The Impact of Disease-Associated Mutations on UHRF1-Mediated Localization of DNA Methyltransferase 1
Date of Thesis
Epigenetic modifications, such as DNA methylation, are a central mechanism of regulating gene expression. DNA methylation is catalyzed by a family of enzymes called DNA methyltransferases (DNMTs). DNMT1 is primarily responsible for methylating hemimethylated DNA after replication to facilitate the inheritance of methylation patterns. DNMT1 requires a system of intra- and intermolecular regulators to ensure faithful methylation transfer. The RFTS domain is an N-terminal regulatory domain of DNMT1 that binds to the active site and potently inhibits methyl transfer. Intermolecular regulators can bind to the RFTS domain, removing it from the active site, to allow for efficient methyl transfer. These protein-protein interactions also serve to recruit DNMT1 to hemimethylated loci. UHRF1, an E3 ubiquitin ligase, is one such regulator required for the accurate maintenance of DNA methylation patterns in cells. Mutations in the RFTS domain of DNMT1 are associated with adult-onset neurodegenerative diseases characterized by aberrant DNA methylation patterns. Three mutations, G589A, V590F, and A554V, are associated with one such neurodegenerative disease called autosomal dominant cerebellar ataxia – deafness and narcolepsy (ADCA-DN). The present work aimed to investigate the structural impacts of these mutations on the RFTS domain and their effects on UHRF1-mediated DNMT1 localization. Circular dichroism spectroscopy analyses found the mutant and wild-type RFTS domains had similar secondary structures and thermal stabilities indicating the mutations do not significantly perturb the folding of the RFTS domain. Functional impacts on the proposed model of UHRF1-mediated DNMT1 localization were investigated using a fluorescence polarization-based competition assay. No impairment in this regulatory step was observed for the mutant RFTS domains. Taken together, these findings indicate the ADCA-DN-associated mutations do not significantly alter the structure of the RFTS domain nor its interaction with UHRF1. These findings have important implications for the function of DNMT1 carrying the ADCA-DN-associated mutations in humans.
DNMT1, UHRF1, DNA Methylation, Enzyme, Enzyme Regulation, Epigenetics
Honors Thesis (Bucknell Access Only)
Bachelor of Science
Hewett, Geoff, "The Impact of Disease-Associated Mutations on UHRF1-Mediated Localization of DNA Methyltransferase 1" (2022). Honors Theses. 628.