A Biochemical Characterization of the DNA Glycosylase DEMETER

Abstract

The methylation state of DNA is important for gene expression, gene imprinting, X-chromosome inactivation, and transposon silencing in mammals and plants. DNA methylation is established by methyltransferases to mark a silenced gene in the form of 5-methylcytosine (5mC). The mechanism of 5mC removal in mammals remains poorly understood, but recent evidence indicates that DNA glycosylases, which function to remove toxic and mutagenic lesions to DNA, may also function in gene regulation by removing methylated cytosines. This dissertation includes a review of the structural mechanisms of DNA glycosylases, as the 3-dimensional structures of these enzymes have yielded significant insight into substrate specificity and function within the base excision repair (BER) pathway. In Arabidopsis thaliana, DEMETER (DME) is a 5mC DNA glycosylase that activates expression of the maternally imprinted MEDEA gene. Thus, DME has evolved what is normally a DNA repair function to remove the non-toxic 5mC. This dissertation focuses on a structure-function analysis of DME to understand the basis for this unique activity. DME contains a conserved iron-sulfur cluster-containing DNA glycosylase domain, as well as two flanking domains necessary for base excision activity but whose structures and functions are unknown. A homology model of DME constructed from EndoIII was used as a guide for mutational analysis of base excision and DNA binding to identify several residues important for DME activity. Recent literature indicates that removal of 5mC may proceed by removal of oxidation derivatives of 5mC by thymine DNA glycosylase. DME has reduced activity for 5-hydroxymethylcytosine, limited activity for 5-carboxyctyosine, and no activity for 5-formylcytosine. The Arabidopsis DME paralog, DML3, was also studied and compared to reports of the activity of DME paralog ROS1 in order to understand the rationale for high redundancy of 5mC excision in plants.