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.     

DNA circles with cruciforms from Isospora (Toxoplasma) gondii

We have isolated a closed circular duplex DNA fraction from the unicellular parasite Isospora (Toxoplasma) gondii and examined the purified DNA by electron microscopy. A major part of this circular DNA consists of 12-micron circles containing a cruciform with 0.5-micron tails. We also found 23-micron circles with the properties expected of head-to-tail dimers of the 12-micron circles. Some of these dimers have two cruciforms with 0.4-micron tails, some have one cruciform with 0.8-micron tails. When ethidium bromide was diffused into the DNA solution, circles with tails were replaced by twisted circles without tails. Direct mixing of the DNA with high ethidium bromide concentrations (5 micrograms/ml) gave rise to highly twisted circles with tails. This proves that the tailed circles are covalently continuous and indicates that ethidium bromide blocks branch migration. The 0.5-micron tails are part of a 1.7-micron palindrome, which was visualized by spreading denatured DNA under snap-back conditions. We argue that the cruciform is not present in vivo and that the 12-micron circles may represent the mitochondrial DNA of Toxoplasma.     

A procedure for the quantitation of relaxed closed circular DNA in the presence of superhelical DNA: an improved fluorometric assay for nicking-closing enzyme.

The procedure described here takes advantage of the recently discovered single-strand-specific endonuclease activity of snake-venom phosphodiesterase to convert supercoiled PM2 DNA (DNA I′), but not relaxed DNA (DNA I′), to open forms of DNA. The DNA I' was quantitated using a fiuorometric method for covalently closed circular DNA (A. R. Morgan and D. E. Pulleyblank, 1974, Biochem. Biophys. Res. Commun.61, 396–403). The percentage of DNA I′ in mixtures of DNAs I and I′ can be determined to ±l%. The procedure was used as an assay for a nicking-closing enzyme activity partially purified from simian virus 40-infected monkey cells. The assay is linear from 0 to 0.4 μg DNA I′ produced and reproducible to ±0.01 μg DNA I′.     

Electron microscopic studies of bacteriophage M13 DNA replication.

Intracellular forms of M13 phage DNA isolated after infection of Escherichia coli with wild-type phage have been studied by electron microscopy and ultracentrifugation. The data indicate the involvement of rolling-circle intermediates in single-stranded DNA synthesis. In addition to single-stranded circular DNA, we observed covalently closed and nicked replicative-form (RF) DNAs, dimer RF DNAs, concatenated RF DNAs, RF DNAs with single-stranded tails (theta, rolling circles), and, occasionally, RF DNAs with theta structures. The tails in theta molecules are always single stranded and are never longer than the DNA from mature phage; the proportion of theta to other RF molecules does not change significantly with time after infection. The origin of single-stranded DNA synthesis has been mapped by electron microscopy at a unique location on RF DNA by use of partial denaturation mapping and restriction endonuclease digestion. This location is between gene IV and gene II, and synthesis proceeds in a counterclockwise direction on the conventional genetic map.     

Covalently closed circles of human adenovirus DNA are infectious

Replication of the linear adenovirus DNA molecule is thought to result from semiconservative synthesis off linear templates, starting from origins at either end of the genome. Recently, however, it has been shown that in cells infected with adenovirus type 5 (Ad5) a significant fraction of the ends of viral DNA molecules become joined head-to-tail due at least in part to the formation of covalently closed circles. Circular DNA is not present in virions but joining of the ends of viral DNA is detectable shortly after infection, well before the onset of viral DNA replication. To learn more about the structure and possible function of these circular forms of viral DNA, I have cloned Ad5 circles as plasmids replicating in Escherichia coli. Two plasmids have been analyzed in detail and shown to generate infectious virus with an efficiency comparable with that of virion DNA following transfection into human cells. These results suggest that circles are not totally inert or functionless but that, once formed, they are capable of re-entering the pool of replicating molecules to generate linear progeny.     

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.     

