Base Flipping: Detection, Structures and Energetics

Base flipping is the movement of a DNA base from an intrahelical, base stacked position to an extrahelical, solvent exposed position. As there are favorable interactions for an intrahelical base, both hydrogen bonding and base stacking, base flipping is expected to be energetically prohibitive for an undamaged DNA duplex. For damaged DNA bases, however, the energetic cost of base flipping may be considerably lower. Using a selective, non-covalent assay for base flipping, the sequence dependence of base flipping in DNA sequences containing an abasic site has been studied. The dissociation constants of the zinc-cyclen complex to small molecules and single strands of DNA as well as the equilibrium constants for base flipping have been determined for these sequences. Molecular dynamics simulations of the zinc-cyclen complex bound to both single and double stranded DNA have been performed. The results are compared to previous studies of base flipping in DNA containing an abasic site. A specific DNA damage of interest is the cis,syn-cyclobutane pyrimidine dimer (CPD), also known as the thymine dimer, which is formed via a photochemical [2+2] cycloaddition between two adjacent thymine bases in DNA. The structure of the thymine dimer within the DNA helix has been previously reported. To date, there has been only one report of the structure of an extrahelical thymine dimer. In this work the structure of both the flipped-in and the flipped-out CPD lesion in duplex DNA is reported. These structures are then used to define the starting and ending points for the base flipping process for the CPD lesion. Using a complex, two-dimensional pseudodihedral coordinate the potential of mean force (PMF) for the base flipping process was calculated using novel methodology. Using the novel method, the energetics of base flipping in various DNA sequences containing a thymine dimer have been calculated. Significant disruptions of the base pairs to the 3' side of the CPD are observed for the flipped-out structures with adjacent A-T pairs, while those with G-C pairs adjacent show no such distortions. The structural differences correlate well with the free energy differences for base flipping calculated using the novel 2D-PMF method.