Considerable attention has been focused recently on endogenous metabolic products as sources of DNA damage. Our laboratory has demonstrated that DNA adducts derived from the lipid oxidation product, malondialdehyde (MDA), are present in genomic DNA of healthy human beings. The major MDA-DNA adduct is a pyrimidopurinone that we call M1G; we and others have detected M1G in DNA from human liver, leukocytes, pancreas, and breast. Its identity in liver and leukocyte DNA has been verified by mass spectroscopy. Site-specific mutagenesis experiments with double-stranded viral genomes demonstrate that M1G induces mutations to T and to A in Escherichia coli and that it is repaired by nucleotide excision repair and by mismatch repair. Recently, we discovered that M1G undergoes reversible hydrolytic ring-opening to N2-oxopropenyl-G when it is present in duplex DNA opposite dC residues. This finding has important implications for the biological responses elicited by M1G, because it establishes that M1G is an electrophile in DNA with a reactive center located in the major groove. Its ring-opening product, N2-oxopropenyl-dG, presents an electrophilic center in the minor groove. Random mutagenesis experiments in bacterial and human cells suggest that other MDA-DNA adducts may be biologically important endogenous lesions, as well. Foremost among these are N6-oxopropenyl-A (M1A) and an enaminoimine N2-G-N2-G interstrand cross-link. We seek to define the chemistry of MDA-DNA adducts and to relate it to the biology that they exert. In the present application, we propose to 1) determine the kinetics of ring-opening and ring-closing of M1G and N2-oxopropenyl-G in different forms of DNA; 2) define the chemistry of of M1G-mediated DNA-DNA and DNA-protein cross-link formation; 3) determine the kinetics and products of bypass of M1G, N2- oxopropenyl-G, M1A, and an N2-G-N2-G trimethylene cross-link by the Klenow fragment of DNA polymerase I and by DNA polymerase V; and 4) determine the effects of M1G, N2-oxopropenyl-G, M1A, and an N2-G-N2-G trimethylene cross-link on DNA replication in bacterial and mammalian cells. The results of these experiments will define the chemistry and biology of a family of structurally dynamic lesions in the genome derived from an endogenous metabolic product of lipid oxidation.
Showing the most recent 10 out of 44 publications
