ALBERT

Notes: The kinetics of the helix-coil transition have been investigated for T2 and T7 phage DNA in a formamide-water-salt mixed solvent using a slow temperature perturbation technique (applicable to kinetic processes with rate constants ≤ 3 min-1). In this solvent degradation of the DNA is effectively suppressed. Complex kinetic curves are observed by absorbance and viscosity measurements for the response to denaturing perturbations in the transition region. Analysis of the decay curves indicates that the denaturation reaction in this time range can be treated as a first-order reaction with a variable first-order rate parameter, k, the derivative of the logarithm of the absorbance or viscosity change with respect to time. In the approach to denaturation equilibrium in the transition region, the rate parameter is determined only by the instantaneous extent of denaturation of the molecules. Near equilibrium, the rate parameter assumes a constant value characteristic of the equilibrium state. In this region, where the denaturation reaction proceeds as a simple first-order process, both the decay of absorbance (reflected local conformational change) and the decay of solution viscosity (reflecting macromolecular conformational change) are characterized by the same constant value of k. In 83% formamide, 0.3M Na+, the rate parameter k for T2 DNA decreases from an extrapolated value of 2.0 min-1 at 0% denaturation to 0.11 min-1 at 90% denaturation. Rate parameters determined for T7 DNA at the same counterion concentration and fraction of denaturation are approximately five times as large as those cited for T2 DNA, indicating an inverse proportionality of rate constant to molecular length. On the other hand, simple first-order kinetic responses with constant k are obtained for renaturing perturbations within the transition, indicating that the mechanism of rewinding differs, in most cases, from that of unwinding. Only in the limit of very small perturbations about a given equilibrium position are the rate constants k obtained from denaturing and renaturing perturbations equal. For perturbations of finite size, it appears possible that an intramolecular initiation or nucleation event may precede rewinding and limit the rate of this reaction. The rate parameters again are approximately inversely proportional to molecular weight. The one exception to the first-power dependence on molecular weight appears when temperature jumps are made upward into the post-transition region. Here the molecular-weight dependence is second power, but complications arising from the different strand-separation properties of T2 and T7 DNA's make interpretation difficult. The previously used model of friction-limited unwinding appears to fit all the observations except for the molecular-weight dependence.

Notes: Strand separation of T2 DNA has been investigated in a helix-destabilizing solvent. Temperature-shift experiments in which the conformation of the DNA is monitored by its viscosity, sedimentation behavior, and kinetics of helix formation show that a well-defined strand-separation transition follows the helix-coil transition usually observed by changes in absorbance. For T2 DNA, this strand-separation transition is 70% as broad as the helix-coil transition, and is characterized by extremely slow kinetics of conformational change in the population. Strand separation requires the expansion of the two-stranded coil observed at the end of the helix-coil transition. This expansion is apparently coupled with the disurption of the last remaining base pairs in the molecule. The expansion process increases the viscosity, and can be readily followed as a function of time and/or temperature. Subsequent separation of the expanded form into complementary strands results in a viscosity decrease, the net result of a reduction in hydrodynamic volume and the halving of the molecular weight. Only under conditions where the driving force for strand separation is large are these events at all synchronous in the population. When the kinetics of conformational change are complete in the strand-separation transition, a mixture of expanded forms and separate strands is observed; the breadth of the transition reflects differences in stability with respect to strand separation among the molecules in the population. The transition exhibits hysteresis and is not a reversible equilibrium between double-stranded and single-stranded forms. It appears that renucleation is kinetically forbidden within the strand-separation region.

Notes: The strand-separation transition of T7 DNA has been investigated by temperature shift and viscosity measurements in two formamide-water solvents. The strand-separation region is quite narrow, and follows directly at the end of the denaturation transition observed by absorbance. The kinetics of strand separation of T7 DNA are slow and complex in the strand-separation transition. Similarities and differences in the behavior of T2 and T7 DNA in strand separation are indicated and discussed. Briefly, the time course of strand separation and the conformational changes observed in the population undergoing strand separation are similar for the two molecules. However, the transition breadths and the interval between the helix-coil transition and the strand-separation transition differ markedly. Both DNA molecules exhibit hysteresis in the strand-separation region. For both molecules, it appears that strand separation involves the coupled denaturation and disentanglement of the two-stranded form found at the end of the helix-coil transition.