Domain structure of the DEMETER 5-methylcytosine DNA glycosylase.
DNA glycosylases initiate the base excision repair (BER) pathway by excising damaged, mismatched, or otherwise modified bases. Animals and plants independently evolved active BER-dependent DNA demethylation mechanisms important for epigenetic reprogramming. One such DNA demethylation mechanism is uniquely initiated in plants by DEMETER (DME)-class DNA glycosylases. Arabidopsis DME family glycosylases contain a conserved helix-hairpin-helix domain present in both prokaryotic and eukaryotic DNA glycosylases as well as two domains A and B of unknown function that are unique to this family. Here, we employed a mutagenesis approach to screen for DME residues critical for DNA glycosylase activity. This analysis revealed that amino acids clustered in all three domains, but not in the intervening variable regions, are required for in vitro 5-methylcytosine excision activity. Amino acids in domain A were found to be required for nonspecific DNA binding, a prerequisite for 5-methylcytosine excision. In addition, mutational analysis confirmed the importance of the iron-sulfur cluster motif to base excision activity. Thus, the DME DNA glycosylase has a unique structure composed of three essential domains that all function in 5-methylcytosine excision.
Department of Plant Science and Plant Genomics and Breeding Institute, Seoul National University, Seoul 151-921, Korea.
Young Geun
YG
Mok
Rie
R
Uzawa
Jiyoon
J
Lee
Gregory M
GM
Weiner
Brandt F
BF
Eichman
Robert L
RL
Fischer
Jin Hoe
JH
Huh
D044503:5-Methylcytosine / Q000378:metabolism*; D000596:Amino Acids; D017360:Arabidopsis / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics*; D045647:DNA Glycosylases / Q000737:chemistry* / Q000235:genetics; D019175:DNA Methylation; D004260:DNA Repair; D007506:Iron-Sulfur Proteins; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary
D000596:Amino Acids; D029681:Arabidopsis Proteins; D007506:Iron-Sulfur Proteins; D044503:5-Methylcytosine; D045647:DNA Glycosylases