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PubMed1978

Defective herpes simplex virus DNA: circular and circular-linear molecules resembling rolling circles.

The formation of defective herpes simplex virus (HSV) in BSC-1 cells and the synthesis of defective virus DNA was studied. The fourth consecutive passage of undiluted virus yielded defective DNA that was 0.008 g/ml more dense than wild type (w.t.) virus DNA. The amount of defective DNA increased at passage 6 concomitantly with the decrease in infectious virus progeny. The synthesis of defective DNA was always accompanied by w.t. virus DNA synthesis. Defective DNA from both infected nuclei and defective virions had a mol. wt. of 100 X 10(6) and was linear as determined by electron microscopy. Electron microscopy of defective virus DNA at passage 6 revealed circular molecules varying in size in addition to linear DNA molecules with the length of intact virion DNA. The circular DNA molecules had contour lengths of 10, 5, 2.5 and less than 2.5 micron. The smallest circular DNA molecules had a contour length of 0.3 micron, possibly one virus gene. In addition, circular-linear DNA molecules were observed in which both the circular and the linear components varied in length. Most of these DNA molecules had circular components of either 2.5 or 5.0 micron, and linear components varying in length from less than 1 to 50 micron. Based on the present study, it is proposed that the S component of w.t. virus DNA is fragmented into small circular molecules that serve as templates for DNA synthesis, possibly by the rolling circle mechanism.

Cell Line↗
PubMed1992

Closed circular DNA as a probe for protein-induced structural changes.

Biological systems are replete with examples of DNA formed into a closed loop structure, either alone or in close association with proteins. Such closed circular DNA molecules are subject to a topological constraint that modifies, often in a major way, the structure and reactivity of the DNA. The topological constraint also permits closed circular DNAs to be used as analytical tools to learn about the structure of DNA-protein complexes.

DNA Probes↗
PubMed1983

The unwinding of circular DNA by intercalating agents as determined by gel electrophoresis.

The conventional counting of electrophoretically resolvable topoisomers is an attractive technique for determining the number of superhelical turns in a closed circular DNA molecule. The method can be extended in order to determine the unwinding produced by a drug, if its binding constants are known under similar environmental conditions. Ethidium bromide was found to unwind a DNA molecule derived from the plasmid pBR322 by 26.0 degrees in a magnesium-containing buffer. The method is convenient for investigating the possible effects of different environmental changes (such as ionic strength, ionic species, or temperature) on the unwinding angle produced by a particular drug. It can also give an early indication of multiple modes of binding.

Animals↗
PubMed1993

Characterization of human bone marrow-derived closed circular DNA clones.

Because of interest in mechanisms of recombination involved in chromosomal deletions in neoplastic disease, and their relation to possible rearrangements in normal tissues, we are studying circular DNA molecules from human tissue with a long-term goal of investigating them as possible by-products of physiologically relevant intrachromosomal recombination events. Covalently closed circular (ccc) DNA from human bone marrow was cloned in bacteriophage vectors, and fourteen clones chosen randomly from the cccDNA-derived library were characterized. Five clones originated from chromosome-specific centromeric alpha-satellite DNA; two clones carried highly repetitive sequences probably derived from interspersed repetitive elements; six clones were derived from single-copy chromosome-specific sequences which detected homologous rodent sequences; and one clone (EPM10) was derived from a small chromosome 11-specific sequence family which localized to chromosome regions 11cen and 11q14. Oligonucleotide primers derived from the cccDNA clones were used in polymerase chain reaction studies to show that (1) the EPM10 clone carried the circular junction, (2) several of the single-copy products could be detected in three different bone marrow cccDNA preparations, and (3) the Alu-PCR profile for bone marrow cccDNA showed distinct bands which were similar in four bone marrow cccDNA preparations.

Acute Disease↗
PubMed1988

Large-scale isolation of covalently closed circular DNA using gel filtration chromatography.

The isolation of covalently closed circular (ccc) DNA free of contamination by RNA and other forms of DNA is fundamental to molecular biology. A variety of methods have been explored but CsCl density-gradient centrifugation remains the method most widely used for preparative scale resolution. The process is expensive, time-consuming, requires the use of large amounts of the carcinogen ethidium bromide, and is subject to considerable variation in yield and purity. To avoid these problems, we have devised a procedure for the preparation of cell lysates which results in consistently good yields of biologically active ccc DNA minimally contaminated with chromosomal DNA fragments and RNA. Lysates are deproteinized, precipitated with CaCl2 to remove rRNA, concentrated by ethanol precipitation, and applied to a Sephacryl S-1000 column which resolves chromosomal fragments, open circular plasmid DNA, and residual RNA from the ccc DNA. We have found that substituting the gel filtration column for CsCl density-gradient centrifugation results in substantially better purification as well as reducing processing time, cost, and degree of difficulty. The time required from harvest of cells to final recovery of DNA is about 16 h. We have used the method to isolate plasmids from 4.4 to 12 kb and, with slight modifications, recombinant M13 replicative form DNAs.

