(Applicant's Description) The central theme of this Program Project will remain focused on the relationship between molecular structure, thermodynamics, and biological function. By developing novel biological systems that reflect the mutagenic specificity of a single DNA adduct and by using NMR spectroscopy and thermodynamic analysis to establish three-dimensional coordinates and physical properties of duplex DNA containing these adducts, fundamental relationships between structure, energetics, and biological activity can be established. This information will enable us to elucidate and explain mechanisms of mutagenesis and DNA repair at the molecular level. In turn, biological observation raise structural and thermodynamic questions that bring the process of inquiry full circle. Important correlations between structure, energetics, and biological activity have begun to emerge from this research. Insights have been gained into structural and conformational changes which lead to miscoding and/or misalignment of templates during DNA replication. Establishing the three dimensional structure and physical properties of chemically-modified DNA has allowed us to propose mechanisms by which DNA repair enzymes recognize and excise lesions from damaged DNA. Accurate structural and mechanistic information is central to understanding DNA replication and mutagenesis. Mutagenesis produced by endogenous DNA damage, including DNA adducts and oxidized bases is related microscopically to molecular structure and macroscopically to thermodynamics and kinetics, thereby providing a mechanistic rationale for this interdisciplinary research program. The long term goals of this Program include (I) developing new synthetic methods that allow defined lesions to be introduced site-specifically into DNA, (ii) characterizing thermodynamic and extra-thermodynamic impacts of mutagenic lesions on conformational preferences, temperature-dependent transitions, and melting cooperativities of DNA duplexes containing lesions of interest, (iii) determining site-specifically mutational spectra of DNA adducts in human cels and elucidating molecular mechanisms involved in mutagenesis and DNA repair, (iv) relating biological and physical properties of damaged DNA to their three dimensional structure, as determined by 2D NMR and computer-assisted molecular modeling techniques.
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