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The fluoroquinolone ciprofloxacin, an inhibitor of eubacterial DNA gyrase, induces single- and double-stranded DNA breaks in the plasmid pGRB-1 from the halophilic archaebacterium Halobacterium GRB when the cells are treated by this drug in a magnesium-depleted medium. This reaction is prevented by a dose of novobiocin known to specifically inhibit DNA gyrase. Cleavage of pGRB-1 DNA induced by either ciprofloxacin or the antitumoral drug etoposide (VP16) produces DNA fragments of identical lengths. These results indicate that ciprofloxacin, novobiocin, and etoposide have a common target in Halobacterium GRB: an archaebacterial type II DNA topoisomerase. The similarity of DNA cleavage patterns induced by ciprofloxacin and etoposide is a new and strong argument that quinolone and epipodophyllotoxins have the same mode of interaction with the DNA-DNA topoisomerase II complexes. The plasmid pGRB-1 could be used to prescreen in the same system both antibiotics that inhibit bacterial gyrase and antitumoral drugs that inhibit eukaryotic DNA topoisomerase II.

The topoprofile of 1.7 kb plasmids from the archaebacterium Halobacterium GRB was analysed from cells growing with or without VP16 (etoposide). This drug interferes with the breakage-reunion reaction of eukaryotic DNA topoisomerase II by inhibiting the ligase activity of this enzyme. Addition of VP16 to the culture medium of Halobacterium GRB cells results in the introduction of single- and double-strand DNA breaks in part of the plasmid population, with proteins covalently associated at their 5' ends. While some of the remaining covalently closed circular DNA molecules are relaxed, VP16 treatment also gives rise to the production of positively supercoiled 1.7 kb plasmids. In contrast to adriamycin, VP16 does not intercalate into the 1.7 kb plasmid DNA in vivo. These results suggest that the VP16 target in halobacteria is a DNA topoisomerase II. Three major cleavage sites were detected on the 1.7 kb plasmid after VP16 treatment in vivo.

We have examined the presence of methylated adenine at GATC sequences (Dam phenotype) in the DNA of 23 eubacteria and 13 archaebacteria by using isoshizomer restriction enzymes. We have found a completely Dam+ phenotype in bacteria of nine genera related to the families Enterobacteriaceae, Parvobacteriaceae, and Vibrionaceae, and in the five cyanobacteria tested. We have found a partial Dam+ phenotype in the two archaebacteria Halobacterium saccharovorum and Methanobacterium sp. strain Ivanov. All of the other archaebacteria (three genera) and eubacteria (nine genera) tested were Dam-. Phylogenetic analysis, based on the evolutionary tree of Fox et al. (Science 209:457-463, 1980), indicates that dam methylation in the Escherichia coli lineage appeared recently in bacterial evolution and is restricted to a small range of closely related bacteria.

The archaebacteria constitute a group of prokaryotes with an intermediate phylogenetic position between eukaryotes and eubacteria. The study of their DNA polymerases may provide valuable information about putative evolutionary relationships between prokaryotic and eukaryotic DNA polymerases. As a first step towards this goal, we have purified to near homogeneity a DNA polymerase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. This enzyme is a monomeric protein of 100 kDa which can catalyze DNA synthesis using either activated calf thymus DNA or oligonucleotide-primed single-stranded DNA as a template. The activity is optimal at 70 degrees C and the enzyme is thermostable up to 80 degrees C; however, it can still polymerize up to 200 nucleotides at 100 degrees C. These remarkable thermophilic properties and thermostability permit examination of the mechanism of DNA synthesis under conditions of decreased stability of the DNA helix. Furthermore, these properties make S. acidocaldarius DNA polymerase a very efficient enzyme to be used in DNA amplification by the recently developed polymerase chain reaction method (PCR) as well as in the Sanger DNA sequencing technique.

The halophilic archaebacterium Halobacterium strain GRB harbours a multicopy plasmid of 1.7 kb which is negatively supercoiled. After addition of novobiocin to culture medium all 1.7 kb plasmid molecules become positively supercoiled. Positive supercoiling occurs at the same dose of novobiocin inhibiting the eubacterial DNA gyrase in vitro. Novobiocin also induces positive supercoiling of pHV2, a 6.3 kb plasmid from Halobacterium volcanii. These results indicate the existence of a mechanism producing positive superturns in halobacteria. The 1.7 kb plasmid from Halobacterium GRB could be used to produce high amounts of pure positively supercoiled DNA for biophysical and biochemical studies.

