A unified theory of nucleation-rate-limited DNA renaturation kinetics

Abstract

DNA renaturations under nucleation-rate-limiting conditions on simple DNA such as bacterial and bacteriophage DNA show significant deviation from ideal second-order kinetics when followed by optical density measurements at 260 nm. Ideal second-order kinetics yield linear plots when the data is plotted in the standard reciprocal second-order (RSO) manner. The observed deviations from ideal second-order behavior take the form of steadily downward-curving RSO plots. In this paper, experiments are presented for E. coli and T2 DNA documenting this non-ideal behavior. Since experiments using T4, T5 and B, subtilis DNA yield identical non-ideal behavior, this behavior appears to be a property of DNA renaturation followed by optical density, not a peculiarity of a particular DNA. Identical non-ideal behavior is also seen in kinetics followed by S1 nuclease assay. A theory is developed to explain this deviation from ideal second-order kinetics. The theory also explains why kinetics followed by hydroxyapatite chromatography show nearly ideal second-order kinetics. In contrast to the approach taken by others in developing equations that describe S1 nuclease monitored reactions, our view is that nonideal second-order kinetics are fundamentally due to the reacton of free single strands to yield partially helical duplex species. Later reactions of these species tend to reduce the deviations from non-ideal second-order kinetics.