ENZYMATIC REPAIR OF CARCINOGENIC DAMAGE TO HUMAN DNA

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Cells are continuously exposed to chemical and physical agents that can alter the structure and coding properties of DNA. Alkylating chemicals represent the largest class of DNA- damaging agents found in the environment and can result in cellular carcinogenesis, mutagenesis and lethality. Three predominant lesions, 7-methylguanine, O6-methylguanine and 3- methyladenine, are formed in DNA exposed to simple monofunctional alkylating agents. Studies have shown that O6- methylguanine is a mutagenic lesion in DNA, while 3- methyladenine results in cell lethality. Mammalian cells have evolved efficient mechanisms for recognizing and repairing alkylation-induced DNA damages. This proposal describes a comprehensive study of the human 3-methyladenine-DNA glycosylase; the enzyme responsible for the excision of 3- methyladenine from alkylated DNA. The glycosylase will be purified to homogeneity from human lymphoblasts and the substrate specificity against alkylated DNA characterized by high pressure liquid chromatography. Using this purified enzyme as a probe, the effects of DNA base sequence on the formation of 3- methyladenine and its subsequent in vitro and in vivo repair will be quantitated. The cDNA coding for the human 3-methyladenine -DNA glycosylase will be isolated and from that the amino acid sequence of the protein deduced. Further studies will be performed at the molecular level to examine the cellular regulation of gene expression in response to low level exposure to alkylating agents. The repair of alkylation damage in cells from patients with genetic disorders will be analyzed by alkaline sucrose density gradients and compared to that of normal controls to analyze cellular sensitivity to toxic doses of alkylating chemicals. The results of these studies should lead to a better understanding of the molecular processes that constitute the human cellular response to alkylating agents.