KINETOPLAST DEOXYRIBONUCLEIC ACID OF THE HEMOFLAGELLATE TRYPANOSOMA LEWISI

Cesium chloride centrifugation of DNA extracted from cells of blood strain Trypanosoma lewisi revealed a main band, ρ = 1.707, a light satellite, ρ = 1.699, and a heavy satellite, ρ = 1.721. Culture strain T. lewisi DNA comprised only a main band, ρ = 1.711, and a light satellite, ρ = 1.699. DNA isolated from DNase-treated kinetoplast fractions of both the blood and culture strains consisted of only the light satellite DNA. Electron microscope examination of rotary shadowed preparations of lysates revealed that DNA from kinetoplast fractions was mainly in the form of single 0.4 µ circular molecules and large masses of 0.4 µ interlocked circles with which longer, often noncircular molecules were associated. The 0.4 µ circular molecules were mainly in the covalently closed form: they showed a high degree of resistance to thermal denaturation which was lost following sonication; and they banded at a greater density than linear DNA in cesium chloride-ethidium bromide gradients. Interpretation of the large masses of DNA as comprising interlocked covalently closed 0.4 µ circles was supported by the findings that they banded with single circular molecules in cesium chloride-ethidium bromide gradients, and following breakage of some circles by mild sonication, they disappeared and were replaced by molecules made up of low numbers of apparently interlocked 0.4 µ circles. When culture strain cells were grown in the presence of either ethidium bromide or acriflavin, there was a loss of stainable kinetoplast DNA in cytological preparations. There was a parallel loss of light satellite and of circular molecules from DNA extracted from these cells.     

Retention of helical structure of pBR322 covalently closed circular duplex DNA at high alkaline pH

Denaturation of covalently closed circular duplex replicative form (RF) I at high pH yields a form with high sedimentation coefficient even after neutralization. This form allowed less ethidium bromide to be intercalated but yielded a circular dichroic spectrum which had reduced magnitude of both positive circular dichroism at 273 nm and negative circular dichroism at 245 nm. The circular dichroic spectrum of this form is similar to that of RFV DNA. Gel electrophoretic analysis of this DNA revealed that, although part is retained in the groove, another part appeared as a faster-moving band, which we designated as RF I_d. This faster-moving form is cleaved by the restriction endonuclease BamHI at a single site giving a single RF III, comigrating with the RF III obtained from RF I by BamHI cleavage. This signifies that the two strands of RF I did not slide over one another during the formation of RF I_d as suggested previously.     

Enzymatic end-to end joining of DNA molecules

A way to join naturally occurring DNA molecules, independent of their base sequence, is proposed, based upon the presumed ability of the calf thymus enzyme terminal deoxynucleotidyltransferase to add homopolymer blocks to the ends of double-stranded DNA. To test the proposal, covalently closed dimer circles of the DNA of bacteriophage P22 were produced from linear monomers. It is found that P22 DNA as isolated will prime the terminal transferase reaction, but not in a satisfactory manner. Pre-treatment of the DNA with λ exonuclease, however, improves its priming ability. Terminal transferase can then be used to add oligo(dA) blocks to the ends of one population of P22 DNA molecules and oligo(dT) blocks to the ends of a second population, which enables the two DNAs to anneal to one another to form dimer circles. Subsequent treatment with a system of DNA repair enzymes converts the circles to covalently closed molecules at high efficiency. It is demonstrated that the success of the joining system does not depend upon any obvious unique property of the P22 DNA.The joining system yields several classes of by-products, among them closed circular molecules with branches. Their creation can be explained on the basis of the properties of terminal transferase and the DNA repair enzymes.     

Replication of kinetoplast DNA of Crithidia acanthocephali. I. Density shift experiments using deuterium oxide

