Abstract
Active DNA demethylation processes play a critical role in shaping methylation patterns, yet our understanding of the mechanisms involved is still fragmented and incomplete. REPRESSOR OF SILENCING 1 (ROS1) is a prototype member of a family of plant 5-methylcytosine DNA glycosylases that initiate active DNA demethylation through a base excision repair pathway. As ROS1 binds DNA non-specifically, we have critically tested the hypothesis that facilitated diffusion along DNA may contribute to target location by the enzyme. We have found that dissociation of ROS1 from DNA is severely restricted when access to both ends is obstructed by tetraloops obstacles. Unblocking any end facilitates protein dissociation, suggesting that random surface sliding is the main route to a specific target site. We also found that removal of the basic N-terminal domain of ROS1 significantly impairs the sliding capacity of the protein. Finally, we show that sliding increases the catalytic efficiency of ROS1 on 5-meC:G pairs, but not on T:G mispairs, thus suggesting that the enzyme achieves recognition and excision of its two substrate bases by different means. A model is proposed to explain how ROS1 finds its potential targets on DNA.
Highlights
DNA methylation at carbon 5 of cytosine 5-methylcytosine (5-meC) is a reversible epigenetic mark that influences chromatin structure and is usually associated with gene silencing [1], its effects may vary in different genomic contexts [2]
Our previous work revealed that REPRESSOR OF SILENCING 1 (ROS1) binds unmethylated and methylated DNA with similar affinity [21], suggesting that facilitated diffusion along DNA may importantly contribute to target location by ROS1
We have found that ROS1 shows end-dependent dissociation that is severely restricted when access to the DNA ends is obstructed by tetraloops blocks
Summary
DNA methylation at carbon 5 of cytosine 5-methylcytosine (5-meC) is a reversible epigenetic mark that influences chromatin structure and is usually associated with gene silencing [1], its effects may vary in different genomic contexts [2]. DNA methylation is involved in vital physiological processes such as genomic imprinting, X chromosome inactivation, defence against parasitic mobile elements and the establishment of developmental programs [3]. In agreement with such essential roles, aberrant DNA methylation has important consequences for cells and is a crucial component in many forms of human disease, including cancer [4,5]. Active DNA demethylation is initiated by a group of DNA glycosylases, typified by Arabidopsis REPRESSOR OF SILENCING (ROS1) and DEMETER (DME) [7,8]. Animals apparently lack 5-meC DNA glycosylases, but several lines of evidence suggest that demethylation involves excision of de-aminated and/or oxidized derivatives of 5-meC [reviewed in [12]]
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