The first step of the base excision repair (BER) pathway of DNA damage is the recognition and excision of damaged bases by lesion-specific N-glycosylases. The glycosylases that remove alkylation-damaged bases from DNA have an unusually broad catalytic specificity to counteract the promiscuous alkylation of DNA bases by a variety of metabolites and environmental toxins. X-ray structures of these enzymes are being determined complexed to alkylated DNA substrates. Their broad catalytic specificities are not fully explained by the available crystal structures. Catalytic specificity might be manifested during the steps of the reaction, before or after binding of a flipped out nucleotide substrate in the catalytic pocket. For example, alkylated bases might be more easily flipped out of the DNA helix than normal bases, resulting in more exposure of alkylated bases for binding to the enzyme. We are measuring the kinetic and thermodynamic parameters of DNA binding, nucleotide flipping, cleavage of the glycosylic bond, and product release for alkylated and undamaged DNA substrates, in order identify the selectivity determining steps of the reaction that have not been captured in the crystal structures. BER is functionally linked to other DNA repair and recombination processes, which together maintain the chemical and structural integrity of the genome. We are interested in the physical interactions between the damage sensors and the proteins that transmit signals causing the cessation of cell growth and coordination of different DNA repair processes. We have begun crystallographic studies of proteins that are defective in Fanconi Anemia (FA) patients. Cells from FA patients are very sensitive to DNA crosslinking agents, exhibiting severe cytogenetic anomalies after exposure to psoralen or mitomycin C. Available evidence suggests the FANC proteins function in a damage response pathway. They are not homologous to proteins of known function and their structures are likely to provide important clues about this signaling pathway and about the repair of DNA interstrand crosslinks. The silent information regulators (SIR proteins) of yeast form a protein complex that has histone deacetylase activity that is required for transcriptional silencing. The SIR proteins have less well characterized roles in the joining of nonhomolgous DNA ends and DNA repair. Interactions of the yeast Sir2p, Sir3p, and Sir4p proteins with one another and with histone proteins are being studied biochemically and crystallographically.
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