Acrylic Resins↗
PubMed2005

Proximity extension of circular DNA aptamers with real-time protein detection.

Multivalent circular aptamers or 'captamers' have recently been introduced through the merger of aptameric recognition functions with the basic principles of DNA nanotechnology. Aptamers have strong utility as protein-binding motifs for diagnostic applications, where their ease of discovery, thermal stability and low cost make them ideal components for incorporation into targeted protein assays. Here we report upon a property specific to circular DNA aptamers: their intrinsic compatibility with a highly sensitive protein detection method termed the 'proximity extension' assay. The circular DNA architecture facilitates the integration of multiple functional elements into a single molecule: aptameric target recognition, nucleic acid hybridization specificity and rolling circle amplification. Successful exploitation of these properties is demonstrated for the molecular analysis of thrombin, with the assay delivering a detection limit nearly three orders of magnitude below the dissociation constants of the two contributing aptamer-thrombin interactions. Real-time signal amplification and detection under isothermal conditions points towards potential clinical applications, with both fluorescent and bioelectronic methods of detection achieved. This application elaborates the pleiotropic properties of circular DNA aptamers beyond the stability, potency and multitargeting characteristics described earlier.

Aptamers, Nucleotide↗
PubMed1978

A rapid method for the isolation of circular DNA using an aqueous two-phase partition system.

A method of isolating circular plasmid DNA from cleared lysates of E. coli is described. Purification is achieved by virtue of the rapid re-annealing kinetics or supercoiled DNA. After a brief denaturation step, double stranded plasmid DNA is separated from denatured chromosomal DNA and RNA in a two-phase partition system using dextran and polyethylene glycol. The method is much more rapid than the conventional dye-centrifugation technique and plasmid DNA of comparable purity and yield is obtained.

DNA, Bacterial↗
PubMed2000

Atomic force and electron microscopy of high molecular weight circular DNA complexes with synthetic oligopeptide trivaline.

Intramolecular compact structures formed by high molecular weight circular superhelical DNA molecules due to interaction with synthetic oligopeptide trivaline (1) were studied by atomic force and electron microscopy. Three DNA preparations were used: plasmids pTbol, pRX10 and cosmid 27,877, with sizes 6,120 bp, 10,500 bp and 44,890 bp respectively. Plasmid pTbo1 and pRX10 preparations along with monomers contained significant amount of dimers and trimers. Main structures in all preparations observed were compact particles, which coincide in their appearance and compaction coefficient (3,5-3,7) with triple rings described earlier. The size and structure characteristics of triple rings and other compact particles on atomic force images in general coincide with those obtained by EM (2). AFM (3) images allow to get additional information about the ultrastructural organization and arrangement of DNA fibers within the compact structures. Along with triple rings in pTbol and pRX10-TVP complexes significant amount of compact structures were observed having the shape of two or three compact rings attached to each other by a region of compact fibre. Basing on the data of contour length measurements and the shape of the particles it was concluded that these structures were formed due to compaction of dimeric and trimeric circular DNA molecules. Structures consisting of several attached to each other triple rings were not found for pTbol, pRX10 monomers or cosmid preparations--TVP complexes where only single triple rings were observed. The conclusion is made that initiation of compact fibre formation within the circular molecules depends on the primary structure and for dimeric or trimeric circular molecules two or three compaction initiation points are present, located in each monomer unit within one circular DNA molecule. The nucleotide sequence dependent compaction mechanism providing independent compaction of portions of one circular molecule can be of interest for understanding of DNA compaction processes in vivo.

Cosmids↗
PubMed1997

Kinetic analysis of drug cleavage of closed-circular DNA.

Various cleavage agents interact with circular double-stranded DNA molecules to convert the closed-circular form (form-I) to the open-circular (form-II) and linear (form-III) forms, and ultimately to small DNA fragments. The various cutting processes which take place in the DNA pool are here analyzed kinetically, and, by solving the kinetic equations, expressions are derived for the amounts of the closed-circular, open-circular, and linear forms of DNA as a function of reaction time and concentration of cleavage agent. Conversions between subspecies of forms II and III, differing in numbers of internal cuts, are taken into account. The only assumption required to solve the kinetic equations is that the concentration of cleavage agent obeys [D] = D0 f(t) where the function of time f(t) is independent of D0, the initial concentration of cleavage agent. By choosing parameters in the expressions for the calculated amounts of forms I, II and III to give the best fit to the measured amounts, one obtains information about the rates and rate constants for the conversions. The rate constants in turn give important information about the specificity and mechanism of action of the cleavage agents. The analysis is applied to the cleavage of pBR322, SV-40 and PM2 DNAs by DNase I, Fe-EDTA, and the antitumor agents calicheamicin and bleomycin. Cleavage rate constants are derived and discussed for these systems.