The protozoan Crithidia acanthocephali contains, within a modified region of a mitochondrion, a mass of DNA known as kinetoplast DNA (kDNA). This DNA consists mainly of an association of approximately 27,000 covalently closed 0.8-mum circular molecules which are apparently held together in a definite ordered manner by topological interlocking. After culturing of C. acanthocephali cells for 25 generations in medium containing 75% deuterium oxide, both nuclear DNA (rhonative, nondeuterated=1.717 g/cm3) and kDNA (rhonative, nondeuterated=1.702 g/cm3) increased in buoyant density by 0.012 g/cm3. The replication of the two DNAs was studied by cesium chloride buoyant density analysis of DNAs from exponentially growing cells taken at 1.0, 1.4, 2.0, 3.0, and 4.0 cell doublings after transfer of cells from D2O- containing medium into medium containing only normal water. The results obtained from analysis of both native and denatured nuclear DNAs indicate that this DNA replicates semiconservatively. From an analysis of intact associations of kDNA, it appears that this DNA doubles once per generation and that the newly synthesized DNA does not segregate from parental DNA. Fractions of covalently closed single circular molecules and of open circular and unit length linear molecules were obtained from associations of kDNA by sonication, sucrose sedimentation, and cesium chloride-ethidium bromide equilibrium gradient centrifugation. Buoyant density profiles obtained from these fractions indicate that: (a) doubling of the kDNA results from the replication of each circular molecule rather than from repeated replication of a small fraction of the circular molecules; (b) replication of kDNA is semiconservative rather than conservative, but there is recombination between the circles at an undefined time during the cell cycle.     

Sedimentation velocity properties of catenated DNA molecules from HeLa cell mitochondria

Catenated molecules of closed circular DNA have been isolated from the mitochondrial DNA of HeLs cells. The sedimentation coefficients of several purified species have been investigated. The catenated dimer, made up of two interlocked duplex circles, sediments at 51 S in its superhelical (closed) form. Treatment with pancreatic DNase to relax the duplex circles converts the 51 S doubly closed dimer to a 42 S singly open species, then to a 36 S doubly open catenated dimer. The triply closed trimer sediments at 63 S and is converted to a 45 S triply open form by DNase. Electron microscopy of the DNA samples before and after DNase treatment shows that under the conditions used DNase does not change the catenated nature of the DNA. The measured sedimentation coefficients, have been compared with those estimated from previously proposed correlations of sedimentation coefficient and molecular weight, and with the sedimentation coefficients for catenated DNA presented by Wang. When all the interlocked circles in a catenane are relaxed, the DNA sediments about 5–10% faster than a relaxed multiple-length circular molecule of the same molecular weight. The sedimentation coefficient, 36 S, of the fully relaxed catenated dimer is 1.4 times that of the relaxed monomer.     

Entrapment of plasmid DNA by liposomes and their interactions with plant protoplasts.

Lecithin and lecithin/cholesterol liposomes formed in aqueous solutions of DNA entrap covalently closed circular, open circular and linear DNA molecules of size up to at least 13 kilobases. The sequestered DNA molecules are efficiently protected against exogenous deoxyribonuclease action although nicking and linearization of circular DNA can be observed. The size of these liposomes ranges from approximately 0.5 to 7.5 mu with an average of 2.5--4 mu. DNA filled liposomes strongly interact with plant protoplasts under conditions inducing protoplast fusion. Results suggest that sequestered plasmid DNA can be transferred to protoplast nuclei.     

Biological activity of different forms of bacteriophage lambda DNA]

Hershey circles and linear tandem aggregated forms of DNA have been obtained in vitro and treated with polynucleotide ligase to form phosphodiester bond. Using zone centrifugation in glycerol gradient covalently closed circles and linear dimers have been purified and their biological activity investigated. It was found that closed circular molecules lost most, if not all, of their activity in CaCl2-dependent system. In order to investigate the biological activity of tandem dimer molecules, hybrid dimers consisting of DNA's from lambda C1857 and lambda 1434 have been obtained. In plaque assay with the appropriate non-permissive strains of E. coli the efficiency of infectivity of hybrid dimers was measured. Biological activity of dimer molecules sealed with ligase was about 5% of the activity of linear monomers. Ig has been suggested that tandem dimers of lambda DNA joined by phosphodiester bond are able to penetrate into the CaCl2-treated host cells and both components of dimers are active during subsequent multiplication.     

Preparation and isolation of covalently closed circular rDNA molecules from DNA of Xenopus laevis.

We describe a method leading to the formation of closed circles of rDNA starting from total DNA of Xenopus laevis. Linear DNA molecules were digested with exonuclease 3 and self-annealed. Open circles were enriched and covalently closed by the simultaneous use of polynucleotide kinase, DNA polymerase and polynucleotide ligase. Closed circles of rDNA1 were shown to be alkali-resistant, to have higher density than linear molecules in cesium chloride density gradients containing ethydium bromide, and to have the sedimentation constant expected for a single repeat unit of rDNA comprehensive of its spacer.     

