Synthesis, structure and biological effects of carcinogen/drug-induced bulky, intercalatable N7-alkylguanine lesions

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Covalent modification of DNA by alkylating mutagens and carcinogens is closely associated with cancer development. A wide variety of alkylating agents are known to attack DNA to produce N7-alkylguanine (N7- alkylG) and alkyl formamidopyrimidine (alkyl-FapyG) lesions. Despite the major advances in the characterization of chemical, biochemical, and/or mutagenic properties of the lesions, our molecular-level understanding of structural and biological effects of carcinogen/drug-induced bulky intercalatable N7-alkylG and alkyl-FapyG adducts is still limited, except for a few lesions such as aflatoxin B1-N7G adducts. This knowledge gap had been due in part to a technical limitation in generating sufficient quantities of DNA containing site-specific incorporated N7-alkylG; although N7-alkylG lesions in duplex DNA have half-lives of several hours to days, the lesions in nucleosides are chemically extremely unstable to rapidly undergo spontaneous depurination, thereby precluding the use of the solid-phase method for the synthesis of N7- alkylG-containing DNA. We previously developed a transition-state destabilization strategy to solve the chemical instability problem and reported the first crystal structure of an N7-alkylG-containing DNA. Our central hypothesis of the proposed research is that bulky, intercalatable N7-alkylG and alkyl-FapyG lesions affect DNA structure and biological processes including DNA replication and mutagenesis. Our long-term research goal is to elucidate the structural and biological effects of carcinogen/drug-induced N7-alkylG and N3-alkyladenine lesions. The objectives here are to elucidate the impacts of N7-alkylG and alkyl-FapyG lesions on DNA structure, replication and mutagenesis and to dissect the DNA repair mechanism of the lesions. To accomplish this objective, we propose synthesis, structure determination, and biochemical evaluation of N7-alkylG and alkyl-FapyG lesions that are induced by potent carcinogens/drugs including N- methylbenzyl nitrosamine, safrole, ptaquiloside, acridine half-mustard ICR-191, nitrogen half-mustard, and a platinum-based “alkylating-like” agent. As a next step for achieving our long-term goals, we have designed three Specific Aims that are 1) Evaluating the impact of the N7-alkylG and alkyl-FapyG lesions on the structure and stability of duplex DNA; 2) Elucidating the mutagenesis mechanisms of the lesions; and 3) Delineating the DNA repair mechanism for the lesions. Our expectation is that the accomplishment of the proposed research would advance our molecular-level understanding of the impact of intercalatable N7-alkylG and alkyl-FapyG adducts on DNA structure, replication and mutagenesis and the repair mechanism of the lesions, thereby providing new insights into the etiology of alkylation-induced mutagenesis and carcinogenesis. In addition, crystal structures of N7-alkylG-containing DNA would facilitate a structure-based design and development of novel alkylating agents that can alter DNA structure and biological processes.