The structure and energetics of damaged DNA alter normal cellular functions and induce DNA repair. The presence of oxidative DNA damage and exocyclic DNA lesions leads to miscoding during DNA replication, a process associated with mutagenesis, cancer and other age-related diseases. The conformation of mutagenic intermediates, together with the properties of DNA polymerases, determines the ultimate outcome of this process. The goal of this project is to perform the structural characterization of lesion-containing DNA and, in so doing, advance our knowledge of damage recognition by DNA giycosylases and of translesion synthesis catalyzed by DNA polymerases.
The specific aims of Project 4 involve (a) the use of multidimensional solution-state NMR spectroscopy coupled with restrained molecular dynamics simulations to establish three-dimensional structures of DNA duplexes containing exocyclic and oxidative DNA lesions; (b) the application of specifically labeled [15N] and [13C] exocyclic adducts to explore dynamic processes related to translesion synthesis and to identify functional groups in the lesion involved in the recognition of oxidative damage; (c) the performance of unrestrained molecular dynamics simulations to elucidate mechanisms of lesion recognition by DNA glycosylases and the energetics of damaged-base eversion from DNA duplexes. Our structural studies are designed to generate information at the molecular level with respect to structural determinants in the recognition of damaged DNA, and to inform the analysis of translesion synthesis events, complementing mutagenesis, crystallographic, and thermodynamic studies conducted in this Program. These interactions are expected to provide new insights on molecular mechanisms associated with lesion formation, DNA repair, and mutagenesis.
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