Date of Thesis
The addition of a methyl group to cytosine at CpG sites in DNA is a major epigenetic mechanism in humans. Faithful replication of epigenetic markers through successive rounds of DNA replication is crucial for preservation of cell specific gene expression. Failure to maintain methylation patterns, the primary role of human DNA methyltransferase 1 (DNMT1), has been linked to the development of a spectrum of neurodegenerative disorders. Specifically, mutations in the regulatory replication focus targeting sequence (RFTS) domain of DNMT1 have been linked to the neurodegenerative disorder hereditary sensory and autonomic neuropathy type 1E (HSAN1E). HSAN1E is characterized by sensory neuropathy, hearing loss, and cognitive decline, on average presenting at 37.7 years of age with survival to 53.6 years of age. The objective of this thesis is to investigate how three RFTS point mutants (C353F, P491L, and I531N) identified in HSAN1E kinships contribute to the associated phenotype of global hypomethylation and site-specific hypermethylation. The RFTS domain is known to act in an autoinhibitory manner, binding the catalytic domain and preventing DNA binding; we hypothesized that proteins containing RFTS mutations would have decreased overall stability and partial relief of autoinhibition leading to increased activity. Two of the mutant proteins (C353F and I531N) were purified to homogeneity; the third (P491L) was not soluble. The purified enzymes exhibited increased heparin affinity, reduced thermal stability, and increased DNA binding activity as compared to the wildtype enzyme. The mutant C353F also exhibited increased temperature-dependent aggregate formation. Combined, these results support the claim that these disease-causing mutations in the RFTS domain result in proteins that are less stable but more active than wildtype DNMT1, contributing to a lab-wide effort to better understand the molecular mechanisms of disease formation.
Enzyme, DNA Methylation, DNA Methyltransferase 1 (DNMT1), hereditary sensory and autonomic neuropathy type 1E (HSAN1E)
Honors Thesis (Bucknell Access Only)
Bachelor of Science
Minor, Emphasis, or Concentration
Dr. Rebecca Fagan Switzer
Francomacaro, Lisa M., "Functional Analysis of Replication Focus Targeting Sequence Mutations in DNA Methyltransferase 1" (2018). Honors Theses. 440.