The action of novobiocin and coumermycin (two coumarins which interact with the gyrB subunit of eubacterial DNA gyrase) and ciprofloxacin (a fluoroquinolone which interacts with the gyrA subunit of DNA gyrase) was tested on several archaebacteria, including five methanogens, two halobacteria, and a thermoacidophile. Most strains were sensitive to doses of coumarins (0.02 to 10 micrograms/ml) which specifically inhibit DNA gyrase in eubacteria. Ciprofloxacin inhibited growth of the haloalkaliphilic strain Natronobacterium gregoryi and of the methanogen Methanosarcina barkeri. In addition, ciprofloxacin partly relieved the sensitivity to coumarins (and vice versa). Novobiocin inhibited DNA replication in Halobacterium halobium rapidly and specifically. Topological analysis has shown that the 1.7-kilobase plasmid from Halobacterium sp. strain GRB is negatively supercoiled; this plasmid was relaxed after novobiocin treatment. These results support the existence in archaebacteria of a coumarin and quinolone target related to eubacterial DNA gyrase.

Toluene-treated Escherichia coli can synthesize DNA in the presence of precursors and ATP [Moses, R.E. & Richardson, C.C. (1970) Proc. Natl Acad. Sci. U.S.A. 67, 674--681]. The replacement of ATP by another NTP or dNTP leads to the premature arrest of the reaction. Residual synthesis in the presence of an NTP or dNTP other than ATP differs from the complete reaction in the presence of ATP because it is less sensitive to nalidixic acid and novobiocin and because its maximal activity can be obtained with lower concentrations of dNTP or shorter times of toluene treatment. However, like the complete reaction, residual synthesis occurs at the replication fork pre-existing in vivo at the time of toluenization, produces short and long pieces of DNA, is inhibited by arabinosyl-adenine triphosphate, azide or mitomycin C, and is dependent on the dnaE, DNAB and dnaG gene products. We conclude from these data that ATP is specifically required for a step in DNA replication which involves the activity of DNA gyrase, the target of nalidixic acid and novobiocin [Higgins, N.P., Peebles, C.L., Sugino, A. & Cozzarelli, N.R. (1978) Proc. Natl Acad. Sci. U.S.A. 75, 1773-1777]. In the absence of DNA gyrase activity, short DNA pieces are formed and sealed but only a limited amount of the chromosome can be replicated (residual synthesis). In the presence of DNA gyrase activity, DNA synthesis can occur on a longer portion of the chromosome (complete synthesis).

We have purified to near homogeneity a DNA polymerase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. Sodium dodecyl sulfate gel electrophoresis of the purified enzyme revealed a polypeptide of 100 kDa. On the basis of a Stokes radius of 4.2 nm and a sedimentation coefficient of 6 S, the purified enzyme has an estimated molecular mass of 109 kDa. These results are consistent with the enzyme being a monomer of 100 kDa. In addition a polyclonal antiserum, obtained by injection of the electroeluted 100-kDa polypeptide into a rabbit, specifically neutralized the DNA-polymerase activity. The enzyme is sensitive to both N-ethylmaleimide and 2',3'-dideoxyribosylthymine triphosphate and resistant to aphidicolin. The purified DNA polymerase has neither exonuclease nor primase activities. In our in vitro conditions, the enzyme is thermostable up to 80 degrees C and is active between 55 degrees C and 85 degrees C in the presence of activated calf-thymus DNA.

We have examined the replication of a dam methylated and non-methylated ori-C plasmid in an in vitro ori-C dependent replication system. The results show that the non-methylated plasmid is 50% to 80% less efficient in the initiation of DNA synthesis; that the methylation state of the plasmid does not change the site of initiation at ori-C, and that in both cases initiation at this region requires the presence of exogenously furnished dnaA protein.

Treatment of Halobacterium GRB cells with the DNA topoisomerase II inhibitor novobiocin induces the accumulation of a circular single-stranded DNA form of the plasmid pGRB-1. This form corresponds to the transcribed strand of pGRB-1. A tiny amount of this form is detectable in untreated cells. The induction of single-stranded pGRB-1 molecules by novobiocin is abolished when cells are pretreated with aphidicolin or anisomycin, which inhibit halobacterial DNA replication and protein synthesis, respectively. These results suggest that the single-stranded form of pGRB-1 is generated in the course of plasmid replication.

