Malondialdehyde (MDA) and other aldehydic products of oxidative stress represent an important class of endogenous DMA damaging agents universally generated in human beings. These bifunctional electrophiles generate exocyclic adducts to deoxynucleoside bases that prevent Watson-Crick base pairing, block DNA replication, and induce mutations. Our laboratory has defined the chemistry and biology of the pyrimidopurinone adduct to deoxyguanosine, M1dG, which is the major product of DNA damage by MDA and base propenals. Duplex DNA catalyzes a dynamic equilibrium between M1dG and its ring-opened derivative, N2-(3-oxopropenyl)-deoxyguanosine (OPdG). OPdG is less blocking than M1dG to DNA replication and less mutagenic than stable exocyclic analogs of M1dG . We recently discovered that M1dG induces sequence-dependent frameshift mutations in reiterated d(CG)n sequences in E. coli and mammalian cells. We hypothesize that the ability of M1dG to induce frameshift mutations is due to its conversion to OPdG and we propose to test this hypothesis by directly comparing the ability of a series of structural analogs of M1dG and OPdG to induce frameshift mutations in d(CG)n sequences. We recently developed a general synthesis of MDA-DNA adducts in olignucleotides and we propose to use it to explore the chemistry and biology of the other major DNA adduct, N6-(3-oxopropenyl)-deoxyadenosine (OPdA). We will determine the mutation spectrum of OPdA in mammalian cells and we will test the hypothesis that OPdA and other deoxyadenosine products of oxidative stress induce frameshift mutations in reiterated dAn sequences. This type of mutation is commonly observed in critical growth regulating genes that contribute to human cancers and its occurrence has been associated with oxidative stress. MDA-induced DNA-protein cross-links have been reported to exist in cells exposed to oxidants but the identity of these cross-links and the chemistry of their formation is very poorly defined. We propose to explore the chemistry of reaction of M1dG, OPdG, and OPdA with amino acids, peptides, and proteins. Particular attention will be given to the identification of conjugates with DMA-binding proteins such as restriction endonucleases, nucleotide excision repair enzymes, and histones. These experiments will provide critical chemical information with which to evaluate the hypothesis that MDA-induced DNA-protein cross-links are important products of oxidative damage to cells.
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