DNA damage is an important cause of genetic diseases. The chemical events that lead to DNA damage include hydrolysis, oxidation and electrophilic attack. These reactions are triggered by exposure of cells to exogenous chemicals (e.g., environmental agents, food constituents, etc.) or they can result from endogenous metabolic processes. Exogenous chemicals were once thought to be the major sources of DNA damage in human beings, but advances in analytical chemistry reveal the existence of diverse and abundant types of damage resulting from endogenous sources. The integration of synthetic chemistry with molecular biology provides strategies for the construction of small genomes containing single lesions at defined sites for evaluation of mutagenic potential and repair. And the generation of organisms bearing targeted deletions in specific DNA repair genes makes it possible to evaluate the mutational consequences of increased steady-state levels of particular types of lesions.

Our laboratory has focused on DNA damage by aldehydes produced endogenously in mammalian cells as a result of lipid peroxidation. Malondialdehyde is the major mutagenic product of lipid peroxidation and is produced essentially ubiquitously in animal and human tissues. It reacts with DNA to form a series of adducts that we and others have identified. The major adduct is a pyrimidopurinone that we have abbreviated M₁G. This adduct possesses a blocked Watson-Crick base-pairing region so one anticipates that it is mutagenic. We have evaluated its mutagenicity by synthesizing viral genomes containing M₁G at defined positions. Following transfection into bacterial or mammalian hosts, the replicated genome is interrogated to determine the outcome of replication at the site of the adduct. These experiments indicate that M₁G is indeed mutagenic. We have used a variation of this approach to establish that M₁G is repaired by nucleotide excision repair. To support and extend these in vivo studies, we conduct experiments utilizing adduct-containing duplex DNA molecules or template-primers as substrates for purified DNA polymerases or repair enzymes. These investigations provide more detailed information about the structural and functional basis for induction of mutation. Our laboratory has had a long-standing collaboration with the Stone laboratory at Vanderbilt, which has provided precise information about the structural perturbations introduced into DNA by adducts such as M₁G.

Recent work from our group revealed that M₁G is a chemically reactive electrophile in DNA. It is capable of adding nucleophiles to the pyrimidine ring. We are investigating the importance of this reaction in altering the function of DNA, e.g., by forming DNA-DNA or DNA-protein cross-links. These studies led to the discovery that duplex DNA accelerates the hydrolytic ring-opening of M₁G to N²-OPG by atleast four orders of magnitude. We are currently investigating the molecular basis of this impressive rate acceleration and its impact on the mutagenicity of M₁G.

Recent Leading Articles:

L.J. Marnett and J.P. Plastaras "Endogenous DNA Damage and Mutation," Trends in Genetics, 17, 214-221 (2001).

J.P. Plastaras, P.C. Dedon, and L.J. Marnett "Effects of DNA Structure on Oxopropenylation by the Endogenous Mutagens, Malondialdehyde and Base Propenal," Biochemistry, 41, 5033-5042 (2002).

N.C. Schnetz-Boutaud, S. Saleh, L.J. Marnett, and M.P. Stone "The Exocyclic 1,N2-Deoxyguanosine Pyrimidopurinone M₁G is a Chemically Stable DNA Adduct When Placed Opposite a Two-Base Deletion in the (CpG)3 Frameshift Hotspot of the Salmonella typhimurium hisD3052 Gene," Biochemistry 40, 15638-15649 (2001).

L.A. VanderVeen, M.F. Hashim, L.V. Nechev, T.M. Harris, C.M. Harris, and L.J. Marnett "Evaluation of the Mutagenic Potential of the Principal DNA Adduct of Acrolein," J.Biol.Chem., 276, 9066-9070 (2001).

N.Schnetz-Boutaud, J.S. Daniels, M.F. Hashim, P. Scholl, T. Burrus, and L.J. Marnett "Pyrimido[1,2-a]purin-10(3H)-one: A reactive electrophile in the genome," Chem.Res.Toxicol., 13, 967-970 (2000).