Effects of the antitumor drug VP16 (etoposide) on the archaebacterial Halobacterium GRB 1.7 kb plasmid in vivo.

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.     

Coumarin and quinolone action in archaebacteria: evidence for the presence of a DNA gyrase-like enzyme.

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.     

Ciprofloxacin and etoposide (VP16) produce a similar pattern of DNA cleavage in a plasmid of an archaebacterium

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.     

Novobiocin induces accumulation of a single strand of plasmid pGRB-1 in the archaebacterium Halobacterium GRB.

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.     

DNA gyrase can supercoil DNA circles as small as 174 base pairs.

DNA gyrase introduces negative supercoils into closed-circular DNA using the free energy of ATP hydrolysis. Consideration of steric and thermodynamic aspects of the supercoiling reaction indicates that there should be a lower limit to the size of DNA circle which can be supercoiled by gyrase. We have investigated the supercoiling reaction of circles from 116-427 base pairs (bp) in size and have determined that gyrase can supercoil certain relaxed isomers of circles as small as 174 bp, dependent on the final superhelix density of the supercoiled product. Furthermore, this limiting superhelical density (-0.11) is the same as that determined for the supercoiling of plasmid pBR322. We also find that although circles in the range 116-152 bp cannot be supercoiled, they can nevertheless be relaxed by gyrase when positively supercoiled. These data suggest that the conformational changes associated with the supercoiling reaction can be carried out by gyrase in a circle as small as 116 bp. We discuss these results with respect to the thermodynamics of DNA supercoiling and steric aspects of the gyrase mechanism.     

DNA topology in hyperthermophilic archaea: reference states and their variation with growth phase, growth temperature, and temperature stresses

In order to address the dynamics of DNA topology in hyperthermophilic archaea, we analysed the topological state of several plasmids recently discovered in Thermococcales and Sulfolobales. All of these plasmids were from relaxed to highly positively super-coiled in vitro, i.e. they exhibited a significant linking excess compared to the negatively supercoiled plasmids from mesophilic organisms (both Archaea and Bacteria). In the two archaeai orders, plasmid linking number (Lk) decreased as growth temperature was lowered from its optimal value, i.e. positively super-coiled plasmids were relaxed whereas relaxed plasmids became negatively supercoiled. Growth temperatures above the optimum correlated with higher positive supercoiling in Sulfolobales (Lk increase) but with relaxation of positive supercoils in Thermococcus sp. GE31. The topological variation of plasmid DNA isolated from cells at different growth phases were found to be species specific in both archaeai orders. In contrast, the direction of topological variation under temperature stress was the same, i.e. a heat shock correlated with an increase in plasmid positive supercoiling, whilst a cold shock induced negative supercoiling. The kinetics of these effects were analysed in Sulfolobales. In both temperature upshift (from 80 to 85C) and downshift (from 80 to 65C), a transient sharp variation of Lk occurred first, and then DNA supercoiling progressively reached levels typical of steady-state growth at the final temperature. These results indicate that DNA topology can change with physiological states and environmental modifications in hyperthermophilic archaea.     

DNA supercoiling and thermal regulation of unsaturated fatty acid synthesis in Bacillus subtilis

Bacillus subtilis growing at 37° C synthesizes, almost exclusively, saturated fatty acids. However, when a culture growing at 37°C is transferred to 20°C, the synthesis of unsaturated fatty acids is induced. The addition of the DNA gyrase inhibitor novobiocin specifically prevented the induction of unsaturated fatty acid synthesis at 20° C. Furthermore, it was determined that plasmid DNA isolated from cells growing at 20°C was significantly more negatively supercoiled than the equivalent DNA isolated from cells growing at 37°C. The overall results agree with the hypothesis that an increase in DNA supercoiling associated with a temperature downshift could regulate the unsaturated fatty acids synthesis in B. subtilis.     

Identification of a DNA supercoiling activity in Saccharomyces cerevisiae.

A relaxed plasmid DNA is shown to become positively supercoiled in cell extracts from top1 strains of Saccharomyces cerevisiae. This positive supercoiling activity is dependent on the presence of bacterial DNA topoisomerase I and ATP (or dATP), and the positive supercoils generated in this reaction are not constrained by protein(s). Non-hydrolyzable ATP analogs cannot substitute for ATP in this supercoiling reaction, and the supercoiling activity is not due to RNA synthesis. The presence of an ARS sequence in the DNA does not alter the activity. Furthermore, this activity is equally active against UV irradiated or intact DNA. Extracts prepared from rad50 and rad52 mutant cells exhibited the same activity. Partial purification of this activity suggests that a protein factor with a native molecular weight of approximately 150 kDa is primarily responsible for the activity. The possibility that this supercoiling activity may be due to tracking of a protein along the intact duplex DNA is discussed.     

Plasmid migration using orthogonal-field-alternation gel electrophoresis.

The migration properties of a series of supercoiled plasmids ranging in size from 4 to 16 kilobases (kb) have been analyzed by orthogonal-field-alternation gel electrophoresis (OFAGE). These circular DNAs enter the gel and are well resolved. Unlike linear DNA molecules, the relative mobilities of these plasmids are constant over a wide range of pulse times, from 10 to 120 seconds, as well as over a broad range of total running times, from 6 to 24 hours. Electrophoresis of supercoiled, relaxed, and nicked open circular forms as well as topoisomers of pBR322 shows that the extent of supercoiling has a dramatic effect on plasmid migration on OFAGE. Several practical applications for exploiting the different migration properties of circular and linear DNA molecules on OFAGE are presented.     

