The 23S twisted circular form of ColE1 DNA has been isolated from Escherichia coli as a tightly associated DNA-protein complex with a sedimentation coefficient of approximately 24S. Treatment of this complex with pronase, trypsin, sodium dodecyl sulfate, Sarkosyl, or heat results in a conversion to a slower sedimenting form of 17S or 18S, as determined by centrifugation in neutral sucrose gradients. These treatments do not alter the sedimentation properties of noncomplexes supercoiled ColE1 DNA even in the presence of the ColE1-protein complex. Electron microscopic analyses indicate that the decrease in sedimentation rate of the ColE1-protein complex after treatment with these various agents is largely owing to an induced transition of ColE1 DNA from the supercoiled to the open circular state.

https://www.pnas.org/content/pnas/62/4/1159.full.pdf

SUPERCOILED CIRCULAR DNA-PROTEIN COMPLEX IN Escherichia coli: PURIFICATION AND INDUCED CONVERSION TO AN OPEN CIRCULAR DNA FORM

A preparative method for isolating high-molecular-weight DNA from animal cells is described. This method is based on the use of proteinase K, a powerful proteolytic enzyme with a broad action spectrum, which is very active in the presence of sodium dodecylsulfate and ethylene-diamine tetraacetate. The DNA preparation is free of RNA, protein and degrading enzymes. The number-average molecular weight of the native DNA is 190 × 106, whereas it is 90 × 106 for single-stranded DNA, indicating that the DNA molecules do not contain single-stranded nicks. The native DNA molecules range in molecular weight from 40 × 106 to more than 500 × 106.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1973.tb02881.x

Isolation of high-molecular-weight DNA from mammalian cells.

Single linear DNA molecules were bound at multiple sites at one extremity to a treated glass cover slip and at the other to a magnetic bead. The DNA was therefore torsionally constrained. A magnetic field was used to rotate the beads and thus to coil and pull the DNA. The stretching force was determined by analysis of the Brownian fluctuations of the bead. Here, the elastic behavior of individual λ DNA molecules over- and underwound by up to 500 turns was studied. A sharp transition was discovered from a low to a high extension state at a force of ∼0.45 piconewtons for underwound molecules and at a force of ∼3 piconewtons for overwound ones. These transitions, probably reflecting the formation of alternative structures in stretched coiled DNA molecules, might be relevant for DNA transcription and replication.

https://science.sciencemag.org/content/sci/271/5257/1835.full.pdf

The Elasticity of a Single Supercoiled DNA Molecule

Covalently closed circular duplex DNA’s are now known to be widespread among living organisms. This DNA structure, originally identified in polyoma viral DNA,1’2 has been assigned to the mitochondrial DNA’s in ox3 and sheep heart,4 in mouse and chicken liver,3 and in unfertilized sea urchin egg.5 The animal viral DNA’s—polyoma, SV40,6 rabbit7 and human8 papilloma—the intracellular forms of the bacterial viral DNA’s—φX174,9, 10 lambda,11, 12 M13,13 and P2214—and a bacterial plasmid DNA, the colicinogenic factor E2,15 have all been shown to exist as closed circular duplexes. Other mitochondrial DNA’s16, 17 and a portion of the DNA from boar sperm18 have been reported to be circular, but as yet have not been shown to be covalently closed.

A dye-buoyant-density method for the detection and isolation of closed circular duplex DNA: the closed circular DNA in HeLa cells.

The method of alkaline elution provides a sensitive measure of DNA single-strand length distribution in mamalian cells and is applicable to a variety of problems concerning DNA damage, repair, and replication. The physical basis of the elution process was studied. The kinetics of elution above the alkaline transition pH were found to occur in two phases: an initial phase in which single-strand length is rate limiting, followed by a phase in which elution is accelerated due to the accumulation of alkali-induced strand breaks. The range of DNA single-strand lengths that can be discriminated by elution above the alkaline transition pH was estimated by calibration relative to the effects of x ray, and was found to be 5 X 10(8)-10(10) daltons. Shorter DNA strands elute within the pH transition zone, which extended from pH 11.3 to 11.7 when tetrapropylammonium hydroxide was used as base. This elution was relatively rapid, but was sharply limited by pH, according to the length of the strands: the length of the strands eluted increased with increasing pH. Alkaline elution was inhibited by treatment of cells with low concentrations of nitrogen mustard, a bifunctional alkylating known to cross-link DNA. On investigation of the possibility that DNA subclasses may differ in their elution behavior, satellite L strands were found to elute more slowly from cells exposed to a low dose of x ray than did the bulk DNA.

Fractionation of DNA from mammalian cells by alkaline elution

The major part of the DNA from polyoma virus has been shown to consist of circular base-paired duplex molecules without chain ends1–3 The intertwined circular form accounts for the ease of renaturation4 of this DNA and the failure of the strands to separate in strand-separating solvents1–3

P Laipis

Papers

The twisted circular form of polyoma viral DNA.