) This project provides a biological focus for this program. In it, we explore molecular and cellular events involved in mutagenesis and DNA repair, relating our findings to the structure and thermodynamic properties of damaged DNA. Our experiments focus on exocyclic DNA adducts and oxidative lesions, endogenous forms of DNA damage that may play an important role in the initiation of human cancer. Using a novel double strand shuttle vector system containing a single defined DNA adduct, we propose to establish in human cells the mutagenic specificity of site-specifically placed lesions and to investigate the effects of sequence context on nucleotide misincorporation during translesion synthesis. We also will explore the repair and mutagenicity of complex DNA damages, including interstrand crosslinks and bistrand abasic sites. Increased rates of nucleotide misincorporation have been observed up to five bases away from the site of damage, suggesting these events take place in a thermodynamically destabilized region of DNA. Using purified DNA polymerases, we plan to establish efficiency and fidelity of DNA synthesis at positions remote from the lesion site, relating these effects to thermodynamic and structural properties of damaged DNA. We also will investigate the effects of proofreading on translesion synthesis. Accurate repair of DNA damage is critical to the survival of living organisms. We will examine mechanisms by which DNA repair enzymes recognize damaged DNA by determining the crystal structures of selected DNA glycosylases complexed to non-hydrolyzable substrates. In addition, we hope to isolate and characterize novel DNA glycosylases that selectively excise exocyclic adducts from DNA.
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