Domain structure of the DEMETER 5-methylcytosine DNA glycosylase

Abstract

DNA glycosylases initiate the base excision repair (BER) pathway by excising damaged, mismatched, or otherwise modified bases. Animals and plants independently evolved active BER-dependent DNA demethylation mechanisms important for epigenetic reprogramming. One such DNA demethylation mechanism is uniquely initiated in plants by DEMETER (DME)-class DNA glycosylases. Arabidopsis DME family glycosylases contain a conserved helixhairpin-helix domain present in both prokaryotic and eukaryotic DNA glycosylases as well as two domains A and B of unknown function that are unique to this family. Here, we employed a mutagenesis approach to screen for DME residues critical for DNA glycosylase activity. This analysis revealed that amino acids clustered in all three domains, but not in the intervening variable regions, are required for in vitro 5-methylcytosine excision activity. Amino acids in domain A were found to be required for nonspecific DNA binding, a prerequisite for 5-methylcytosine excision. In addition, mutational analysis confirmed the importance of the iron-sulfur cluster motif to base excision activity. Thus, the DME DNA glycosylase has a unique structure composed of three essential domains that all function in 5-methylcytosine excision.DNA repair | helix-hairpin-helix protein | mutagenesis | mixed charge cluster T he modified base 5-methylcytosine (5mC) is a stable epigenetic mark that silences gene expression and plays an important role in many developmental processes such as gene imprinting, X-chromosome inactivation, and transposon silencing (1-4). DNA methylation primarily occurs at symmetric CG sequences in animals, whereas DNA methylation in plants occurs in all sequence contexts: CG, CHG, and CHH (where H ¼ A, T, or C) (5). The overall CG DNA methylation pattern in the genome is faithfully inherited to daughter cells by maintenance DNA methyltransferases, which convert hemimethylated sites generated by DNA replication to fully methylated sites. When maintenance methyltransferases are absent or down-regulated, DNA methylation is progressively lost during replication, which is referred to as passive DNA demethylation. By contrast, active DNA demethylation that is independent of DNA replication requires alternative pathways.Base excision repair (BER), which normally functions to repair damaged and mispaired bases, is also required for active DNA demethylation and epigenetic reprogramming in eukaryotes (3, 6-9). BER is initiated by DNA glycosylase enzymes that catalyze the hydrolysis of the N-glycosidic (base-ribose) bond. In plants, the DEMETER (DME) family of DNA glycosylases functions to remove 5mC, which is then replaced by unmethylated cytosine (10, 11), resulting in transcriptional activation of target genes (10,12). Arabidopsis has three other DME-like (DML) genes-ROS1, DML2, and DML3 (12-14) (Fig. S1). DME is essential for plant reproduction and influences the endosperm DNA methylation profile (15, 16), whereas DMLs function in vegetative tissues to prevent inappropriate gene silencing and maintain ...

Genomic DNA methylation analyses reveal the distinct profiles in castor bean seeds with persistent endosperms

“…Similar to the amino acid sequences in Arabidopsis, identified DNA methyltransferases (including MET1, DRM, CMT, and Dnmt2) and demethylases (including DME, ROS1, DML2, and DML3) from castor bean shared the conserved domains (Supplemental Figs. S1-S5), as reported previously in other plants (Finnegan and Dennis, 1993;Mok et al, 2010;Qian et al, 2014). Based on the structural features of these genes, in particular the presence and distribution of domains and motifs (Supplemental Fig.…”

Targeted DNA demethylation in human cells by fusion of a plant 5-methylcytosine DNA glycosylase to a sequence-specific DNA binding domain

“…GBD contains a Cys 6 -zinc cluster motif 26 and, as other zinc-coordinating DNA-binding proteins is expected to bind non-specifically to DNA while searching for its target site among an enormous amount of non-specific sites. 27 Furthermore, it has been reported that GBD is able to recognizes a variety of UAS sequences that adjust to the general formula (A/C)GGN [10][11][12] CCG and that DNA binding specificity is more stringent in vivo than in vitro. 28 We have shown that transient expression of GBD-ROS1_CD in HEK293 cells significantly increases transcription of a methylated 5xUAS-TK-Luc reporter gene.…”

“…Convincing biochemical and genetic evidence indicates that proteins belonging to a plant-specific family of DNA glycosylases directly excise 5mC from DNA and initiate active DNA demethylation through a base excision repair (BER) pathway. [7][8][9] Arabidopsis thaliana REPRESSOR OF SILENCING 1 (ROS1) is a representative member of this 5mC DNA glycosylase family, whose members are uniquely characterized by a discontinuous DNA glycosylase domain, 10 a conserved C-terminal domain that is essential for the catalytic activity, 11,12 and a basic amino-terminal domain that mediates nonspecific binding to DNA but it is not required for catalysis. 13 The understanding of active DNA demethylation mechanisms may have broad implications for epigenetic editing, a new discipline aimed to develop molecular tools to modulate gene expression.…”

Current status and prospects of epigenetic information in sexual reproductive processes of plants

“…DME 유전자는 비교적 큰 단백질을 코드하지만, C-말단 측 에 염기 제거복구에 관련된 DNA glycosylase와 비슷한 domain 구조를 가지고 있다 (Mok et al 2010). 애기장대에서 이와 상 동성이 높은 DME 유전자, DML2 유전자, DML3 유전자, ROS1 유전자 등 4개를 분리하였다고 하였다 (Zhu et al 2009).…”