The exocyclic pyrimidopurinone DNA adduct, M1dG, is the major product of the reaction of DNA with malondialdehyde or base propenals, which are endogenously generated during the oxidation of polyunsaturated fatty acids or the deoxyribose moieties of DNA, respectively. M1dG gives rise to base substitution and frameshift mutations, and its levels in human and rodent DNA correlate with exposure to oxidative stress in some model systems. We have discovered that M1dG in genomic DNA is oxidized by an as yet unknown nuclear enzyme to the stable and distinct DNA adduct, 6-oxo-M1dG. In cells studied to date, oxidation to 6-oxo-M1dG occurs more rapidly than M1dG repair. Unlike M1dG, which can ring-open to the less mutagenic N2-oxopropenyl-dG when placed opposite dC in duplex DNA, 6-oxo-M1dG is predicted to retain its exocyclic ring rendering it more locally disruptive to the DNA double helix and therefore more mutagenic than M1dG. We will test this hypothesis by (1) using NMR spectroscopy to elucidate the structure of duplex oligonucleotides containing 6-oxo-M1dG, (2) determining the mutagenicity of 6-oxo-M1dG during in vitro replication by translesion polymerases and in vivo replication in E. coli, and (3) identifying, purifying, expressing, and characterizing the enzyme responsible for the oxidation of M1dG. Support of the hypothesis that 6-oxo- M1dG is more mutagenic than M1dG will provide the first demonstration of the metabolic activation of an adduct in genomic DNA. This entirely new concept regarding the disposition of DNA damage lays the foundation for important future studies of how the metabolic products of specific DNA adducts lead to mutations and the resulting genomic dysfunction that is the hallmark of cancer.
The DNA adduct, M1dG, is the most common form of damage resulting from the reaction of the endogenous mutagen malondialdehyde with DNA. We have discovered that M1dG in genomic DNA is oxidized to a new stable adduct 6-oxo-M1dG more quickly than it is repaired. We propose to test the hypothesis that formation of 6-oxo-M1dG represents metabolic activation of a DNA adduct to a more mutagenic adduct, a process that may have important implications for understanding how DNA damage contributes to carcinogenesis.
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