title: 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.
authors.affiliation: Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road, Atlanta, GA 30322, USA; Molecular and Systems Pharmacology Graduate Program, Emory University School of Medicine, 1510 Clifton Road, Atlanta, GA 30322, USA. Electronic address: shong27@emory.edu.
authors.forename: Samuel
authors.initials: S
authors.lastname: Hong
authors.affiliation: Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road, Atlanta, GA 30322, USA. Electronic address: hhashi3@emory.edu.
authors.forename: Hideharu
authors.initials: H
authors.lastname: Hashimoto
authors.affiliation: Department of Radiation Oncology, Emory University School of Medicine, 1510 Clifton Road, Atlanta, GA 30322, USA. Electronic address: ykow@emory.edu.
authors.forename: Yoke Wah
authors.initials: YW
authors.lastname: Kow
authors.affiliation: Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road, Atlanta, GA 30322, USA. Electronic address: xzhan02@emory.edu.
authors.forename: Xing
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road, Atlanta, GA 30322, USA. Electronic address: xcheng@emory.edu.
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Cheng
mesh_terms: D044503:5-Methylcytosine / Q000378:metabolism*; D000818:Animals; D017360:Arabidopsis / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D045647:DNA Glycosylases / Q000235:genetics / Q000378:metabolism*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000378:metabolism*; D051379:Mice; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary
chemical_list: D029681:Arabidopsis Proteins; D009687:Nuclear Proteins; C471271:ROS1 protein, Arabidopsis; D044503:5-Methylcytosine; C093335:5-methylcytosine-DNA glycosylase; D045647:DNA Glycosylases; C061738:mutY adenine glycosylase; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase
keywords: 5-methylcytosine; DNA 5mC glycosylase; DNA demethylation; epigenetic regulation; repressor of silencing