Date of Thesis

Fall 2025

Description

Nearly every cell in a multicellular organism contains the same genetic information; however, not all cells need to express the same proteins for proper function. Thus, cells require regulatory mechanisms to ensure the correct genes are expressed. One form of this regulation occurs via DNA methylation, the process by which methyl groups are added to specific regions of DNA. DNA methylation is accomplished by a family of enzymes called DNA methyltransferases (DNMTs). DNMT1, the most abundant methyltransferase in humans, is a large multidomain protein, containing an autoinhibitory domain called the replication foci-targeting sequence (RFTS) domain. This domain must be removed from the active site for DNMT1 to bind and methylate hemimethylated DNA for proper regulation of gene expression. Mutations within the RFTS domain of DNMT1 are associated with the development of two separate adult-onset neurodegenerative disorders: Autosomal Dominant Cerebellar Ataxia, Deafness, and Narcolepsy (ADCA-DN) and Hereditary Sensory and Autonomic Neuropathy Type 1E (HSAN1E). Previous work has shown that mutations associated with development of ADCA-DN relieve normal RFTS-mediated autoinhibition, resulting in hyperactive mutant enzymes. This research aims to elucidate the structural and functional consequences of the Y524D mutation, an RFTS domain mutation that is causal for HSAN1E. To address this question, Y524D DNMT1 was recombinantly expressed and purified for biochemical analysis. Electrophoretic mobility shift assays indicate that Y524D DNMT1 exhibits increased DNA binding ability in comparison with wild-type (WT) DNMT1. Additionally, methylation assays show that Y524D DNMT1 exhibits increased activity relative to WT DNMT1. Finally, protein aggregation assays indicate that Y524D DNMT1 is more prone to form aggregates in solution than the WT enzyme. Taken together, this evidence supports the hypothesis that the disease-associated mutation Y524D relieves autoinhibition, resulting in a hyperactive, yet less stable enzyme.

Keywords

DNA methylation, enzyme function, mutation

Access Type

Honors Thesis (Bucknell Access Only)

Degree Type

Bachelor of Science

Major

Cell Biology/Biochemistry

First Advisor

Rebecca Switzer

Second Advisor

Marie Pizzorno

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