| DNA methylation is a stable epigenetic modification associated with repressed chromatin and gene silencing. Gene imprinting is monoallelic expression according to the parent-of-origin and often involves allele-specific differential methylation. Many imprinted genes play an important role during reproduction in mammals and angiosperms. The DEMETER (DME) gene is responsible for maternal allele hypomethylation and expression of the imprinted genes in the central cell and endosperm. DME has bifunctional DNA glycosylase/AP-lyase activity and can excise 5-methylcytosine in vitro, regardless of the sequence context. DME also possesses mismatch base excision activity on both thymine and uracil when paired with guanine. When expressed in E. coli, DME shows cytotoxicity perhaps by removing 5-methylcytosine in the genome leading to the formation of abasic sites and/or nicks. Cytosine methylation-deficient strains are resistant to DME expression. Abasic sites opposite 5-methylcytosine inhibit DME activity and may prevent DME from generating double-stranded DNA breaks. Deletion and point mutation analyses revealed that the conserved domains and the Fe-S cluster are important for DME enzyme activity. Taking advantage of DME cytotoxicity to E. coli cells, mutations which disrupt the enzyme activity have been identified through random mutagenesis. These results suggest that DME, a DNA repair enzyme, activates gene transcription by removing 5-methylcytosine. |