Antibiotics, Antineoplastic↗
PubMed2004

Localization and dynamics of small circular DNA in live mammalian nuclei.

While genomic DNA, packaged into chromatin, is known to be locally constrained but highly dynamic in the nuclei of living cells, little is known about the localization and dynamics of small circular DNA molecules that invade cells by virus infection, application of gene therapy vectors or experimental transfection. To address this point, we have created traceable model substrates by direct labeling of plasmid DNA with fluorescent peptide nucleic acids, and have investigated their fate after microinjection into living cells. Here, we report that foreign DNA rapidly undergoes interactions with intranuclear structural sites that strongly reduce its mobility and restrict the DNA to regions excluding nucleoli and nuclear bodies such as PML bodies. The labeled plasmids partially co-localize with SAF-A, a well characterized marker protein for the nuclear 'scaffold' or 'matrix', and are resistant towards extraction by detergent and, in part, elevated salt concentrations. We show that the localization and the low mobility of plasmids is independent of the plasmid sequence, and does not require the presence of either a scaffold attachment region (SAR) DNA element or a functional promoter.

Animals↗
PubMed1980

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, PhiX174, or lambdab(2)b(5)c 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. The closed circular DNA of bacteriophage PM2 has properties distinct from those of the other DNAs of this study in that it has been shown to contain fractions which exist in the base-paired duplex form after neutralization of strongly alkaline solutions of this DNA incubated at ambient temperature, while no duplex DNA is observed after exposure to alkali and neutralization of solutions of closed circular DNA from other sources. (1,2) The fraction of denaturation-resistant PM2 DNA is shown in the present work to depend upon the temperature and time of incubation in alkali, but not upon the superhelix density of the starting DNA. PM2 closed circular DNA also behaves anomalously with respect to its kinetics of accumulation of forms renaturing upon neutralization of alkaline, EtdBr-containing solutions. Evidence is presented that the translocation of one of the strands of a closed circular molecule relative to the other, which is required for the molecule to exist in the denatured form at neutral pH, is a process to which PM2 DNA is less labile than the other closed circular DNAs of this study.

Bacteriophages↗
PubMed1982

Ethidium-bromide-inhibited restriction endonucleases cleave one strand of circular DNA.

We analyzed the effect of ethidium bromide (EtBr) on the cleavage of closed circular pBR322 DNA molecules by six restriction enzymes which make staggered or flush cuts (EcoRI, HindIII, BglI, PstI, HincII, PvuII). EtBr concentrations and reaction temperatures were determined at which DNA molecules with single-strand breaks were the major reaction product of digestion by all the enzymes. However, the amounts of intermediates which could be isolated differed for various enzymes. The results extend previous studies, showing that sequential cleavage of the DNA strands probably is a general property of restriction endonucleases.

DNA Restriction Enzymes↗
PubMed1998

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.

DNA Ligases↗
PubMed1998

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 vito 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.

DNA Ligases↗
PubMed1973

Isolation of circular DNA molecules from whole cellular DNA by use of ATP-dependent deoxyribonuclease.

A technique is described for isolation of plasmid DNA in closed and open circular double-stranded forms from bacterial cells, by use of ATP-dependent deoxyribonuclease purified from Micrococcus luteus. This DNase, acting only upon linear DNA molecules, degrades all bacterial chromosomal DNA extracted in the linear form. Circular plasmid DNAs are left intact, and are then separated by sedimentation through a sucrose gradient. Unlike previous techniques for analysis of plasmid DNA, this technique can be used to isolate not only closed circular DNA but also open circular DNA. Several plasmids, such as those from phage (lambdadv1 and lambdadv21), a colicinogenic factor (Col E2), a sex factor (F(8)' gal), and "minicircles" in Escherichia coli 15, in both the open and closed circular forms, were well separated from chromosomal DNA by this technique.

Adenosine Triphosphate↗
PubMed1976

Circular DNA of a yeast episome with two inverted repeats: structural analysis by a restriction enzyme and electron microscopy.

Small circular DNA molecules from genetically characterized clones of Saccharomyces cerevisiae have been studied by restriction endonuclease analysis and electron microscopy. The circular monomers (6000 bases) are shown to contain two inverted repeats of the same sequence (600 bases) situated opposite each other along the perimeter. Four endonuclease EcoRI fragments are obtained in 1:1:1:1 stoichiometry, and their sum gives a length of about 12,000 bases. The two large fragments and the two small ones differ from each other by 200 bases. We propose a model for the structure of the monomer molecule. Two classes of monomers can be generated by intramolecular recombinations within inverted repeats; they differ by the relative orientation of nonrepeated segments. The structure of dimers as predicted by the model is verified by self-renaturation of single-stranded circles. Inverted repeats in circular molecules may be related to the insertion release faculty of II episome in the chromosomes.

Base Sequence↗