The role of DNA polymerase I-associated 5'-exonuclease in replication of coliphage M13 replicative-form DNA.

The conversion of both parental- and progeny-nascent open circular M13 RF DNA into covalently closed RF I is drastically reduced in an $\text{E. coli}$ mutant deficient in the 5′ → 3′ exonuclease associated with DNA polymerase I. The nascent progeny RF DNA also contains a significant proportion of fragments of smaller than unit length.     

Uptake of circular deoxyribonucleic acid and mechanism of deoxyribonucleic acid transport in genetic transformation of Streptococcus pneumoniae.

Deoxyribonucleic acid (DNA) from the covalently closed circular DNA molecules of Pseudomonas phage PM2 was found to enter normally transformable cells of Streptococcus pneumoniae as readily as linear bacterial DNA. In a mutant of S. pneumoniae that lacks a membrane nuclease and is defective in DNA entry, as many molecules of PM2 DNA as of linear DNA were bound on the outside of cells at equivalent DNA concentrations. Bound DNA suffered single-strand breaks, but circular DNA with preexisting breaks was bound no better than closed circles. In the presence of divalent cations, DNA bound to cells of a leaky nuclease mutant showed double-strand breaks. At least the majority of PM2 DNA that entered normal cells was single stranded. These results are consistent with a mechanism for DNA entry in which DNA is first nicked on binding, then a double-strand break is formed by cleavage of the complementary strand, and continued processive action of the membrane nuclease facilitates entry of the originally nicked strand. Although the bulk of circular donor DNA appeared to enter in this way, the results do not exclude entry of a small amount of donor DNA in an intact form.     

Mode of replication of the conjugative R-plasmid RSF1040 in Escherichia coli.

Replicating deoxyribonucleic acid (DNA) molecules of plasmid RSF1040, a deletion mutant of the conjugative R plasmid R6K, appear in the electron microscope as partially supercoiled structures with two open circular branches of equal size, although open structures with three branches, two branching points and no supercoiled regions (theta structures) were also found at a lower frequency. The partially supercoiled molecules sediment more rapidly than native covalently closed circular DNA in neutral sucrose gradients and band at a position intermediate between covalently closed circular and open circular DNA in CsClethidium bromide gradients. Electron microscope measurements of the linear EcoRI-treated replicative intermediates indicate that replication can be initiated at two sites (origins) on the plasmid DNA molecule located at about 23% (alpha) and 39% (beta) of the total genome length from an EcoRI end designated arbitrarily as the "left-hand" end of the molecule. The overall replication of RSF1040 is asymmetrically bidirectional. Replication from the alpha origin proceeds first to the "right" to a unique termination site located some 55% of the total genome length from the left-hand end of the molecule. At this point replication proceeds from the alpha origin to the "left" (i.e., opposite to the original direction of replication) until replication of the molecule is completed. Replication also proceeds from the beta origin asymmetrically to the unique terminus site.     

Mitochondrial DNA recombination in Brassica campestris

The structure of the mitochondrial genome in plants is unclear, but appears to consist of mostly linear DNA with some other structures, including branched molecules and subgenomic circles. Mitochondrial DNA (mtDNA) recombination was analyzed in Brassica campestris, which has one of the smallest mitochondrial genomes (218 kb) in higher plants. Field-inversion gel electrophoresis (FIGE) separated mtDNA into discrete populations that each represents the entire genome. Electron microscopy revealed large, mostly linear molecules trapped in the wells, slower migrating populations with mostly linear DNA and a low level of circular and networked mtDNA molecules of 10–140 kbp, and a fast migrating population of 10–50 kbp linear mtDNA. Some smaller than genome size circular molecules and circles with tails were observed, and may represent recombination or rolling circle replication intermediates. Hybridization of end-labeled mtDNA suggests there may be specific ends (or recombination hotspots) for some linear molecules. Analysis of mtDNA enriched by BND-cellulose and separated by two-dimensional agarose gel electrophoresis shows the presence of complex recombination structures and the presence of significant single-stranded regions in mtDNA. These findings provide further evidence that DNA recombination contributes to the complex structure of mtDNA in plants.     

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.