Dynamic aspects of native DNA structure: Kinetics of the formaldehyde reaction with calf thymus DNA

Abstract

In this paper we outline the use of formaldehyde as a chemical probe to study the dynamic structure of native calf thymus DNA at temperatures well below the melting transition zone. It is demonstrated that the over-all reaction can be formulated as an equilibrium conformational “opening” step, followed by a slow chemical reaction of formaldehyde with nucleotide amino-groups normally involved in interchain hydrogen bonding. The kinetics of the latter reaction are calibrated as a function of temperature, formaldehyde concentration, pH and NaClO 4 concentration, using heat-denatured calf thymus DNA (after correction for residual base-pairing) as a model for the “transiently-open” regions in native DNA. The initial rate of formylation of native DNA is found to be equal to the product of K conf and the initial rate of formylation of denatured DNA under the same conditions, where K conf represents the fraction of the total base-pairs open (and unstacked) under the particular experimental conditions. Thus, we obtain a direct measure of this parameter. By studying the temperature dependence of K conf, we show that the enthalpy of the co-operative opening process which precedes the formation of the formaldehyde adduct ( ΔH conf) is temperature independent and corresponds to a co-operative opening unit of 9 ±1 base-pairs over a temperature range from just below T m to temperatures at least 30 deg. C lower. Increasing the concentration of the helix destablizing agent, sodium perchlorate, markedly diminishes the temperature dependence of K conf, and thus also the magnitude of ( ΔH conf. A number of lines of evidence are invoked to show that this decrease in ΔH confis not associated with a diminution in the size of the co-operative opening unit, but rather with a decrease in the enthalpy of melting per base-pair. Finally, we consider the possible biological significance of transiently open segments of this length in otherwise native DNA.