Investigating the Impact of Disease-Associated Mutations on DNA Methyltransferase 1 Function

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Project Summary In humans, the most common epigenetic DNA modification is methylation of cytosines, predominantly in CpG dinucleotides. Disruption of the normal DNA methylation pattern is known to play a role in several diseases. DNA methyltransferase 1 (DNMT1) is primarily responsible for maintenance of the DNA methylation pattern through multiple rounds of cell division. DNMT1 is a multidomain protein with a C-terminal catalytic methyltransferase domain and a large N-terminal regulatory region. The Replication Focus Targeting Sequence (RFTS) domain, found in the N-terminal regulatory region, is a key regulator of DNMT1 activity in vivo. The RFTS domain binds to the DNA binding site and prevents association with DNA. In addition, the RFTS domain is involved in several protein-protein interactions that serve to localize and activate DNMT1 for catalysis. Recently, mutations in the RFTS domain have been identified that result in two different adult onset neurodegenerative disorders. Affected individuals exhibit modified DNA methylation patterns with global hypomethylation and site-specific hypermethylation. The molecular mechanisms that lead to this altered methylation pattern are still unclear and little is known about the biochemical consequences of these mutations. In this proposal, we seek to understand how these changes in amino acid sequence are impacting the structure and function of DNMT1. For specific aim 1, melting temperatures and fluorescence anisotropy will be used to examine changes in protein stability and dynamics induced by the disease-associated mutations. For specific aim 2, RFTS-mediated autoinhibition will be assessed in the mutant enzymes. Both DNA binding affinity and DNA methylation activity will be examined in wild-type and mutant enzymes to determine if the mutations relieve normal autoinhibition. For specific aim 3, the impact of the mutations on key intermolecular interactions will be examined. The RFTS domain is known to bind to UHRF1 (ubiquitin-like, containing PHD and RING finger domains protein 1) and modified histone H3 tails. Isothermal titration calorimetry will be used to investigate the impact of RFTS mutations on these regulatory protein-protein interactions. Our preliminary work shows that disease-associated mutations G589A and V590F reduce thermal stability of the protein while also increasing DNA binding affinity and catalytic activity, indicating at least partial relief of normal RFTS- mediated autoinhibition in these mutant enzymes. Collectively, these studies represent an excellent training opportunity for undergraduate students. Undergraduates will engage in all aspects of this research and gain hands-on experience designing experiments, collecting and analyzing data, and interpreting results. We expect our biochemical studies to yield key insights into the consequences of the disease-associated mutations that will ultimately aid in our understanding of the molecular mechanisms of disease formation in affected individuals.