The broad hypothesis driving the proposed studies is that the products of deoxyribose oxidation in DNA function as a source of endogenous DNA and protein adducts and thus affect the cellular responses to oxidative stress. The basis for these studies is our observation that, like the lipid peroxidation product malondialdehyde, base propenal derived from deoxyribose 4'-oxidation reacts with dG and DNA to form the mutagenic M1G adduct. The problem is approached with four specific aims: 1) Base propenal and M1G. The contribution of base propenal to the cellular burden of MIG will be assessed in two ways: (1) by quantifying M1G in human cells treated with DNA-directed and non-specific oxidants; (2) by quantifying M1G in model cells containing variable polyunsaturated fatty acid content or [13C]-labeling of deoxyribose in DNA. 2) 3'-Phosphoglycolaldehyde residues and glyoxal adducts. These studies build on our observation that phosphoglycolaldehyde residues react to form glyoxal and its dG adducts. We will extend these studies by (1) comparing the formation of phosphoglycolaldehyde and glyoxal adducts with different oxidizing agents in vitro and in cells; and (2) defining the role of deoxyribose oxidation in the cellular burden of glyoxal adducts. 3) DNA adducts derived from products of deoxyribose 5'-oxidation. Having demonstrated the formation of trans-1,4-dioxo-2-butene in gamma-irradiated DNA, we will quantify this lesion in isolated DNA and cells exposed to different oxidants. We have also shown that cis- and trans-1,4-dioxo-2-butene reacts rapidly with dC to form a novel adduct, so we will develop LC/MS technology to quantify this adduct in cells. Finally, we will investigate the 2-phosphoryl-1,4-dioxobutane residue as a precursor to trans-1,4-dioxo-2-butene. 4) Histone adducts derived from 3'-formylphosphate residues. The reversible acetylation of lysine in histones and other chromatin proteins is recognized as an important control of gene expression. We have obtained evidence consistent with an analogous formylation of histones by formylphosphate residues derived from deoxyribose 5'-oxidation. Given the potential for affecting the physiology of chromatin proteins, we propose to characterize the chemistry and biology of protein formylation in cells by (1) quantifying N6-formyllysine residues in nuclei treated with DNA oxidants; (2) defining the source of N6-formyllysine residues; and (3) characterizing the reaction of histone deacetylases with N6-formyllysine residues.
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