Date of Thesis

Spring 2026

Description

While almost all cells in a multicellular organism have the same genetic information, they do not need to express every protein at once. Certain proteins are needed in certain amounts at certain times, so cells require mechanisms to regulate gene expression. One form of gene expression regulation is through DNA methylation, in which methyl groups are added to CpG dinucleotides in DNA. A group of enzymes known as DNA methyltransferases (DNMTs) are responsible for DNA methylation. DNMT1 is the most abundant human DNMT, as it is required for maintenance methylation after DNA replication. DNMT1 is a large multidomain protein containing an autoinhibitory domain called the replication foci-targeting sequence (RFTS) domain. The RFTS domain autoinhibits DNMT1 activity, as it sits in the catalytic cleft of the methyltransferase domain, preventing unwanted methylation. Mutations in the RFTS domain are associated with two adult-onset neurodegenerative disorders: Hereditary Sensory and Autonomic Neuropathy Type 1E (HSAN1E) and Autosomal Dominant Cerebellar Ataxia, Deafness, and Narcolepsy (ADCA-DN). Previous studies have shown that mutations causal for ADCA-DN occur only in the RFTS domain and relieve RFTSmediated autoinhibition of DNMT1 activity, resulting in aberrant methylation. C580R is a mutation that has been found in ADCA-DN patients, but the biochemical consequences of this mutation are unknown. To investigate the effects of the C580R mutation on the structure, stability, and function of DNMT1, C580R DNMT1 was recombinantly expressed and purified for biochemical analysis. Electrophoretic mobility shift assays suggest that C580R DNMT1 has a higher binding affinity for DNA than WT DNMT1, ix suggesting that the mutated RFTS domain does not function as an autoinhibitory domain, and does not block DNA binding. Methylation assays show that C580R DNMT1 exhibits increased activity in relation to the wild-type (WT) protein. Circular dichroism spectroscopy indicates slight changes in the secondary structure of the mutant RFTS domain, notably a loss of helical content, suggesting that the mutation causes structural changes that potentially destabilize the protein. Finally, protein aggregation assays suggest that the C580R RFTS domain is less stable in solution than the WT RFTS domain. Collectively, these findings reveal that the mutation C580R generates a hyperactive yet unstable mutant DNMT1 enzyme, suggesting a potential link between this mutation and the onset of the disease.

Access Type

Honors Thesis (Bucknell Access Only)

Degree Type

Bachelor of Science

Major

Cell Biology/Biochemistry

First Advisor

Rebecca Switzer

Second Advisor

Sarah Smith

Third Advisor

Ellen Chamberlin

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