Interaction of covalently closed circular PM-2 DNA and hedamycin.

The interaction of hedamycin with covalently closed circular PM-2 DNA was examined. Hedamycin produced strand breakage detectable in alkaline sucrose gradients. Under neutral conditions hedamycin inhibited ethidium bromide binding and induced conformational changes in PM-2 DNA.     

Escherichia coli DNA topoisomerase I catalyzed linking of single-stranded rings of complementary base sequences

Eco DNA topoisomerase I (E. coli omega protein) has been observed to catalyze the formation of double-stranded, covalently closed DNA from complementary single-stranded DNA rings, a novel reaction which is topologically forbidden without the enzyme-catalyzed breakage and rejoining of DNA backbone bonds. Incubation of a mixture of single-stranded PM2 DNA rings of complementary base sequences with omega yields a species with a sedimentation coefficient in an alkaline medium characteristic of a covalently closed circular double-stranded DNA. Buoyant density measurements in CsCl at alkaline pH also identify the product as a covalently closed duplex ring. If the omega-catalyzed reaction is stopped short of completion, highly negatively supercoiled molecules are formed which sediment more slowly in an alkaline medium than the final duplex product. As the reaction proceeds the mean sedimentation rate of the intermediates increases. This is in agreement with the expectation that the linking number between the two complementary rings increases gradually during the course of the reaction from zero to that of a relaxed covalently closed circular DNA duplex. The possible role of DNA topoisomerases in genetic recombination is discussed.     

Pisum sativum endonuclease. Studies on substrate specificity and possible use as a biochemical tool

An endonuclease purified from germinating pea (Pisum sativum) seeds has been shown to catalyze the hydrolysis of heat-denatured single-stranded DNA. Since P. sativum endonuclease shows appreciable activity in the presence of DNA destabilizing agents and, unlike many similar endonucleases, significant activity at neutral pH, it is a potentially valuable tool for studies of the secondary structure of nucleic acids. The residual hydrolysis of duplex DNA is directed towards partially denatured, A,T-rich areas in native DNA. The rate of hydrolysis of deoxypolynucleotides was in the order poly(dT) greater than denatured DNA greater than poly(dA) greater than poly(dA-dT) = native DNA. Neither poly(dC), poly(dG) nor poly(dC).poly(dG) were attacked by the enzyme. Supercoiled, covalently closed circular phage PM2 form I DNA is converted to singly hit nicked circular form II and doubly hit linear from III duplexes. Prolonged treatment with enzyme does not further cleave the linear form III DNA. Addition of increasing concentrations of NaCl in the incubation mixture suppresses the conversion of form I to form II, but not the conversion of form II to form III, which is enhanced with the increasing ionic strength. The enzymatically relaxed circular form, I degree, obtained by unwinding of supercoiled DNA with a DNA-relaxing protein, is resistant to the action of the enzyme. Molecules with intermediate superhelix densities do not serve as substrates. The sites of cleavage of P. sativum endonuclease in PM2 DNA occur within regions that are readily denaturable in a topologically constrained superhelical molecule.     

Photonuclease activity of Taylor's blue.

Taylor's blue (1,9-dimethylmethylene blue, DMMB+) associates with DNA, at least in part, through intercalation as is evidenced from the red shift in the absorption maximum, diminution of the fluorescence, and induced circular dichroism in the presence of nucleic acid. Irradiation of DMMB+/covalently closed circular supercoiled phiX174 phage DNA complex at lambda > 520 nm leads to DNA nicking in a dose-dependent manner.     

A new method for the purification and identification of covalently closed circular DNA molcules.

A new technique has been developed for the rapid isolation of covalently closed circular DNA molecules. The procedure is a selective extraction based on differences in the partitioning of covalently closed circular DNA molecules and noncovalently closed species between phenol and water at acid pH and low ionic strength. Under the conditions described, linear as well as nicked circular DNA is extracted into phenol, while covalently closed circular DNA molecules remain in the water phase. The method permits the quantitative isolation of covalently closed circular DNA from either total cellular DNA or partially purified preparations, to a degree of purity comparable with buoyant density procedures.     

