The proposed experiments are aimed at providing a more complete understanding of the manner by which alkylation damage to DNA is influenced by the repair protein, O6-alkylguanine-DNA alkyltransferase (AGT). AGT is a major factor in preventing the toxicity of certain alkylating agents. AGT is highly unusual in DNA repair mechanisms since it apparently acts alone to directly remove damage and restore the DNA structure in a single step. The repair reaction uses up the protein since it can act only once. There are five interrelated specific aims. Most of the experimental techniques and reagents needed including mutant AGT proteins and vectors for expressing them are already available in my laboratory. Our recent progress, which includes the derivation of the crystal structure of the human AGT (hAGT) protein, the production of many mutants in hAGT that affect its ability to repair different DNA adducts and development of an in vitro system to study the degradation of the alkylated form of hAGT protein have provided new tools to address these aims which are: (1) To investigate the mechanism by which the toxicity of dibromoethane is paradoxically enhanced by human AGT and the nature of the toxic/mutagenic adducts formed. Mutant forms of hAGT that do not activate dibromoethane will of particular value in this analysis. (2) To fully characterize a novel form of AGT, cAGT-2, which appears to be a more diverse member of the AGT family. It is planned to study the its substrate specificity of cAGT-2 in detail and to examine the functional significance of a long C-terminal extension in this protein. (3) To determine the factors regulating the release from DNA, localization and turnover of the alkylated form of hAGT and forms modified by nitric oxide. Mutants of hAGT including C145S, which is a stable, inactive analog of WT, and C145F, which resembles alkyl- hAGT in susceptibility to degradation, will be used in these studies. (4) To understand structural features of AGTs that determine adduct substrate recognition and alkyl transfer with particular reference to species-specific features that allow recognition of O4-alkylthymines and of bulky O6-alkylguanine adducts such as O6-[4-oxo-(3- pyridyl)butyl]guanine. (5) To characterize and evaluate the biological significance of the differences in hAGT activity imparted by different polymorphic forms of the protein.
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