The Carboxy-Terminal Domain of ROS1 Is Essential for 5-Methylcytosine DNA Glycosylase Activity

Abstract

Arabidopsis thaliana Repressor of silencing 1 (ROS1) is a multi-domain bifunctional DNA glycosylase/lyase, which excises 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) as well as thymine and 5-hydroxymethyluracil (i.e., the deamination products of 5mC and 5hmC) when paired with a guanine, leaving an apyrimidinic (AP) site that is subsequently incised by the lyase activity. ROS1 is slow in base excision and fast in AP lyase activity, indicating that the recognition of pyrimidine modifications might be a rate-limiting step. In the C-terminal half, the enzyme harbors a Helix-hairpin-Helix DNA glycosylase domain followed by a unique C-terminal domain. We show that the isolated glycosylase domain is inactive for base excision, but retains partial AP lyase activity. Addition of the C-terminal domain restores the base excision activity and increases the AP lyase activity as well. Furthermore, the two domains remain tightly associated and can be co-purified by chromatography. We suggest that the C-terminal domain of ROS1 is indispensable for the 5mC DNA glycosylase activity of ROS1.

Expression of the C-Terminal Domain of Mammalian <i>TET</i>3 DNA Dioxygenase in <i>Arabidopsis thaliana</i> Induces Heritable Methylation Changes at <i>rDNA</i> Loci

“…The latter explanation, however, becomes less likely in view of the presence of 5 hmC marks in TET3c containing lines. Plant DNA glycosylases have been shown to process 5 hmC in vitro [27]- [29] but our data argue against an efficient 5 hmC conversion rate in vivo. While 5 hmC levels are higher in the hypermethylated line B compared to the hypomethylated line A, both lines have comparable 5 hmC/5 mC ratios, with 33% for line A and 43% for line B.…”

“…This is surprising considering that in vitro assay has shown a lack of 5 fC-specific excision activity for ROS [29] and for DME and DML3 [28].…”

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.…”

The Mechanisms of Generation, Recognition, and Erasure of DNA 5-Methylcytosine and Thymine Oxidations

“…the deamination products of 5mC and 5hmC) when paired with a guanine, leaving an apyrimidinic site that is subsequently incised by the lyase activity (75). ROS1 is slow in base excision and fast in apyrimidinic lyase activity in vitro (71,76), indicating that the recognition of pyrimidine modifications might be a rate-limiting step or that other cofactors might be needed to stimulate ROS1 enzymatic activity (in a manner similar to that described for Dnmt3a activity stimulation by Dnmt3L (77)). Mammalian DNA glycosylases that excise 5mC or 5hmC have not been identified, but such activities have been reported (78,79).…”