The Renaturation of Denatured DNA

Abstract

The kinetics of renaturation of heat-denatured DNA from E. coli and pneumococcus have been examined by ultraviolet absorption measurements. The molecularity of the reaction was assessed by three independent treatments of the data, and all lead to the conclusion that renaturation is essentially first order at 60 degrees ; at 70 degrees and 80 degrees there is an increasing second order component, resulting in simultaneous unimolecular and bimolecular kinetics. The unimolecular kinetics rule out reaction between two, kinetically separate strands, indicating rather the zippering-up of a single, denatured entity. The bimolecular kinetics can be attributed to the complexing of two such entities; thus, the genetic or density-labeled complexes that have been observed by other investigators can be accounted for without invoking strand separation. Since renaturation at best is never complete, the free ends of two renatured molecules permit sufficient bimolecular reaction to produce density hybrids. The observed kinetics can be accounted for if the hydrogen bonds of DNA are broken during heat denaturation but the strands do not separate. Light scattering supports this by showing that the molecular weight is unchanged by denaturation. Since there is no existing evidence that is inconsistent with this hypothesis, it is reasonable to conclude that heat denaturation does not completely separate the entangled strands of the DNA molecule.