Date of Thesis

Spring 2020


DNA methylation is a form of eukaryotic epigenetic gene regulation. A family of proteins called DNA methyltransferases (DNMTs) add methyl groups to cytosines in DNA thereby causing gene repression. The DNMT isozyme primarily responsible for maintenance methylation, DNMT1, preferentially methylates the hemi-methylated DNA that forms as a result of cell division. DNMT1 is a multidomain protein containing a C terminal catalytic methyltransferase domain and an N-terminal regulatory region that contains the Replication Foci Targeting Sequence (RFTS) domain. This domain acts as an inhibitor of DNMT1 activity by binding to the active site and preventing DNA binding. In addition, this domain is involved in many intermolecular protein-protein interactions that serve to localize and activate DNMT1. Thus, the RFTS domain is an important hub for intra- and intermolecular binding interactions that regulate DNMT1 activity in cells. Point mutations in the RFTS domain have been shown to be causal for adult-onset neurodegenerative disorders, though little is known about how these mutations affect the structure and function of DNMT1. My research is focused on two disease-causing mutations: G589A and V590F. These mutations significantly decrease the thermal stability of the RFTS domain. The mutant domains exhibit at least a 2.5 °C decrease in observed melting temperature as compared to the wild-type domain. I also examined DNMT1 activity using a fluorescence-based DNA methylation assay. Adding wild-type RFTS domain to RFTS-lacking DNMT1 is known to inhibit DNA methylation. I accessed the ability of mutant RFTS domains to inhibit RFTS-lacking DNMT1. Under identical assay conditions, the IC50 values obtained for wild-type, G589A, and V590F RFTS domains were 0.9 ± 0.1 µM, 3.6 ± 0.6 µM, and 6.5 ± 0.9 µM respectively. Thus, the mutations weakened the observed inhibition. In addition, I used isothermal titration calorimetry to study the binding interaction between the RFTS domain of DNMT1 and the SRA domain of UHRF1, an important binding partner that activates DNMT1. Early experiments indicate a Kd of approximately 1.8 µM for this interaction. This lays the groundwork for studying how the disease-associated mutations impact this regulatory binding interaction. Taken together, these data suggest that the disease-causing mutations G589A and V590F decrease overall protein stability and, at least partially relieve normal RFTS-mediated autoinhibition of DNMT1. In addition, this work will serve as a starting point for investigating how these disease-associated RFTS mutations affect intermolecular binding interactions.


DNMT1, DNA methyltransferase 1, epigenetics, RFTS, replication foci targeting sequence domain

Access Type

Honors Thesis (Bucknell Access Only)

Degree Type

Bachelor of Science



Minor, Emphasis, or Concentration


First Advisor

Rebecca Switzer