DNA topoisomerase I mutants. Increased heterogeneity in linking number and other replicon-dependent changes in DNA supercoiling

Plasmid pBR322 DNA isolated from Salmonella typhimurium supX (topoisomerase I) mutants exhibits a novel supercoiling distribution characterized by extreme heterogeneity in linking number and the presence of highly negatively supercoiled topoisomers. The most negatively supercoiled topoisomers isolated from one supX mutant have more than twice the wild-type level of supercoiling; the distribution as a whole has a median superhelix density about 1.3 times that of wild type. Surprisingly, the supercoiling distribution of plasmid pUC9 DNA isolated from supX mutants differs from that of pBR322. Escherichia coli topoisomerase I mutants have been shown to acquire compensatory mutations that reduce bacterial chromosome supercoiling to below the wild-type level even in the absence of topoisomerase I. We find that such a compensatory mutation in an E. coli topoisomerase I deletion mutant does not reduce pBR322 DNA supercoiling to a level below that of wild type. Thus, the effects of topoisomerase mutations on supercoiling depend on the replicon.     

Reverse gyrase functions as a DNA renaturase: annealing of complementary single-stranded circles and positive supercoiling of a bubble substrate

Reverse gyrase is a hyperthermophile-specific enzyme that can positively supercoil DNA concomitant with ATP hydrolysis. However, the DNA supercoiling activity is inefficient and requires an excess amount of enzyme relative to DNA. We report here several activities that reverse gyrase can efficiently mediate with a substoichiometric amount of enzyme. In the presence of a nucleotide cofactor, reverse gyrase can readily relax negative supercoils, but not the positive ones, from a plasmid DNA substrate. Reverse gyrase can completely relax positively supercoiled DNA, provided that the DNA substrate contains a single-stranded bubble. Reverse gyrase efficiently anneals complementary single-stranded circles. A substoichiometric amount of reverse gyrase can insert positive supercoils into DNA with a single-stranded bubble, in contrast to plasmid DNA substrate. We have designed a novel method based on phage-mid DNA vectors to prepare a circular DNA substrate containing a single-stranded bubble with defined length and sequence. With these bubble DNA substrates, we demonstrated that efficient positive supercoiling by reverse gyrase requires a bubble size larger than 20 nucleotides. The activities of annealing single-stranded DNA circles and positive supercoiling of bubble substrate demonstrate that reverse gyrase can function as a DNA renaturase. These biochemical activities also suggest that reverse gyrase can have an important biological function in sensing and eliminating unpaired regions in the genome of a hyperthermophilic organism.     

Large scale preparation of positively supercoiled DNA using the archaeal histone HMf.

A technique to prepare relatively large quantities (>/=100 microg) of highly positively supercoiled DNA is reported. This uses a recombinant archaeal histone (rHMfB) to introduce toroidal supercoils, and an inexpensive chicken blood extract to relax unrestrained superhelical tension. Preparation of positively supercoiled pUC19 DNA molecules, >50% of which have linking number changes ranging from+8 to+17, is demonstrated. Advantages include the high degree of positive supercoiling that can be achieved, control over the extent of supercoiling, easy production of relatively large quantities of supercoiled DNA, and low cost.     

Internal Dynamics of Supercoiled DNA Molecules

The intramolecular diffusive motion within supercoiled DNA molecules is of central importance for a wide array of gene regulation processes. It has recently been shown, using fluorescence correlation spectroscopy, that plasmid DNA exhibits unexpected acceleration of its internal diffusive motion upon supercoiling to intermediate density. Here, we present an independent study that shows a similar acceleration for fully supercoiled plasmid DNA. We have developed a method that allows fluorescent labeling of a 200-bp region, as well as efficient supercoiling by Escherichia coli gyrase. Compared to plain circular or linear DNA, the submicrosecond motion within the supercoiled molecules appears faster by up to an order of magnitude. The mean-square displacement as a function of time reveals an additional intermediate regime with a lowered scaling exponent compared to that of circular DNA. Although this unexpected behavior is not fully understood, it could be explained by conformational constraints of the DNA strand within the supercoiled topology in combination with an increased apparent persistence length.     

Raman spectroscopy of supercoiled and nicked ColE1 plasmid

Native supercoiled and nicked ColE1 DNA were examined using laser Raman spectroscopy. ColE1 contains 6646 base pairs (bp) and, when supercoiled, approximately 47 negative supercoils. An analytical buoyant density gradient centrifugation technique developed by Burke and Bauer was scaled to preparative quantities, and used to isolate the supercoiled plasmid fraction from its nicked counterpart. This procedure allowed enriched fractions of the supercoiled plasmid to be extracted without the use of the optical contaminant ethidium bromide. The intensities of several Raman bands were altered between the spectra of the two topological forms. Notably absent were any changes in bands arising from cytosine and guanine vibrations. The observed changes are interpreted in terms of the polymorphic structures which have been observed in many DNA structural studies. The results of this study suggest that accommodation of supercoiling takes place chiefly in A-T base pairs and backbone moieties, without substantial modification of G-C base-pair structure. Premelting effects may account for the observed changes, including a slight shift to lower frequency of a band known to be responsive to base-pair disruption. Heteronomous ribose sugar pucker is evident in both supercoiled and nicked plasmid species. No gross conformational transitions were detected for native supercoiled DNA, and consequently, subtle rearrangements appear sufficient to absorb the supercoiling deformations.