Abstract
DNA cytosine methylation is an epigenetic mark that promotes gene silencing and performs critical roles during reproduction and development in both plants and animals. The genomic distribution of DNA methylation is the dynamic outcome of opposing methylation and demethylation processes. In plants, active demethylation occurs through a base excision repair pathway initiated by 5-methycytosine (5-meC) DNA glycosylases of the REPRESSOR OF SILENCING 1 (ROS1)/DEMETER (DME) family. To gain insight into the mechanism by which Arabidopsis ROS1 recognizes and excises 5-meC, we have identified those protein regions that are required for efficient DNA binding and catalysis. We have found that a short N-terminal lysine-rich domain conserved in members of the ROS1/DME family mediates strong methylation-independent binding of ROS1 to DNA and is required for efficient activity on 5-meC.G, but not for T.G processing. Removal of this domain does not significantly affect 5-meC excision from short molecules, but strongly decreases ROS1 activity on long DNA substrates. This region is not required for product binding and is not involved in the distributive behavior of the enzyme on substrates containing multiple 5-meC residues. Altogether, our results suggest that methylation-independent DNA binding allows ROS1 to perform a highly redundant search for efficient excision of a nondamaged, correctly paired base such as 5-meC in long stretches of DNA. These findings may have implications for understanding the evolution of structure and target specificity in DNA glycosylases.
Highlights
That initiate formation of repressive chromatin [2]
We have recently reported that REPRESSOR OF SILENCING 1 (ROS1) activity is facilitated at mismatched 5-meC residues, but strongly inhibited by replacement of the C5 methyl group by halogen derivatives, even if these substituents decrease the strength of the scissile C1Ј-Nglycosidic bond [21]
ROS1 Binds with Similar Affinity to Both Methylated and Nonmethylated DNA—We used the Electrophoretic Mobility Shift Assay (EMSA) to compare the capability of ROS1 to bind methylated and unmethylated
Summary
That initiate formation of repressive chromatin [2]. In higher eukaryotes, methylation of specific loci allows transmission of stable states of gene expression and plays essential roles in the establishment of developmental programs and in genome defense against parasitic mobile elements [3]. We show that a lysine-rich N-terminal domain conserved across the ROS1/DME family allows ROS1 to bind DNA in a methylation-independent manner, selectively favors 5-meC over T excision, and significantly facilitates initiation of DNA demethylation in long DNA substrates.
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