DNA repair enzymes detect damaged DNA bases in the dramatically different contexts of naked duplex DNA as well as nucleosomes and chromatin. These fascinating and essential molecular recognition processes require dynamic motions of the DNA base pairs to expose damaged bases to the enzyme, and also, facilitated diffusion of the enzyme along the DNA chain so that stretches of the DNA chain can be meticulously interrogated in a single bimolecular encounter event. A fundamental understanding of damage recognition in the contexts of naked DNA and nucleosomes requires a paradigm system that is especially amenable to study using a wide variety of biophysical tools. One such system is the uracil base excision repair pathway (UBER). This multienzyme pathway is initiated by the enzyme uracil DNA glycosylase (UNG), which efficiently locates and excises uracil bases from DNA. The biomedical relevance of UBER arises from the central role of this pathway in the adaptive immune response, in providing innate immunity against viruses, and in mediating the therapeutic effects of fluoropyrimidine anticancer drugs. In addition, aberrant UBER has been related to carcinogenesis. The characterization of the molecular nature of these interactions provides the basis for identifying new targets for biomedical intervention in the immune response, cancer therapy and viral pathogenesis. The focus of this proposal is to: (1) Elucidate the mechanism and molecular interactions that allow human UNG to execute intramolecular facilitated transfer between uracil sites in duplex DNA. Using NMR paramagnetic relaxation enhancement (PRE) methods, we will characterize transient and poorly populated binding modes of hUNG to nonspecific and specific DNA that are important for intramolecular site transfer. Based on unique structural insights, we will mutate specific residues on UNG, and make discrete perturbations of functional groups on the DNA, to uncover enzyme-DNA interactions important for intramolecular transfer. (2) Elucidate the role of nucleosomal DNA dynamics in uracil excision by hUNG. We will use newly conceived NMR and biophysical methods to explore the dynamic properties of DNA in mononucleosomes for the first time, and we will elucidate the mechanism by which UNG locates and repairs uracil sites imbedded in nucleosomal DNA. (3) Determine the mechanism and efficiency of intramolecular facilitated transfer by hUNG between uracil sites embedded in nucleosomes. The significance and mechanism of intramolecular site transfer in the context of mononucleosomes will be elucidated using novel biophysical and chemical approaches. In particular, we will discern whether the nucleosome surface provides a pathway for intramolecular transfer, whether the DNA scaffold is the primary conduit, or if site location occurs by diffusion from bulk solution.
The biomedical relevance of uracil base excision repair (UBER) arises from the central role of this pathway in the adaptive immune response, in providing innate immunity against viruses, and in mediating the therapeutic effects of fluoropyrimidine anticancer drugs. In addition, aberrant UBER has been related to carcinogenesis. The characterization of the molecular interactions in this pathway provides the basis for identifying new targets for biomedical intervention in the immune response, cancer therapy and viral pathogenesis.
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