This research will be done primarily in Russia as an extension of NIH grant # R0l CA 20851. The methylation of DNA plays an important role in the control of gene expression in mammalian cells. The enzymes involved in this process are DNA methyltransferases (MTases) which recognize CpG sequences in DNA and catalyze the transfer of methyl group from S-adenosyl-L-methionine to form 5-methylcytosine. In different types of tumors, aberrant or accidental methylation of CpG islands in the promoter region has been observed for many cancer-related genes resulting in the silencing of their expression. The polycyclic aromatic hydrocarbons (PAR) such as benzo[a]pyrene or dibenzo[a,1]pyrene are ubiquitous environmental pollutants. They are metabolized in vivo to highly genotoxic dihydrodiol epoxides (DE). Furthermore these diol epoxides bind to cellular DNA causing mutations, thus contributing to the initiation of tumorigenesis. The question how MTases function when carcinogen lesions are present in DNA is still open. It is generally assumed that the target cytosine flipping-out-mechanism, first found for HhaI Mtase occurs in the case of all cytosine-C5-DNAMtases from pro- and eukaryotes. Hence, one of the possibilities may be that distortions of DNA produced by bulky carcinogens which favor extrahelical position of cytosine (displacement of bases, bending, and destabilisation of the double helix) should facilitate DNA methylation. The purpose of this project is to reveal the relationship between DNA methylation and carcinogenic DNA modifications such as PAH lesions, taking prokaryotic cytosine-C5-DNA Mtases EcoRII, SssI and HhaI (recognition sites CCA/TGG, CG, and GCGC, respectively) as models. The general hypothesis to test is that PAR DE-modified DNA can be methylated by DNA MTases with different efficiencies as compared to unmodified DNA.
Specific aim I is to obtain experimental evidences of base-flipping mechanism for EcoRII and SssI Mtases using kinetic and fluorescence studies of the interaction of these enzymes with 2-aminopurine or 2-pyrimidinone- containing substrates analogs.
Specific aim 2 has several components: (i) synthesize a number of stereoisomeric carcinogen-modified substrate analogs of EcoRII, SssI and HhaI Mtases with PAH-guanine and PAH-adenine lesions positioned within the recognition sites of the enzymes, (ii) study PAR-DNA adduct conformations and thermodynamic stabilities, (iii) examine the binding of the adducts to the enzymes, and (iv) investigate the susceptibilities of carcinogen-modified sequences to methylation by the enzymes. The longer range goal of this project is to discover the relationship between the structural features of carcinogen-DNA lesions and the modified DNA sequences, the catalytic mechanisms of DNA methylation, and the susceptibilities of PAH-modified DNA sequences to methylation. The results should provide novel insights into the mechanisms of carcinogenesis associated with the formation of the stereoisomeric PAR diol epoxide-DNA lesions and their impact on biological methylation.
