The objective of this proposal is to understand in detail how DNA adducts on the O6-position of guanine and the O4 position of thymine are repaired in mammalian cells and the extent to which such repair protects cells from the toxic and mutagenic effects of alkylating agents. Experiments will focus on the human protein, O6-alkylguanine-DNA alkyltransferase [AGT] and an E. coli equivalent, Ada-C, which are able to repair such adducts in a single step by transferring an alkyl group from DNA onto themselves at a cysteine receptor site. The alkylating agents to be studied include both potential environmental carcinogens and cancer chemotherapeutic agents. There are 6 specific aims: (1) to obtain the crystal structure of AGT and of Ada-C with an attached substrate. In order to prevent reaction, inactive C145A mutant AGT or a non-metabolizing substrate analog will be used. (2) to study the structure and function of AGT and Ada-C in the repair of O6-alkylguanine adducts. Mutations have already been identified that change the ability of the proteins to react with O6-benzylguanine [b6G] as a free base and in oligodeoxynucleotide substrates. Comparisons of these and other mutants and with AGT's from other species will be used to define the components of the active site that allow the binding and repair of larger adducts. The ability of these proteins to protect E. coli from killing and the formation of G-C to A-T transition mutations by methylating, ethylating, chlorethylating and benzylating agents will be examined. (3) To study the features in AGT needed for the efficient repair of O4-alkylthymine. This is a less favored substrate of the AGT but is repaired more rapidly by alkyltransferases from other species. These studies will indicate which residues are important in the efficient repair of this adduct. (4) To study the DNA binding properties of AGT and Ada-C. Experiments will be carried out to define the DNA binding domain of these proteins, to measure the affinity of binding to DNA with and without an O6-adduct, and to assess the importance of a conformational change in response to DNA binding on the reaction mechanism. (5) To study the fate and possible function of the alkylated form of the AGT protein. Studies will be carried out in intact cells after exposure to methylated DNA or by b6G and in an in vitro system in which the role of ubiquitin and the effect of mutations that mimic the effect of the S-alkylcysteine can be investigated. (6) To study the cellular localization of the AGT protein and its ability to protect mammalian cells from alkylation damage. Cells transfected with AGT, AGT mutants that may affect nuclear localization and with Ada-C will be examined for distribution of AGT and for protection from alkylating agents.
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