Journal Article

Demethylation initiated by ROS1 glycosylase involves random sliding along DNA

María Isabel Ponferrada-Marín, Teresa Roldán-Arjona and Rafael R. Ariza

in Nucleic Acids Research

Volume 40, issue 22, pages 11554-11562
Published in print December 2012 | ISSN: 0305-1048
Published online October 2012 | e-ISSN: 1362-4962 | DOI: http://dx.doi.org/10.1093/nar/gks894

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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.

Journal Article.  5396 words.  Illustrated.

Subjects: Chemistry ; Biochemistry ; Bioinformatics and Computational Biology ; Genetics and Genomics ; Molecular and Cell Biology

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