Conformation and dynamics of abasic sites in DNA investigated by time-resolved fluorescence of 2-aminopurine

Abasic sites are highly mutagenic lesions in DNA that arise as intermediate s in the excision repair of modified bases. These sites are generated by th e action of damage-specific DNA glycosylases and are converted into downstr eam intermediates by the specific activity of apurinic/apyrimidinic endonuc leases. Enzymes in both families have been observed in crystal structures t o impose deformations on the abasic-site DNA, including DNA kinking and bas e flipping. On the basis of these apparent protein-induced deformations, we propose that altered conformation and dynamics of abasic sites may contrib ute to the specificity of these repair enzymes. Previously, measurements of the steady-state fluorescence of the adenine analogue 2-aminopurine (2AP) opposite an abasic site demonstrated that binding of divalent cations could induce a conformational change that increased the accessibility of 2AP to solute quenching [Stivers, J. T. (1998) Nucleic Acids Res. 26, 3837-44]. We have performed time-resolved fluorescence experiments to characterize the states involved in this conformational change. Interpretation of these stud ies is based on a recently developed model attributing the static and dynam ic fluorescence quenching of 2AP in DNA to aromatic stacking and collisiona l interactions with neighboring bases, respectively (see the preceding pape r in this issue). The time-resolved fluorescence results indicate that diva lent cation binding shifts the equilibrium of the abasic site between two c onformations: a "closed" state, characterized by short average fluorescence lifetime and complex decay kinetics, and an "open" state, characterized by monoexponential decay with lifetime approximately that of the free nucleos ide. Because the lifetime and intensity decay kinetics of 2AP incorporated into DNA are sensitive primarily to collisional interactions with the neigh boring bases, the absence of dynamic quenching in the open state strongly s uggests that the fluorescent base is extrahelical in this conformation. Con sistent with this interpretation, time-resolved quenching studies reveal th at the open state is accessible to solute quenching by potassium iodide, bu t the closed state is not. Greater static quenching is observed in the abas ic site when the fluorescent base is flanked by 5'- and 3'-thymines than in the context of 5'- and 3'-adenines, indicating that 2AP is more stacked wi th the neighboring bases in the former sequence. These results imply that t he conformation of the abasic site varies in a sequence-dependent manner. U ndamaged sequences in which the abasic site is replaced by thymine do not e xhibit an open state and have different levels of both static and dynamic q uenching than their damaged homologues. These differences in structure and dynamics may be significant determinants of the high specific affinity of r epair enzymes for the abasic site.