This proposal focuses upon the chemical and structural biology of three types of DNA damage. Malondialdehyde (MDA), produced by lipid peroxidation and prostaglandin biosynthesis, reacts with DNA to form an adduct known as OPdG, which is implicated in human cancer. Aflatoxin B (AFB1), a highly mutagenic mycotoxin, is a contaminant of food. Epidemiological evidence suggests that it acts synergistically with the hepatitis B virus to promote hepatic cancer;these cancers often exhibit mutations in the p53 tumor suppressor gene. The cytochrome P450 oxidation product of AFB1 alkylates the N7 atom of deoxyguanosine. Oxidative damage to thymine from ionizing radiation produces 5,6-dihydroxy-dihydro-2'-thymine (thymine glycol;Tg). Tg inhibits replication by prokaryotic and eukaryotic DNA polymerases, and is implicated in the etiology of human cancer, perhaps associated with the generation of double strand breaks in DNA. The emphasis on these three types of DNA damage is predicated upon the observation that when present in DNA, each of these types of damage can undergo further chemistry depending upon whether the DNA exists in duplex or in single-stranded form, and also depending upon the sequence and identity of the complementary nucleotide in duplex DNA. Thus, in each case the biological response to the lesion is anticipated to depend upon its downstream chemistry in DNA. Some of the secondary lesions;e.g., the AFB1-N7-dG FAPy lesions, are more deleterious than are the initial lesions. This research seeks to delineate the complex chemistry of these lesions in DNA, and to define underlying structure-activity relationships, in an effort to understand their interactions with DNA processing enzymes, such as damage bypass polymerases. To accomplish this, a combination of biochemical and biophysical approaches will be utilized. NMR will be used to determine the structures of site-specific DNA damage in oligodeoxynucleotides. Crystallography will be used to determine structures of sitespecifically damaged oligodeoxynucleotides in complex with DNA polymerases. The resulting structural data will be interpreted in collaboration with colleagues at Vanderbilt University and the University of Connecticut. The ultimate goal is to understand complex cellular responses to specific types of DNA damage.
DNA damage to somatic cells, arising from exogenous and endogenous sources, represents an initiating step in cancer etiology. Coordinated cellular responses to DNA damage are necessary for maintenance of genomic stability. Disruptions within pathways responsible for the restoration of homeostasis following damage lead to alterations that result in cell death or cancer. Developing an understanding of structural perturbations introduced into DNA by genotoxic agents, and their resulting interactions with DNA processing enzymes, is critical to developing a comprehensive understanding of the etiology of cancer.
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