Determining DNA supercoiling enthalpy by isothermal titration calorimetry

Abstract

DNA supercoiling plays a critical role in certain essential DNA transactions, such as DNA replication, recombination, and transcription. For this reason, exploring energetics of DNA supercoiling is fundamentally important for understanding its biological functions. In this paper, using a unique property of DNA intercalators, such as ethidium bromide and daunorubicin, which bind to supercoiled, nicked, and relaxed DNA templates with different DNA-binding enthalpies, we determined DNA supercoiling enthalpy of plasmid pXXZ6, a 4.5 kb plasmid to be about 11.5 kcal/mol per linking number change. This determination allowed us to partition the DNA supercoiling free energy into enthalpic and entropic contributions where the unfavorable DNA supercoiling free energy exclusively originated from the large positive supercoiling enthalpy and was compensated by a large, favorable entropy term (TΔS).