N-Nitroso compounds are potent environmental carcinogens that are thought to act via an initial alkylation of DNA. Unlike most carcinogens which react at only one, or at most two, sites, N-nitroso compounds alkylate virtually all the potential nucleophilic sites of DNA. However, different N-nitroso compounds give rise to different profiles of adducts. The inital goal of this proposal is to elucidate the exact nature of the intermediate and reaction mechanism responsible for the diversity of DNA adducts. In fact the DNA alkylation pattern of compounds that are larger than ethyl is not fully documented. It is proposed to characterize the DNA alkylation profile for a series of methyl N-alkyl-n-nitrosocarbamates (alkyl = methyl, ethy, 1-propyl, 2-propyl, 1-butyl, 2-butyl and benzyl). Concurrent with understanding the mechanism of DNA alkylation is the ability to control where on a DNA template alkylation will occur. It is proposed to synthesize N-methyl-N-nitrosoureas covalently tethered to a DNA intercalator to effect significant increases in DNA alkylation, which could be of potential value in the development of anti-neoplastic agents. It is also proposed to synthesize a similar nitrosourea linked to distamycin, a DNA minor groove binder, with the intent of using the inherent DNA recognition site binding of the groove binder to cause site specific alkylation. If this methodology is effective, it may be possible to chemically induce site-specific mutations in complex DNA templates. Complementing the significance of DNA alkylation is the repair of these adducts, since it is thought that this process may play a major role in the organ- and perhaps species-specificity exhibited by N-nitroso compounds. It is known that there is a protein which repairs the 06-alkylguanine lesion (R=methyl and ethyl) via a suicide reaction that transfer the intact alkyl moiety from the 06-position to a cysteine residue on a receptor site. To date, this mode of repair has been unambiguously documented for methyl and ethyl modifications. It is proposed to study the specificity of this repair protein by determining if it can repair DNA modified with a series of 06-alkylguanine groups (alkyl= 1-propyl, 2-propyl, 1-butyl, 2-butyl and benzyl). Because of reports that there is a transferase in bacterial systems which can repair 04-T and phosphate backbone alkylation lesions, the repair of these adducts by the mammalian protein will be evaluated. Finally, it is proposed to determine if cells that express the human form of this protein can ameliorate the mutagenicity of these larger 06-guanine alkylated adducts.
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