SEQUENCE DEPENDENCE OF BASE-PAIR OPENING IN A DNA DODECAMER CONTAINING THE CACA/GTGT SEQUENCE MOTIF

Proton nuclear magnetic resonance spectroscopy is used to characterize the kinetics and energetics of base-pair opening in two self-complementary DNA dodecamer duplexes: [d(CGCACATGTGCG)](2) and [d(CGCAGATCTGCG)](2). The first dodecamer contains two symmetrical CACA/GTGT motifs; in the second dodecamer, each motif is interrupted by a change of the central C.G base pair to a G.C base pair. The opening rates and the equilibrium constants for formation of the open state of each base pair are obtained from the dependence of the imino proton exchange rates on the concentration of ammonia catalyst. The results indicate that the opening rates of the central three base pairs in the CACA/GTGT motif are 3-8-fold larger than the corresponding ones in the CAGA/GTCT sequence. The activation enthalpies and entropies, and the standard enthalpy and entropy changes for formation of the open state, are obtained from the temperature dependence of the opening rates and equilibrium constants, respectively. The results reveal that enthalpy/entropy compensation exists, for all base pairs in both dodecamers, in activation as well as in the equilibria between closed and open states. As a result, the opening rates and equilibrium constants for opening are maintained, in both dodecamers, within a relatively narrow range of values. Nevertheless, large sequence-induced variations are observed for the activation enthalpies and the standard enthalpy changes for opening. The A.T base pair located between the C.G base pairs in the CACA/GTGT motif has a negative enthalpy change for formation of the activated state during opening. This is the first case in which a negative activation enthalpy is observed for opening of a Watson-Crick base pair in DNA. The activation enthalpies for opening of the C.G base pairs in the CACA/GTGT motif are increased by 5 and 9 kcal/mol relative to the corresponding ones in the CAGA/GTCT sequence. Sequence-induced variations are also observed for the standard enthalpy changes associated with the equilibria between closed and open states. For all base pairs in the CACA/GTGT motif, the standard enthalpy changes are 2-7 kcal/mol lower than the corresponding ones in the CAGA/GTCT sequence. Comparison of the activation enthalpies and standard enthalpy changes for opening suggests that a different opening pathway also exists for the central C.G base pair in the CACA/GTGT motif.