The rate of DNA unwinding

Abstract

We report three kinetic effects in the response of large DNA molecules to temperature perturbations ranging in size from 0.25 to 18 deg. C. Experiments were done in the alkaline melting transition, and the rate was followed by observing the change in absorbance. The temperature increase was produced by passage of an electric current through the solution, using the standard temperature-jump technique for times up to one second. For the time range from 1 second to hours a special instrument was constructed, with which the temperature in the cell can be increased by up to 20 deg. C in less than one second, and maintained indefinitely. The fastest kinetic response of the DNA, called “instantaneous”, occurs in less than 20 msec, and increases in amplitude as the size of the temperature jump increases. We associate this process with a rapid structural disorganization which does not require unwinding of the molecule, analogous to melting of a closed double circle of DNA. When the perturbation size is moderate (between 6 and 18 deg. C) the initial instantaneous effect is followed by kinetic curves of homogeneous appearance, which we label the “fast” effect. The characteristic time for this process (determined as a linear average of the component exponential decay times) depends on the 2.3 power of the molecular length. We ascribe rate-determination for this effect to viscous resistance to unwinding the double helix. Because of the strong dependence on molecular weight, kinetic measurements under these conditions can be used to detect small amounts of undegraded DNA in the presence of material that is broken or contains single-strand breaks. Small perturbations within the melting transition show a kinetic component which is nearly independent of molecular weight and, for a DNA like that from T7 bacteriophage, much slower than the fast effect. This process, labeled the “slow” effect, has an apparent activation energy of at least 100 kcal., and is therefore not likely to be primarily friction-limited. We ascribe it to one of the nucleation events in the co-operative melting transition, specifically the disappearance (or de-nucleation) of a helical section separating two coil regions.