2-Aminopurine (2AP) is an analogue of adenine that has been utilized widely as a fluorescence probe of protein-induced local conformational changes in DNA. Within a DNA strand, this fluorophore demonstrates characteristic decreases in quantum yield and emission decay lifetime that vary sensitively with base sequence, temperature, and helix conformation but that are accompanied by only small changes in emission wavelength. However, the molecular interactions that give rise to these spectroscopic changes have not been established. To develop a molecular model for interpreting the fluorescence measurements, we have investigated the effects of environmental polarity, hydrogen bonding, and the purine and pyrimidine bases of DNA on the emission energy, quantum yield, and intensity decay kinetics of 2AP in simple model systems. The effects of environmental polarity were examined in a series of solvents of varying dielectric constant, and hydrogen bonding was investigated in binary mixtures of water with 1,4-dioxane or N,N-dimethylformamide (DMF). The effects of the purine and pyrimidine bases were studied by titrating 2AP deoxyriboside (d2AP) with the nucleosides adenosine (rA), cytidine (rC), guanosine (rG), and deoxythymidine (dT), and the nucleoside triphosphates ATP and GTP in neutral aqueous solution. The nucleosides and NTPs each quench the fluorescence of d2AP by a combination of static (affecting only the quantum yield) and dynamic (affecting both the quantum yield and the lifetime, proportionately) mechanisms. The peak wavelength and shape of the emission spectrum are not altered by either of these effects. The static quenching is saturable and has half-maximal effect at approximately 20 mM nucleoside or NTP, consistent with an aromatic stacking interaction. The rate constant for dynamic quenching is near the diffusion limit for collisional interaction (k(q) approximately 2 x 10(9) M(-1) s(-1)). Neither of these effects varies significantly between the various nucleosides and NTPs studied. In contrast, hydrogen bonding with water was observed to have a negligible effect on the emission wavelength, fluorescence quantum yield, or lifetime of 2AP in either dioxane or DMF. In nonpolar solvents, the fluorescence lifetime and quantum yield decrease dramatically, accompanied by significant shifts in the emission spectrum to shorter wavelengths. However, these effects of polarity do not coincide with the observed emission wavelength-independent quenching of 2AP fluorescence in DNA. Therefore, we conclude that the fluorescence quenching of 2AP in DNA arises from base stacking and collisions with neighboring bases only but is insensitive to base-pairing or other hydrogen bonding interactions. These results implicate both structural and dynamic properties of DNA in quenching of 2AP and constitute a simple model within which the fluorescence changes induced by protein-DNA binding or other perturbations may be interpreted.

Abasic sites are highly mutagenic lesions in DNA that arise as intermediates in the excision repair of modified bases. These sites are generated by the action of damage-specific DNA glycosylases and are converted into downstream intermediates by the specific activity of apurinic/apyrimidinic endonucleases. Enzymes in both families have been observed in crystal structures to impose deformations on the abasic-site DNA, including DNA kinking and base flipping. On the basis of these apparent protein-induced deformations, we propose that altered conformation and dynamics of abasic sites may contribute 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 studies is based on a recently developed model attributing the static and dynamic fluorescence quenching of 2AP in DNA to aromatic stacking and collisional interactions with neighboring bases, respectively (see the preceding paper in this issue). The time-resolved fluorescence results indicate that divalent cation binding shifts the equilibrium of the abasic site between two conformations: 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 nucleoside. Because the lifetime and intensity decay kinetics of 2AP incorporated into DNA are sensitive primarily to collisional interactions with the neighboring bases, the absence of dynamic quenching in the open state strongly suggests that the fluorescent base is extrahelical in this conformation. Consistent with this interpretation, time-resolved quenching studies reveal that the open state is accessible to solute quenching by potassium iodide, but the closed state is not. Greater static quenching is observed in the abasic 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 with the neighboring bases in the former sequence. These results imply that the conformation of the abasic site varies in a sequence-dependent manner. Undamaged sequences in which the abasic site is replaced by thymine do not exhibit an open state and have different levels of both static and dynamic quenching than their damaged homologues. These differences in structure and dynamics may be significant determinants of the high specific affinity of repair enzymes for the abasic site.

Many biological systems have multiple fluorophores that experience multiple depolarizing motions, requiring multiple lifetimes and correlation times to define the fluorescence intensity and anisotropy decays, respectively. To simplify analyses, an assumption often made is that all fluorophores experience all depolarizing motions. However, this assumption usually is invalid, because each lifetime is not necessarily associated with each correlation time. To help establish the correct associations and recover accurate kinetic parameters, a general kinetic scheme that can examine all possible associations is presented. Using synthetic data sets, the ability of the scheme to discriminate among all nine association models possible for two lifetimes and two correlation times has been evaluated. Correct determination of the association model, and accurate recovery of the decay parameters, required the global analysis of related data sets. This general kinetic scheme was then used for global analyses of liver alcohol dehydrogenase anisotropy data sets. The results indicate that only one of the two tryptophan residues in each subunit is depolarized by process(es) independent of the enzyme's rotations. By applying the proper kinetic scheme and appropriate analysis procedures to time-resolved fluorescence anisotropy data, it is therefore possible to examine the dynamics of specific portions of a macromolecule in solution.