A rapid preparation of plasmid DNA fromSaccharomyces cerevisiae

A procedure for extraction of plasmid DNA from Saccharomyces cerevisiae is described. The plasmid DNA of interest is extracted together with 2-micron circular DNA naturally occurring in many yeast strains. Spheroplasts are lyzed at alkaline pH which denatures linear but not covalently closed circular (CCC) DNA. The CCC DNA is recovered by ethanol precipitation and can be detected by gel electrophoresis or used for routine bacterial transformation.     

Characterization of mycoplasmavirus MVL2 DNA.

The size and molecular configuration of mycoplasmavirus MVL2 DNA are presented. The DNA was shown to be a covalently closed circular duplex molecule with a molecular weight of 7.4 X 10(6). In sucrose gradients at neutral pH, the form I and form II DNA molecules have sedimentation values of approximately 29S and 23S, respectively. Under alkaline conditions, the form I and form II have S values of 70 and 20, respectively.     

Isolation of plasmid deoxyribonucleic acid from two strains of Bacteroides.

Two clinical isolates of Bacteroides contained covalently closed circular deoxyribonucleic acid (DNA) as shown by sedimentation in an alkaline sucrose gradient, CsCl ethidium bromide equilibrium centrifugation, and electron microscopy. Bacteriodes fragilis N1175 contained a homogeneous species of plasmid DNA with a molecular weight of 25 x 10(6). Bacteroides ochraceus 2228 contained two distinct, covalently closed circular DNA elements. The larger cosedimented with the covalently closed circular DNA form of the R plasmid, R100, corresponding to a molecular weight of 70 x 10(6); the smaller sedimented as a 58S molecule with a calculated molecular weight of 25 x 10(6). The roles of these plasmids are unknown. Neither strain transferred antibiotic resistance to plasmid-negative Bacteroides or Escherichia coli, and neither produced bacteriocins active against other Bacteroides or sensitive indicator strains of E. coli.     

Mechanism of DNA strand breakage induced by photosensitized tetracycline-Cu(II) complex

Tetracyclines (TCs) in combination with Cu(II) ions exhibited significant DNA damaging potential vis a vis tetracyclines per se. Interaction of tetracyclines with DNA resulted in alkylation at N-7 and N-3 positions of adenine and guanine bases, and caused destabilization of DNA secondary structure. Significant release of acid-soluble nucleotides from tetracycline-modified DNA upon incubation with S(1) nuclease ascertained the formation of single stranded regions in the DNA. Also, the treatment of tetracycline-modified DNA with 0.1 and 0.5M NaOH resulted in 62 and 76% hydrolysis compared to untreated control. Comparative alkaline hydrolysis of DNA modified with tetracycline derivatives showed differential DNA damaging ability in the order as DOTC > DMTC > TC > OTC > CTC. Addition of Cu(II) invariably augmented the extent of tetracycline-induced DNA damage. The alkaline unwinding assay clearly demonstrated the formation of approximately six strand breaks per unit DNA at 1:10 DNA nucleotide/TC molar ratio in the presence of 0.1mM Cu(II) ions. At a similar Cu(II) concentration, a progressive transformation of covalently closed circular (CCC) (form-I) plasmid pBR322 DNA to forms-II and -III was noticed with increasing tetracycline concentrations. The results obtained with the free-radical quenchers viz. mannitol, thiourea, sodium benzoate and superoxide dismutase (SOD) suggested the involvement of reactive oxygen species in the DNA strand breakage. It is concluded that the tetracycline-Cu(II)-induced DNA damage occurs due to (i) significant binding of tetracycline and Cu(II) with DNA, (ii) methyl group transfer from tetracycline to the putative sites on nitrogenous bases, and (iii) metal ion catalyzed free-radical generation in close vicinity of DNA backbone upon tetracycline photosensitization. Albeit, the DNA alkylation and strand cleavage are repairable lesions, but any defect in the critical repair pathway may augment the damage accumulation and mutagenesis.     

Studies on the mechanism of DNA cleavage by ethidium.

