Nucleic Acids Research Advance Access published online on May 13, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp390
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Nucleic Acid Enzymes |
ROS1 5-methylcytosine DNA glycosylase is a slow-turnover catalyst that initiates DNA demethylation in a distributive fashion
Departamento de Genética, Universidad de Córdoba, 14071 Córdoba, Spain
*To whom correspondence should be addressed. Tel: +34 957 218 979; Fax: +34 957 212 072; Email: ge1roarr{at}uco.es
Received March 17, 2009. Revised April 29, 2009. Accepted April 29, 2009.
Arabidopsis ROS1 belongs to a family of plant 5-methycytosine DNA glycosylases that initiate DNA demethylation through base excision. ROS1 displays the remarkable capacity to excise 5-meC, and to a lesser extent T, while retaining the ability to discriminate effectively against C and U. We found that replacement of the C5-methyl group by halogen substituents greatly decreased excision of the target base. Furthermore, 5-meC was excised more efficiently from mismatches, whereas excision of T only occurred when mispaired with G. These results suggest that ROS1 specificity arises by a combination of selective recognition at the active site and thermodynamic stability of the target base. We also found that ROS1 is a low-turnover catalyst because it binds tightly to the abasic site left after 5-meC removal. This binding leads to a highly distributive behaviour of the enzyme on DNA substrates containing multiple 5-meC residues, and may help to avoid generation of double-strand breaks during processing of bimethylated CG dinucleotides. We conclude that the biochemical properties of ROS1 are consistent with its proposed role in protecting the plant genome from excess methylation.