Many alkylating agents react with DNA to afford predominanfiy N7-adducts of deoxyguanosine (dG). The genotoxicity and cytotoxicity of agents that react primarily at N7-dG is often attributed to other less abundant DNA lesions. The overarching hypothesis of this PPG application is the cationic N7-dG adducts are converted into N5-substituted formamidopyrimidine (Fapy-dG) lesions in cells, and the latter lesions contribute significantly to the genotoxicity and cytotoxicity of alkylating agents. This Program Project interactively uses organic synthesis, bioanalytical chemistry, structural biology, enzymology, and molecular biology to elucidate the molecular details by which Fapy-dG adducts derived from endogenous and exogenous electrophiles and chemotherapeutic agents alter DNA replication and repair. Project 1 will utilize bioanalytical mass spectrometry, chemical synthesis, and enzymology to address this hypothesis. Efforts will focus on formamidopyrimidine (Fapy-dG) lesions derived from methylating agents such as temozolomide (MeFapy-dG), chlorooxirane (OxEt-Fapy-dG), thioTEPA (AE-Fapy-dG) and nitrogen mustards (NM-Fapy-dG), including interstrand cross-links (FapyG-NM-FapyG, and FapyG-NMG). Temozolomide, thioTEPA, and nitrogen mustards are clinically used chemotherapeutic agents and we hypothesize that the corresponding N5-substituted Fapy-dG lesion plays an important role in their mechanism of action.
The Specific Aims of Project 1 will establish the presence of N5-substituted Fapy-dG lesions in mammalian cells (Specific Aim 1), chemically synthesized oligonucleotides containing the desired Fapy-dG lesion in a site-specifically manner (Specific Aim 2), and determine the miscoding potential of the Fapy-dG lesion in vitro (Specific Aim 3). Our ability to site-specifically incorporate the Fapy-dG lesions into oligonucleotide is central to the research plans of Projects 1, 2 and 3. Project 1 will work closely with the DNA Synthesis Resource Core.
Fapy lesions have been an understudied class of DNA adduct largely due to their complex chemistry. We propose to develop methods to understand the chemistry of these lesions, which will aid in their detection, preparation, and further study. The result will provide a better understanding of the role of DNA alkylating agents in human cancers and well as the mechanism of action of chemotherapuetic agents.
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