Recognition and excision of nucleosomal uracils by human uracil DNA glycosylase. DNA damage in cells must be repaired to maintain the integrity of the genome and prevent disease- causing mutations. One form of DNA damage is the incorporation of uracil into DNA through polymerase error or cytosine deamination. The main enzyme for removing uracil from DNA is the initiator of the uracil base excision repair pathway, uracil DNA glycosylase (UNG). UNG is conserved in all forms of life and has developed additional roles in antibody diversity and viral defense in eukaryotic cells. Most studies on UNG have examined the excision of uracil from naked DNA substrates, however uracil DNA damage in eukaryotes is also present in nucleosomes.
The first aim of this study is to determine the mechanism by which uracil accesses nucleosomal substrates. It is expected that for uracils that are adjacent to histones rather than solvent facing, will require nucleosomal dynamics to expose the site to the enzyme. The nature of nucleosomal dynamics required such as end unwrapping or local DNA bulging will be determined by examining the nucleosomal position dependence of uracil excision by UNG.
The second aim of this study is to measure how the UNG damage search process changes in the context of nucleosomal. Previous studies on the E.coli UNG and free DNA have shown that the enzyme uses hopping and sliding steps to find uracil damage. The efficiency of transfer between uracils is expected to chain drastically in the protein rich, rigid DNA environment of the nucleosomes. By measuring the intramolecular transfer efficiency of UNG between several pairs of uracils introduced into nucleosomes, the mechanism of the UNG uracil search will be examined.
Uracil DNA glycosylase (UNG) is a ubiquitous enzyme that initiates the uracil base excision repair pathway. In addition to DNA repair functions, the human UNG plays important roles in viral defense, adaptive immunity and 5-flurouracil chemotherapeutic toxicity. The studies proposed in this application will examine this important enzyme in the biological context of the ucleosomes.
