Fapy-dG adducts arise from cellular oxidative damage and exposures to various DNA alkylating agents. The chemistry is complex, involving interconversions between alpha and beta deoxyribose anomers, and conformational rearrangements about the C5-N5 bonds and the formyl bond. All of these interconversions are dependent upon (1) functionality of the Fapy-dG substitutent, (2) DNA sequence and (3) single-strand vs. double strand DNA. Moreover, they may also be dependent upon specific DNA-protein interactions, e.g., during replication bypass by Y-family polymerases. The central goal is to characterize the structural biology of Fapy-dG lesions in DNA, including these configurationally and conformational re-arrangements. Effort will initially focus on the MeFapy-dG lesion, and will be extended to the native (un-substituted) Fapy-dG, oxoethyl Fapy-dG, and nitrogen mustard-derived Fapy-dG lesions, including interstrand DNA cross-links, all of which will be provided by Project 1 and the DNA Synthesis Core. The first specific aim will focus on structures and dynamics of these lesions in DNA, which is relevant to their recognition and repair, e.g., by DNA glycosylases such as human NEIL 1. These studies will primarily involve NMR spectroscopy. We have entered into a collaboration with Prof. Sylvie Doublie (University of Vermont) to examine the structural biology of lesion recognition/repair by NEILI. The second specific aim will focus on error-prone lesion bypass. These studies will primarily involve crystallography, combined with NMR spectroscopy. Structural data, especially targeting human pol eta and pol kappa bypass, will be correlated with biological data regarding site-specific mutagenesis in mammalian cells, siRNA knock-down studies targeting specific polymerases, and replication bypass studies with specific Y-family polymerases, to be obtained by Projects 1 and 2. The third specific aim will focus on the role of Fapy-dG lesions in mediating interstrand DNA cross- linking by nitrogen mustards, an important class of anti-tumor agents. The structures of nitrogen mustard interstrand crosslinks will be determined. These studies will primarily involve NMR spectroscopy, combined with some crystallography.
DNA adduction, and its effects upon genome integrity, are involved in the etiology of cancer. Fapy-dG lesions are an understudied class of highly mutagenic DNA lesions. Additionally, nitrogen mustard-derived Fapy-dG lesions function in cancer chemotherapy as cross-linking agents, and this chemistry remains poorly understood. Understanding how they are recognized, repaired, or subjected to DNA bypass polymerization requires understanding their structural biology in DNA and in the presence of Y-family polymerases.
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