Ethidium causes the cleavage of DNA via a light and oxygen dependent process. Using covalently closed circular DNA as a substrate, the saturation kinetics and the dependence on superhelical density of the cleavage indicate that intercalated ethidium is mainly responsible for nicking DNA. Superoxide dismutase has little effect on the reaction and catalase none. Lowering the pH inhibited the reaction. The reaction mechanism and its use in determining superhelical densities of covalently closed circular DNA's are discussed.     

Covalently closed circular duplex DNA of Epstein-Barr virus in a human lymphoid cell line.

A non-integrated form of Epstein-Barr virus DNA was purified from the Burkitt lymphoma-derived human lymphoid cell line Raji by CsCl density gradient centrifugation and neutral glycerol gradient centrifugation. This intracellular form of the virus DNA sediments at a rate typical of a covalently closed circular DNA molecule of the size of the virus genome in both neutral and alkaline solution. Treatment with low doses of X-rays leads to a discontinuous conversion of the molecules to a form with the sedimentation properties of open circular DNA (a circular duplex molecule containing one or more single-strand breaks). The direct observation of large circular DNA molecules by electron microscopy further confirms the covalently closed circular duplex structure of part of the intracellular viral DNA. Such circular molecules were not detected in corresponding DNA fractions from Epstein-Barr virus-negative human lymphoid cell lines. In ethidium bromide/CsCl density gradient centrifugation experiments, the purified non-integrated virus DNA behaves as twisted, covalently closed DNA circles with the same initial superhelix density as polyoma virus DNA. The latter additional purification technique permits the isolation of intracellular Epstein-Barr virus DNA in > 90% pure form from non-producer cells. The molecular weight of the circular virus DNA from Raji cells, determined by contour length measurements, is the same within experimental error as that of the linear DNA from virus particles.     

Amplification of closed circular DNA in vitro.

The polymerase chain reaction is a powerful technique used to amplify nucleic acids in vitro . The reaction produces linear products, and as of yet, closed circular products have not been possible. Since the replicatively competent form of many DNA molecules is the closed circular form, it would be adventitious to amplify closed circular DNA as closed circular molecules. Until now, these molecules could only be amplified in vivo in appropriate host cells. Here, we describe an in vitro procedure, ligation-during-amplification (LDA), for selective amplification of closed circular DNA using sequence-specific primers. LDA is useful for site-directed mutagenesis, mutation detection, DNA modification, DNA library screening and circular DNA production.     

Ethidium bromide-mediated renaturation of denatured closed circular DNAs. The nature of denaturation-resistant fractions of bacteriophage PM2 closed circular DNA

Addition of the intercalating dye ethidium bromide (EtdBr) to a solution of alkali-denatured double-stranded closed circular PM2, ΦX174, or λb₂b₅c phage DNAs, under conditions such that the solution remains strongly alkaline, can result in the renaturation of up to 100% of the DNA upon neutralization of the solution. For a fixed time of incubation of the alkaline dye-containing solution before neutralization, there exists a minimum concentration of the dye below which no EtdBr-mediated renaturation is observed for each species of closed circular DNA examined. These minimum concentrations increase, for a given DNA, with increasing ionic strength and temperature. The kinetics of accumulation of forms renaturing upon neutralization of alkaline solutions, at fixed concentrations of dye and DNA, are dependent upon the molecular weight and superhelix density of the starting DNA. After extended periods of incubation at a fixed ionic strength and temperature, however, the profiles of percentage of DNA renatured as a function of ethidium concentration become very similar for all the closed circular DNAs tested and display a transition from an absence of dye-mediated renaturation to virtually 100% renaturation upon neutralization over a small range of dye concentration. Circular DNA containing one or more strand scissions remains strand-separated under all the conditions used to effect the renaturation of closed circular DNA. These findings indicate that configurations of closed circular DNA, in which at least some of the complementary bases are apposed, can be selectively stabilized and accumulate in the presence of ethidium in solutions containing 0.19 N hydroxide ion.     

