title: Molecular characterization of a putative plant homolog of MBD4 DNA glycosylase.
abstract: Methyl-CpG-binding domain 4 (MBD4) DNA glycosylase is involved in excision of spontaneous deamination products of cytosine and 5-methylcytosine in animals, but it is unknown whether related proteins perform similar functions in plants. We report here the isolation and biochemical characterization of a putative MBD4 homolog from Arabidopsis thaliana, designated as MBD4L (MBD4-like). The plant enzyme lacks the MBD domain present in mammalian MBD4 proteins, but conserves a DNA glycosylase domain with critical residues for substrate recognition and catalysis, and it is more closely related to MBD4 homologs than to other members of the HhH-GPD superfamily. Arabidopsis MBD4L excises uracil and thymine opposite G, and the presence of halogen substituents at C5 of the target base greatly increases its excision efficiency. No significant activity is detected on cytosine derivatives such as 5-methylcytosine or 5-hydroxymethylcytosine. The enzyme binds to the abasic site product generated after excision, which decreases its catalytic turnover in vitro. Both the full-length protein and a N-terminal truncated version retaining the catalytic domain exhibit a preference for a CpG sequence context, where most plant DNA methylation is found. Our results suggest that an important function of Arabidopsis MBD4L is to protect the plant genome from the mutagenic consequences of cytosine and 5-methylcytosine deamination.
authors.affiliation: Department of Genetics, University of Córdoba/Maimónides Institute for Research in Biomedicine of Córdoba (IMIBIC)/Reina Sofía University Hospital, 14071 Córdoba, Spain.
authors.forename: Ángel
authors.initials: Á
authors.lastname: Ramiro-Merina
authors.forename: Rafael R
authors.initials: RR
authors.lastname: Ariza
authors.forename: Teresa
authors.initials: T
authors.lastname: Roldán-Arjona
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D003596:Cytosine / Q000378:metabolism; D045647:DNA Glycosylases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003641:Deamination; D019143:Evolution, Molecular; D018745:Genome, Plant; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D011485:Protein Binding; D016415:Sequence Alignment; D017385:Sequence Homology; D013379:Substrate Specificity
chemical_list: D029681:Arabidopsis Proteins; D003596:Cytosine; D045647:DNA Glycosylases; C000588848:MBD4L protein, Arabidopsis
keywords: Base excision repair; DNA methylation; Deamination; Mismatches