Endogenous cytosine methylation at 5'-CG-3' sites within the human genome is involved in many physiological processes, including gene expression, host defense, genomic imprinting, and X chromosome inactivation. Interestingly, many tumor types, including smoking-induced lung cancer, are characterized by aberrant DMA methylation patterns. The presence of 5-methylcytosine (MeC) induces small, but noticeable changes in DMA structure and dynamics, leading to altered DNA-protein interactions and chromatin remodeling. Furthermore, MeC is capable of increasing the reactivity of guanine bases in MeCG dinucleotides towards carcinogens. Remarkably, the majority of lung cancer mutational """"""""hot spots"""""""" observed within the p53 tumor suppressor gene are found at endogenously methylated MeCG dinucleotides, e.g. p53 codons 157, 158, 245, 248, and 273. Our previous investigations have revealed that the presence of eC at these sites modulates the reactivity of neighboring guanine bases towards tobacco carcinogens, leading to targeted binding of benzo[a]pyrene diolepoxides (BPDE) to MeCG sequences. In contrast, alkylation of MeCG dinucleotides by reactive metabolites of tobacco specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK), is inhibited. We now propose to determine the structural basis for these effects by conducting stable isotope labeling studies with a series of MeC analogs. We will also test the hypothesis that cytosine methylation status and local DMA sequence context influence guanine adduct formation by reactive oxygen species generated as a result of exposure to tobacco smoke. Finally, any effects of MeC on O6- alkylguanine DMA alkyltransferase-mediated repair of NNK-induced O6-alkylguanine adducts will also be examined. These studies will: 1. Determine the mechanisms by which 5-methylcytosine (MeC) influences the reactivity of neighboring guanine bases towards tobacco carcinogens. 2. Examine the effects of MeC and its structural analogs on the stereochemistry of N2-guanine adducts induced by B[a]P diolepoxides. 3. Map the distribution of oxidative DMA lesions within p53 and K-ras derived DNA sequences. 4. Examine O6-alkylguanine DNA alkyltransferase-catalyzed repair of NNK-induced O6-guanine lesions at methylated and unmethylated CG dinucleotides. The results of this research will identify the mechanisms by which endogenous MeC modulates the reactivity of CG sites towards tobacco carcinogens, providing an insight into the origins of genetic and epigenetic changes observed in lung cancer. Our approach is innovative because our laboratory is employing a novel, mass spectrometry based approach to probe the reactivity of specific bases within DNA duplexes towards alkylating and oxidative agents. The proposed research is significant because of the widespread human exposure to tobacco products and because of their central role in the initiation of lung cancer in humans.
