PubMed1990
In vitro condensation of DNA by multivalent cations can provide useful insights into the physical factors governing folding and packaging of DNA in vivo. We have made a detailed study of hexammine cobalt (III) induced condensation of 2700 and 1350 base pair (bp) fragments of plasmid pUC12 DNA by electron microscopy and laser light scattering. The condensed DNA takes the form of toroids and rods. Both are present in all condensates, but the proportion of toroids is higher with the larger fragments. The intact, closed circular plasmid produces smaller particles than the linear fragments. The size of a particle is independent of DNA fragment length. Two hours after adding the condensing agent, a typical toroid is about 800 A in diameter; the outer radius (R1) is approximately 400 A, and the inner radius (R2) is approximately 140 A for both sets of fragments. These dimensions are relatively stable, but there is sufficient change in both R1 and R2 to produce approximately 50% increase in volume from 2 to 24 h. A typical rod at 2 h is about 1800 A long and 300 A wide. The distribution of rod lengths is similar to that of mean toroid circumferences pi (R1 + R2), and the distribution of rod widths is similar to that of toroidal widths (R1-R2). The 2700-bp fragments show a significantly higher ratio of toroids to rods than the 1350-bp fragments. Both types of particle are multimolecular. The average number of molecules/particle, calculated from the above dimensions, assuming hexagonally packed B-form DNA with a center-to-center spacing of 27 A, is 13 +/- 4 for condensates of 2700-bp fragments and 26 +/- 11 for those of 1350-bp fragments. Monomolecular condensates of much larger DNAs have similar dimensions, suggesting that size is governed primarily by the balance of attractive and repulsive intermolecular forces rather than by the entropic factors associated with incorporation of a number of small particles into a larger one. The similar dimensions and volumes of toroids and rods indicate that the free energy cost of continual bending in toroids, minus that gained by extra net attraction in a cyclic particle, is comparable to that of abrupt bending or kinking in rods. Although the condensed particles are multimeric, their distinct toroidal or rodlike shapes distinguish them from the random aggregates that would be generally expected from the multimolecular association of large, flexible polymers.