Conformational Analysis of DNA Repair Intermediates by Time-Resolved Fluorescence Spectroscopy

DNA repair enzymes are essential for maintaining the integrity of the DNA sequence. Unfortunately, very little is known about how these enzymes recognize damaged regions along the helix. Structural analysis of cellular repair enzymes bound to DNA reveals that these enzymes are able to recognize DNA in a variety of conformations. However, the prevalence of these deformations in the absence of enzymes remains unclear, as small populations of DNA conformations are often difficult to detect by NMR and X-ray crystallography. Here, we used time-resolved fluorescence spectroscopy to examine the conformational dynamics of linear, nicked, gapped, and bulged DNA in the absence of protein enzymes. This analysis reveals that damaged DNA is polymorphic in nature and able to adopt multiple individual conformations. We show that DNA repair intermediates that contain a one-nucleotide gap and bulge have a significant propensity to adopt conformations in which the orphan base resides outside the DNA helix, while DNA structures damaged by a nick or two-nucleotide gap favor intrahelical conformations. Because changes in DNA conformation appear to guide the recognition of DNA repair enzymes, we suggest that the current approach could be used to study the mechanism of DNA repair. Structural analyses of many DNA-enzyme complexes reveal that cellular repair enzymes recognize DNA in a variety o

Spectroscopy of 2-aminopurine: An MCSCF Study

2-aminopurine is a highly fluorescent isomer of adenine that can be incorporated into DNA as a probe of structure, dynamics, and protein-DNA interactions. Interpretation of the fluorescence of 2-aminopurine in DNA requires a model of the electronic structure of this fluorophore in its ground and excited states. To this end, electronic structures and energies of the ground and lowest singlet excited states of 2-amino-9-methylpurine were calculated by the multiconfiguration self-consistent field method supplemented by multiconfiguration perturbation theory. The molecular geometry was optimized in both of these electronic states to permit investigation of both electronic excitation and fluorescence emission. The predicted energies and transition dipoles were in good agreement with experiment. The permanent molecular dipole of 2-amino-9-methylpurine increased upon excitation, suggesting that both the absorption and emission spectra should shift to slightly lower energies in polar solvents. The anomalous spectral shifts observed in water suggest that 2-aminopurine undergoes hydrogen bonding that better stabilizes the ground state than the excited state. From the calculated electrostatic potentials of these two states, the position at which this hydrogen bond forms was predicted. These results form a basis for understanding the excited states and possible intermolecular interactions of 2-aminopurine in DNA

Dynamics of biomolecules: assignment of local motions by fluorescence anisotropy decay.

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