent to those of 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) or 4'-demethylepipodophyllotoxin 9-(4,6-O-ethylidene-beta-D-glucopyranoside) (VP-16). Treatment of a reaction mixture containing saintopin and topoisomerase I or II with either elevated temperature (65 degrees C) or higher salt concentration (0.5 M NaCl) resulted in a substantial reduction in DNA cleavage, suggesting that the topoisomerase I and II mediated DNA cleavage induced by saintopin is through the mechanism of stabilizing the reversible enzyme-DNA "cleavable complex". Consistent with the cleavable complex formation with both topoisomerases, saintopin inhibited catalytic activities of both topoisomerase I and topoisomerase II. The DNA cleavage intensity pattern induced by saintopin with topoisomerase I was different from that by camptothecin. A difference in cleavage pattern was also detected between saintopin and m-AMSA or VP-16 in topoisomerase II mediated DNA cleavage. DNA unwinding assay using T4 DNA ligase showed that saintopin is a weak DNA intercalator like m-AMSA. Thus, saintopin represents a new class of antitumor agent that can induce both mammalian DNA topoisomerase I and mammalian DNA topisomerase II mediated DNA cleavage.     

Topoisomerase I is essential in Cryptococcus neoformans: role In pathobiology and as an antifungal target

Topisomerase I is the target of several toxins and chemotherapy agents, and the enzyme is essential for viability in some organisms, including mice and drosophila. We have cloned the TOP1 gene encoding topoisomerase I from the opportunistic fungal pathogen Cryptococcus neoformans. The C. neoformans topoisomerase I contains a fungal insert also found in topoisomerase I from Candida albicans and Saccharomyces cerevisiae that is not present in the mammalian enzyme. We were unable to disrupt the topoisomerase I gene in this haploid organism by homologous recombination in over 8000 transformants analyzed. When a second functional copy of the TOP1 gene was introduced into the genome, the topoisomerase I gene could be readily disrupted by homologous recombination (at 7% efficiency). Thus, topoisomerase I is essential in C. neoformans. This new molecular strategy with C. neoformans may also be useful in identifying essential genes in other pathogenic fungi. To address the physiological and pathobiological functions of the enzyme, the TOP1 gene was fused to the GAL7 gene promoter. The resulting GAL7::TOP1 fusion gene was modestly regulated by carbon source in a serotype A strain of C. neoformans. Modest overexpression of topoisomerase I conferred sensitivity to heat shock, gamma-rays, and camptothecin. In contrast, alterations in topoisomerase I levels had no effect on the toxicity of a novel class of antifungal agents, the dicationic aromatic compounds (DACs), indicating that topoisomerase I is not the target of DACs. In an animal model of cryptococcal meningitis, topoisomerase I regulation was not critically important to established infection, but may impact on the initial stress response to infection. In summary, our studies reveal that topoisomerase I is essential in the human pathogen C. neoformans and represents a novel target for antifungal agents.     

Positive supercoiling catalysed in vitro by ATP-dependent topoisomerase from Desulfurococcus amylolyticus

A topoisomerase capable of introducing positive supercoils into closed-circular DNA has been isolated from the extremely thermophilic anaerobic archaebacterium Desulfurococcus amylolyticus. This polypeptide has an Mr of 135,000, as determined by electrophoresis under denaturing conditions. The enzyme is active in the temperature range from 65 degrees C to 100 degrees C and catalyzes positive supercoiling both in negatively supercoiled DNA and in relaxed DNA. These reactions require the presence of ATP. The enzyme's action on a single topoisomer has shown the linking number to increase by an integral number upon the relaxation of negative supercoils and the introduction of positive ones. This means that the reverse gyrase from D. amylolyticus is a type I topoisomerase. The presence of an extended AT sequence within the closed-circular DNA enhances the activity of the Desulfurococcus topoisomerase. Even though the enzyme is isolated from a strictly anaerobic bacterium, it is fully active in the presence of oxygen.     

Chloroplast DNA topoisomerase I from cauliflower

An ATP-independent DNA topoisomerase has been isolated from chloroplasts of cauliflower leaves (Brassica oleracea var. botrytis) through DEAE-cellulose, AF-blue Toyopearl, and hydroxyapatite column chromatography. The sedimentation coefficient and Stokes radius of this enzyme are 3.6S and 3.6 nm, respectively, and the molecular weight of native enzyme is estimated to be 54,000. This enzyme changes the linking number in steps of one. The enzyme activity is stimulated by MgCl2, and this enzyme shows optimum activity at 30 degrees C in the range of 3 mM MgCl2 + 100 mM KCl-10 mM MgCl2 + 50 mM KCl. The enzyme activity was reduced remarkably by N-ethylmaleimide, indicating that a free sulfhydryl group is important for the activity; heparin and ellipticine also reduced the activity. Both cauliflower chloroplast topoisomerase and spinach chloroplast topoisomerase can relax positive supercoils as well as negative supercoils. From these properties, cauliflower chloroplast topoisomerase can be classified as a eukaryotic type I DNA topoisomerase.     

Effect of the deletion of the σ32-dependent promoter (P1) of the Escherichia coli topoisomerase I gene on thermotolerance

Topoisomerase I and DNA gyrase are the major topoisomerase activities responsible for the regulation of DNA supercoiling in the bacterium Escherichia coli . The P1 promoter of topA has previously been shown to be a σ³²-dependent heat-shock promoter. A mutant strain with a deletion of P1 was constructed. This mutant is >10-fold more sensitive to heat treatment (52°C) than the wild type. After brief treatment at 42°C, wild-type Escherichia coli acquires an enhanced resistance to the effects of a subsequent 52°C treatment. This is not the case for the P1 deletion mutant, which, and under these conditions, is about 100-fold less thermotolerant than the wild type. The presence of a plasmid expressing topoisomerase I restored the heat-survival level of the mutant to that of the wild type. During heat shock, the superhelical density of a plasmid with the heat-inducible rpoD promoter is increased in the P1 deletion mutant. We also note that the pulse-labelling pattern of proteins at 42°C (displayed on SDS–polyacrylamide gels) is different in the mutant, and, most notably, the amounts of DnaK and of GroEL protein are reduced. A model is proposed in order to unify these observations.     

Winding of the DNA helix by divalent metal ions.

When supercoiled pBR322 DNA was relaxed at 0 or 22 degrees C by topoisomerase I in the presence of the divalent cations Ca2+, Mn2+ or Co2+, the resulting distributions of topoisomers observed at 22 degrees C had positive supercoils, up to an average delta Lk value of +8.6 (for Ca2+at 0 degrees C), corresponding to an overwinding of the helix by 0.7 degrees/bp. An increase of the divalent cation concentration in the reaction mixture above 50 mM completely reversed the effect. When such ions were present in agarose electrophoresis gels, they caused a relaxation of positively supercoiled DNA molecules, and thus allowed a separation of strongly positively supercoiled topoisomers. The effect of divalent cations on DNA adds a useful tool for the study of DNA topoisomers, for the generation as well as separation of positively supercoiled DNA molecules.