Date of Thesis

Spring 2024

Description

DNA methylation is a predominant epigenetic regulatory mechanism in eukaryotes which is catalyzed by a group of enzymes called DNA methyltransferases (DNMTs). DNMT1, the focus of this research, is primarily responsible for “maintenance methylation”. During the S phase of cellular growth, DNMT1 binds to hemimethylated DNA (hm-DNA) and is tasked with preserving phenotypic-specific methylation patterns. DNMT1 activity is regulated by a system of intra- and intermolecular regulatory interactions to prevent aberrant methylation. The N-terminal RFTS domain of DNMT1 is a potent inhibitor of methylation. The RFTS domain binds to the active site, physically occluding DNMT1’s interactions with hm-DNA. To relieve RFTS-mediated autoinhibition and localize DNMT1 onto specific genomic regions, the RFTS domain interacts with the SRA domain of UHRF1, an E3 ubiquitin ligase.

Mutations found in the C-terminal lobe of the RFTS domain have been linked to the neurodegenerative disorder, autosomal dominant cerebellar ataxia, deafness, and narcolepsy (ADCA-DN). Specifically, three point mutations – A554V, G589A, and V590F – have been associated with aberrant methylation patterns in patients that include site-specific hypermethylation and genome-wide hypomethylation. This research aims to elucidate the impact of ADCA-DN mutations on DNMT1’s regulatory binding interaction with UHRF1 and the resulting structural alterations. Pull-down assays were not successful in determining the impact of ADCA-DN mutations on UHRF1-mediated localization. Circular dichroism spectroscopy revealed that disease-associated mutations impacted the structure and stability of DNMT1; however, the changes in secondary structure content observed varied between the mutations. These findings may reshape the initial model used to relate the loss of helical content observed in hyperactive DNMT1 to the presumed helix-to-loop transition of the autoinhibitory linker when the intramolecular binding interaction is weakened.

Keywords

DNMT1, UHRF1, ADCA-DN, DNA methylation, protein binding interaction

Access Type

Honors Thesis (Bucknell Access Only)

Degree Type

Bachelor of Science

Major

Biology

Minor, Emphasis, or Concentration

Chemistry

First Advisor

Rebecca Switzer

Second Advisor

Jordan Lingo

Third Advisor

Aaron Osgood-Zimmerman

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