Conformation and dynamics of normal and damaged DNA.

Abstract

The genetic information that determines the structure and function of living organisms is encoded in the nucleotide sequence of double-stranded DNA molecules. Despite an apparent structural homogeneity displayed by DNA, subtle local variations in structure and dynamics are functionally significant. Short sequences exhibit specificity for regulatory and catalytic proteins, which mediate fundamental processes necessary to the survival of the cell. However, the molecular basis for specific recognition is still incompletely understood. The "indirect readout" mechanism suggests that the relative propensity of DNA to undergo structural deformations induced by the protein contributes to specific protein-DNA recognition. Although the hypothesis was originally formulated to explain recognition of specific nucleic acid sequences by DNA-binding proteins, it may have particular application to the recognition of DNA damage, because damaged sites in DNA have different equilibrium structure and dynamics from undamaged DNA. In this work, we review the approaches that we took to investigate the questions of sequence- and damage-dependent structure and dynamics of DNA. We describe a statistical thermodynamic model that relates DNA configurational flexibility to sequence-specific protein-DNA binding. The model provides a theoretical basis for interpreting experimental measurements of DNA dynamics. We describe results from MCSCF calculations of the excited states of 2-aminopurine (2AP), which provide the theoretical basis for the intramolecular mechanism of quenching as well as the effect of environment on this process. We then describe our investigations of the effect of stacking, base pairing, and base dynamics on the fluorescence of 2-AP in model systems, which allow us to develop the relationships between steady-state and time-resolved fluorescence parameters on the one hand and local structural and dynamic properties of DNA on the other hand. Finally, we describe the application of the experimental approach to study the conformational heterogeneity of DNA abasic sites, a commonly occurring type of DNA damage. We demonstrate the power of the experimental algorithm to characterize the physical differences between undamaged and damaged DNA, as well as the effects of nucleic acid sequence in both of these contexts. Thus, the work described herein comprises a combination of theoretical and experimental approaches to the problem of sequence- and damage-dependent DNA deformation.