The energetics of HMG box interactions with DNA: Thermodynamic descriptionof the target DNA duplexes

The thermal properties and energetics of formation of 10, 12 and 16 bp DNA duplexes, specifically interacting with the HMG box of Sox-5, have been stu died by isothermal titration calorimetry (ITC) and differential scanning ca lorimetry (DSC). DSC studies show that the partial heat capacity of these s hort duplexes increases considerably prior to the cooperative process of st rand separation. Direct extrapolation of the pre and post-transition heat c apacity functions into the cooperative transition zone suggests that unfold ing/dissociation of strands results in no apparent heat capacity increment. In contrast, ITC measurements show that the negative enthalpy of complemen tary strand association increases in magnitude with temperature rise, imply ing that strand association proceeds with significant decrease of heat capa city. Furthermore, the ITC-measured enthalpy of strand association is signi ficantly smaller in magnitude than the enthalpy of cooperative unfolding me asured by DSC. To resolve this paradox, the heat effects upon heating and c ooling of the separate DNA strands have been measured by DSC. This showed t hat cooling of the strands from 100 degrees C to -10 degrees C proceeds wit h significant heat release associated with the formation of intra and inter -molecular interactions. When the enthalpy of residual structure in the str ands and the temperature dependence of the heat capacity of the duplexes an d of their unfolded strands have been taken into account, the ITC and DSC r esults are brought into agreement. The analysis shows that the considerable increase in heat capacity of the duplexes with temperature rise is due to increasing fluctuations of their structure (e.g. end fraying and twisting) and this effect obscures the heat capacity increment resulting from the coo perative separation of strands, which in fact amounts to 200(+/-40) JK(-1) (mof bp)(-1). Using this heat capacity increment, the averaged standard ent halpy, entropy and Gibbs energy of formation of fully folded duplexes from fully unfolded strands have been determined at 25 degrees C as -33(+/-2) kJ (mol bp)(-1), -93(+/-4)J K-1 (mol bp)(-1) and -5.0(+/-0.5)kJ (mol bp)(-1) respectively. (C) 1999 Academic Press.