Rate of unwinding small DNA

Abstract

The kinetics of the helix-coil transition of low molecular weight DNA was investigated using temperature-jump techniques. The material was sonicated or sheared, then fractionated according to molecular weight using differential solubility in an isopropanol-buffer mixture. To prevent irreversible strand separation the samples were cross-linked with mitomycin C. Kinetic observations (266 nm) were made as a function of molecular weight, fraction coil, perturbation size and salt concentration. The Eigen-DeMaeyer apparatus was used for times between 5 μsec and 2 sec; slower effects required a special instrument. Decay times varying with M1.9 down to 6.0 × 105 daltons are attributed to fractionally limited unwinding. Some faster effects are from overtones (melting from ends). The rate of unwinding of DNA with a molecular Weight of 1.0 × 105 daltons appears to be limited by the breakage of hydrogen bonds. From the data we estimate a rate constant of 4 × 106 sec-1 for base pair formation. A very fast decay (~30 μsec) is possibly the nucleation of coil at an A + T rich region. Ascribed to protolytic equilibria and to non-specific base-base interaction is an irresolvable (<5 μsec) optical density change. Also of interest is the appreciable contribution of very slow effects (some slower than 102 sec). These are nearly independent of molecular weight. We believe this originates from the difference in modes of melting for G + C rich and A + T rich regions of amolecule; the former approaches an "all-or-none" transition. The A + T rich regions, because they are less stable, melt first, leaving G + C rich regions between loops. The merging of two of these loopsis statistically favorable but this requires the breakage of a large number of base pairs. Thus, this melting is entropically driven against a large activation energy barrier giving rise to exceedingly slow kinetics. © 1971.