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-compleme ntary 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 eq uilibrium 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 a re 3-8-fold larger than the corresponding ones in the CAGA/GTCT sequen ce. The activation enthalpies and entropies, and the standard enthalpy and entropy changes for formation of the open state, are obtained fro m the temperature dependence of the opening rates and equilibrium cons tants, respectively. The results reveal that enthalpy/entropy compensa tion exists, for all base pairs in both dodecamers, in activation as w ell 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. Neve rtheless, large sequence-induced variations are observed for the activ ation 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 du ring opening. This is the first case in which a negative activation en thalpy 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/G TGT motif are increased by 5 and 9 kcal/mol relative to the correspond ing ones in the CAGA/GTCT sequence. Sequence-induced variations are al so observed for the standard enthalpy changes associated with the equi libria 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 ac tivation 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.