Stabilization of Z-DNA by demethylation of thymine bases: A crystallization and thermodynamic study of d(m(5)CGUAm(5)CG)

This thesis uses the stabilization of Z-DNA by demethylation of thymine as a model to understand solvent-macromolecular interactions and their role in determining macromolecular conformation. The hydration energy of a macromolecule is an estimate of solvent-macromolecular interactions and has successfully been used to explain the relative stabilities of different dinucleotides as Z-DNA (Kagawa et al., 1989). As an extension of this, the instability of d(TA) as Z-DNA was found to be due partly to a decrease in exposed hydrophilic surface of the methyl group of thymine; thus, d(UA) dinucleotides were predicted to be more stable as Z-DNA than the analogous d(TA) dinucleotides. This prediction was confirmed by the finding that the hexamer d(m{dollar}sp5{dollar}CGUAm{dollar}sp5{dollar}CG) crystallized as Z-DNA at 2-fold lower salt concentration than d(m{dollar}sp5{dollar}CGTAm{dollar}sp5{dollar}CG).; The structure of d(m{dollar}sp5{dollar}CGUAm{dollar}sp5{dollar}CG) was solved as Z-DNA to 1.3 A and then compared with the previously published structure of d(m{dollar}sp5{dollar}CGTAm{dollar}sp5{dollar}CG). The stabilization of d(UA) dinucleotides in the Z-conformation was found to be due to a magnesium water complex binding at the major groove and four ordered water molecules binding in the minor groove crevice of the d(UA) dinucleotides. The binding of the magnesium cluster at the major groove was consistent with the predictions from the hydration surface analysis, whereas the binding of waters in the minor groove was proposed to be the result of the binding of the magnesium cluster at the major groove. This model was described as a scissors model.; The stabilization of Z-conformation due to the demethylation of thymine was further analyzed from the crystal structures of d(m{dollar}sp5{dollar}CGUAm{dollar}sp5{dollar}CG) and d(m{dollar}sp5{dollar}CGTAm{dollar}sp5{dollar}CG) by AMBER and hydration energy calculations. The results show that the stabilization of Z-DNA by d(UA) dinucleotides comes from DNA-solvent interactions, not from internal DNA interactions. In addition, the energy minimized structures of d(m{dollar}sp5{dollar}CGUAm{dollar}sp5{dollar}CG) and d(m{dollar}sp5{dollar}CGTAm{dollar}sp5{dollar}CG) in aqueous solution show that modification at the major groove can indeed result in the binding of waters to the minor groove and stabilization of d(UA) in the Z-conformation. This result suggests that solvent interaction is the driving force in determining macromolecular conformation and that solvent macromolecular interactions must be considered to correctly predict the macromolecular conformations.