Cation-induced toroidal condensation of DNA studies with Co3+(NH3)6.

Abstract The polyamines spermidine (3+) and spermine (4+) are known to cause a co-operative intramolecular condensation of T7 or λ phage DNAs in which the DNA assumes a compact toroidal conformation (Gosule & Schellman, 1976,1978). We show here that an inert trivalent metal ion complex, Co 3+ (NH 3 ) 6 , also causes DNA condensation and that DNA condensation in aqueous solution is caused by cations of charge 3+ or more. The DNA products of polyamine-induced and of cobalt hexamine-induced condensation have similar toroidal conformations as judged by electron microscopy and both have the circular dichroism spectrum of DNA B-form, in contrast to the ψ DNA condensates studied by Maniatis et al. (1974). Monovalent and divalent cations (Na + , Mg 2+ ) reverse DNA condensation. Competition between inducing (3+ or 4+) and reversing (1+ or 2+) cations follows the ion-exchange behavior outlined in Manning's (1978) theory of atmospheric cation binding to DNA. Our results are consistent with the suggestion of Manning (1978) and of Wilson & Bloomfield (1979) that DNA condensation can occur when a critical fraction of the DNA phosphate charge has been neutralized by cations adsorbed to the DNA. We suggest further that cation-induced DNA condensation in aqueous solution results from cation crosslinking: electrostatic bridging of adjacent helices by trivalent or higher valence cations. Transition curves for DNA condensation have been measured by the increase in light-scattering, using a photon-counting fluorimeter. To ensure that equilibrium is reached, condensation has been studied in both the forward and reverse directions, by using either Na + or Mg 2+ to reverse the reaction. The kinetics of condensation are slow in the forward direction, in the time range of minutes to hours, and become much slower as the DNA concentration is increased. Reversal of condensation by Na + or Mg 2+ occurs more rapidly, in seconds to minutes, and the transition midpoints are essentially independent of DNA concentration. At DNA concentrations below 1 μ m -phosphate, the kinetics of condensation and of de-condensation are comparable in rate. We suggest that intermolecular DNA contacts compete with, and slow down, intramolecular condensation. Equilibrium data for transition midpoints are obtained in either the forward or reverse direction at sufficiently low DNA concentrations; at higher DNA concentrations, equilibrium is reached in the reverse but not in the forward direction. Phase diagrams for condensation (plots of log [Co 3+ (NH 3 ) 6 ] versus log [Na + ] or log [Mg 2+ ] at the transition midpoint) have been obtained from studies of de-condensation by Na + or Mg 2+ . These plots have a slope of +1 when either Co 3+ (NH 3 ) 6 , spermidine (3+) or spermine (4+) is used to induce condensation. As shown by Wilson & Bloomfield (1979), a slope of +1 is consistent with DNA condensation occurring when a critical fraction of DNA charge has been neutralized, as calculated by Manning's (1978) theory. Two additional results are presented, which bear on the problem of toroidal DNA condensation. (1) Condensation occurs more readily at high temperatures. (2) Restriction fragments as short as 400 base-pairs form toroids by intermolecular condensation, which are similar in diameter and appearance to the intramolecular condensates formed by λ DNA.