A topoisomerase able to introduce positive supercoils in a closed circular DNA, has been isolated from the archaebacterium Sulfolobus acidocaldarius. This enzyme, fully active at 75 degrees C, performed in vitro positive supercoiling either from negatively supercoiled, or from relaxed DNA in a catalytic reaction. In the presence of polyethylene glycol (PEG 6000), this reaction became very fast and highly processive, and the product was positively supercoiled DNA with a high superhelical density (form I+). Very low (5 - 10 micromoles) ATP concentrations were sufficient to support full supercoiling; the nonhydrolyzable analogue adenosine-5' -0-(3-thiotriphosphate) also sustained the production of positive supercoils, but to a lesser extent, suggesting that ATP hydrolysis was necessary for efficient activity. Nevertheless, low residual of positive supercoiling occurred, even in the absence of ATP, when the substrate was negatively supercoiled. Finally, the different ATP-driven topoisomerizations observed, i.e., relaxation of negative supercoils and positive supercoiling, in all cases increased the linking number of DNA in steps of 1, suggesting the action of a type I, rather than a type II topoisomerase.=

Stoichiometric amounts of pure reverse gyrase, a type I topoisomerase from the archaebacterium Sulfolobus acidocaldarius were incubated at 75 degrees C with circular DNA containing a single-chain scission. After covalent closure by a thermophilic ligase and removal of bound protein molecules, negatively supercoiled DNA was produced. This finding, obtained in the absence of ATP, contrasts with the ATP-dependent positive supercoiling catalyzed by reverse gyrase and is interpreted as the result of enzyme binding to DNA at high temperature. Another consequence of reverse gyrase stoichiometric binding to DNA is the formation of a cleavable complex which results in the production of single-strand breaks in the presence of detergent. Like eubacterial type I topoisomerase (protein omega), reverse gyrase is tightly attached to the 5' termini of the cleaved DNA. In the light of these results, a comparison is tentatively made between reverse gyrase and the eubacterial type I (omega) and type II (gyrase) topoisomerases.

We have isolated DNA polymerases and topoisomerases from two thermoacidophilic archaebacteria: Sulfolobus acidocaldarius and Thermoplasma acidophilum. The DNA polymerases are composed of a single polypeptide with molecular masses of 100 and 85 kDa, respectively. Antibodies against Sulfolobus DNA polymerase did not cross react with Thermoplasma DNA polymerase. Whereas the major DNA topoisomerase activity in S. acidocaldarius is an ATP-dependent type I DNA topoisomerase with a reverse gyrase activity, the major DNA topoisomerase activity in T. acidophilum is a ATP-independent relaxing activity. Both enzymes resemble more the eubacterial than the eukaryotic type I DNA topoisomerase. We have found that small plasmids from halobacteria are negatively supercoiled and that DNA topoisomerase II inhibitors modify their topology. This suggests the existence of an archaebacterial type II DNA topoisomerase related to its eubacterial and eukaryotic counterparts. As in eubacteria, novobiocin induces positive supercoiling of halobacterial plasmids, indicating the absence of a eukaryotic-like type I DNA topoisomerase that relaxes positive superturns.

By using hydrophobic interaction as the first chromatographic stage, we purified to homogeneity reverse gyrase, an ATP-dependent DNA topoisomerase I, isolated from the thermoacidophilic archaebacterium Sulfolobus acidocaldrius. This procedure allowed quick and complete separation of reverse gyrase from nucleases and DNA binding proteins present in Sulfolobus. The final product was revealed, by SDS-PAGE, as a unique band with an apparent molecular mass of 128 kDa, and the amino acid composition was determined. Western blotting experiments with antibodies raised against reverse gyrase indicate that no proteolysis occurred during the purification course. Gel filtration and sedimentation data gave a Stokes radius of 42 A and a sedimentation coefficient of 5.7 S, suggesting a monomeric structure for the native enzyme which was confirmed by electron microscopy. Finally, pure reverse gyrase in a monomeric state was still able to promote positive supercoiling of the DNA.

Permeability mutants of Escherichia coli have been used to prescreen antitumor drugs. However, most compounds active against eucaryotic proteins have no effect on isofunctional proteins of eubacteria. In contrast, we show that growth of halophilic archaebacteria, procaryotes as distantly related to eubacteria as to eucaryotes, is inhibited by several drugs known to interact with tubulin, actomyosin and DNA topoisomerase II of eucaryotes. Actually, different types of evidence indicate the presence of analogous proteins in halophilic archaebacteria: (a) a yeast actin probe hybridizes with DNA restriction digests of Halobacterium halobium; (b) antibodies against tubulin and actin from chicken react in a crude extract of H. halobium with polypeptides having Mr of 55,000 and 80,000, respectively; (c) the epipodophyllotoxin VP16, a eucaryotic DNA topoisomerase II inhibitor, induces DNA strand breaks with DNA-protein covalent linkage in H. halobium as in eucaryotes. Besides the evolutionary implications, these data indicate that halophilic archaebacteria can be used to prescreen antitumor drugs active on eucaryotic proteins.