The long-term goal of this project is to determine the mechanisms by which base mispairs are formed during DNA replication, and how the modification of DNA bases increases base mispairing. The focus of this application on three oxidized pyrimidines, 5-formyluracil, 5-hydroxycytosine, and 5-hydroxyuracil. All of these modified bases are known to occur in cellular DNA, and all are suspected mutagenic base analogs. However, the mechanisms by which these oxidized bases induce mispairing during DNA replication is still unknown. The studies proposed here are intended to reveal why these oxidized pyrimidines miscode during DNA replication. In this project, the hydrogen bonding configurations of the oxidized pyrimidines at a model DNA replication fork using state-of-the-art high field NMR methods will be studied. Oligonucleotide complexes containing bases which have been strategically enriched with the stable isotopes 15-N and 13-C will be constructed. Potential mispair formation might be promoted by aberrant hydrogen bonding, ionized bases, or rare tautomeric forms due to the hydroxyl groups present on the oxidized bases. These possibilities will be evaluated in-depth using NMR methods. In conjunction with the experimental studies, a series of theoretical studies involving both high level quantum mechanics of the oxidized pyrimidines and molecular dynamics simulations of the systems studied by NMR, are proposed. The results of these studies are expected to enhance our understanding of the structure and dynamics of aberrant base pairs in DNA, and the ability to predict the types and frequencies of genetic insults resulting from a wide array of DNA damage.
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