Degradation of DNA in cells and extracts of the obligately anaerobic bacterium Roseburia cecicola upon exposure to air.

High-molecular-weight chromosomal DNA from Roseburia cecicola, an oxygen-intolerant anaerobe, could be isolated only when the bacterial cells were kept under anaerobic conditions up to the time of cell lysis. When the cells were exposed to oxygen before lysis, the chromosomal DNA degraded. Likewise, linear but not covalently closed circular DNAs degraded in cell extracts of the organism that were exposed to atmospheres containing O2 but not in extracts that were maintained in a reduced state. Covalently closed circular DNAs were nicked but not degraded in the oxidized extracts.     

Degradation of DNA in cells and extracts of the obligately anaerobic bacterium Roseburia cecicola upon exposure to air

High-molecular-weight chromosomal DNA from Roseburia cecicola, an oxygen-intolerant anaerobe, could be isolated only when the bacterial cells were kept under anaerobic conditions up to the time of cell lysis. When the cells were exposed to oxygen before lysis, the chromosomal DNA degraded. Likewise, linear but not covalently closed circular DNAs degraded in cell extracts of the organism that were exposed to atmospheres containing O2 but not in extracts that were maintained in a reduced state. Covalently closed circular DNAs were nicked but not degraded in the oxidized extracts.     

Catenation of DNA by eucaryotic topoisomerase II associated with simian virus 40 minichromosomes

After incubation of purified SV40 minichromosomes with superhelical DNA molecules either of SV40 or plasmid origin, a catenation of monomeric DNA via dimers and multimers to large networks was observed. The catenation reaction was stimulated by the DNA condensing agent spermidine with ATP as an energy donor and was dependent on the presence of magnesium ions. The reaction could be blocked by inhibitors of topoisomerase II such as novobiocin and nalidixic acid. Relaxed covalently closed circular DNA was catenated to networks in the presence of ATP as the energy donor.     

Extracellular nucleases of Pseudomonas BAL 31. I. Characterization of single strand-specific deoxyriboendonuclease and double-strand deoxyriboexonuclease activities.

The culture medium of Pseudomonas BAL 31 contains endonuclease activities which are highly specific for single-stranged DNA and for the single-stranded or weakly hydrogen-bonded regions in supercoiled closed circular DNA. Exposure of nicked DNA to the culture medium results in cleavage of the strang opposite the sites of preexisting single-strand scissions. At least some of the linear duplex molecules derived by cleavage of supercoiled closed circular molecules contain short single-stranded ends. Single-strand scissions are not introduced into intact, linear duplex DNA or unsupercoiled covalently closed circular DNA. Under these same reaction conditions, 0X174 phage DNA is extensively degraded and PM2 form I DNA is quantitatively converted to PM2 form III linear duplexes. Prolonged exposure of this linear duplex DNA to the concentrated culture medium reveals the presence of a double-strand exonuclease activity that progressively reduces the average length of the linear duplex. These nuclease activities persist at ionic strengths up to 4 M and are not eliminated in the presence of 5% sodium dodecyl sulfate. Calcium and magnesium ion are both required for optimal activity. Although the absence of magnesium ion reduces the activities, the absence of calcium ion irreversibly eliminates all the activities.     

Spectrofluorometry of dyes with DNAs of different base composition and conformation

The increase in fluorescence, upon interaction with several fluorescent dyes was found to depend on the base composition of DNA. 4',6-Diamidino-2-phenylindole-2 HCl and Hoechst 33258 which bind to AT base pairs show a logarithmic relation. This relation is linear when DNAs interact with mithramycin, chromomycin A3, and olivomycin, which bind to GC base pairs. Deviations from these relationships were observed for T2 DNA, containing hydroxymethylcytosine, and for 2C DNA, containing hydroxymethyluracil. On the basis of these data, a simple technique is proposed for determination of base composition. The presence of abnormal bases can be monitored by the use of given fluorophores. Fluorescence intensities were not modified upon linearization of covalently closed circular plasmid pBR322. Denaturation of lambda DNA was accompanied by a decrease of fluorescence, when complexed with the five dyes tested.