Date of Thesis
The most common epigenetic DNA modification in higher eukaryotes is methylation of the 5-carbon of cytosine, almost exclusively at CpG dinucleotides. DNA methyltransferases (DNMTs) are the family of enzymes responsible for the establishment and maintenance of the methylation pattern. As an important epigenetic mark responsible for gene repression, disruption of normal DNA methylation is implicated in several disease states. DNMT1 is the enzyme primarily responsible for maintenance of the methylome. DNMT1 is a multi-domain protein with a C-terminal catalytic methyltransferase domain and a large N-terminal regulatory region. Of special interest is the Replication Foci Targeting Sequence (RFTS) domain found in the N-terminal region; this domain has been shown to be an endogenous inhibitor of DNMT1 activity. Mutations in the RFTS domain were recently shown to be causal for some neurodegenerative diseases. Here, I report on my investigation of V590F and G589A, mutations casual for autosomal dominant cerebellar ataxia, deafness, and narcolepsy (ADCA-DN). I observed a decrease in thermal stability of the two mutant enzymes as compared to wild-type DNMT1. Both mutant proteins showed an increased ability to bind DNA verses the wild-type enzyme. In addition, methylation activity data indicates that the two mutant proteins can methylate DNA faster than the wild-type enzyme. Protein pull-down studies suggest that neither mutation affects the ability of DNMT1 to bind Uhrf1, an important regulatory protein binding partner. Taken together, these studies suggest that DNMT1 mutant enzymes are hyperactive yet destabilized, providing the first biochemical characterization of these disease-causing mutations. A better understanding of the biochemical consequences of these mutations will aid in our understanding of the molecular mechanism of ADCA-DN formation.
protein biochemistry, DNA methyltransferase 1, enzymology, neurodegenerative diseases
Honors Thesis (Bucknell Access Only)
Bachelor of Science
Rebecca L. Switzer
McGinnis, James Henry III, "Determination of the Functional Significance of V590F and G589A Mutations On DNA Methyltransferase 1" (2017). Honors Theses. 399.