An invariant aspartic acid in the DNA glycosylase domain of DEMETER is necessary for transcriptional activation of the imprinted MEDEA gene.
Helix-hairpin-helix DNA glycosylases are typically small proteins that initiate repair of DNA by excising damaged or mispaired bases. An invariant aspartic acid in the active site is involved in catalyzing the excision reaction. Replacement of this critical residue with an asparagine severely reduces catalytic activity but preserves enzyme stability and structure. The Arabidopsis DEMETER (DME) gene encodes a large 1,729-aa polypeptide with a 200-aa DNA glycosylase domain. DME is expressed primarily in the central cell of the female gametophyte. DME activates maternal allele expression of the imprinted MEDEA (MEA) gene in the central cell and is required for seed viability. We mutated the invariant aspartic acid at position 1304 in DME to asparagine (D1304N) to determine whether the catalytic activity of the DNA glycosylase domain is required for DME function in vivo. Transgenes expressing wild-type DME in the central cell rescue seed abortion caused by a mutation in the endogenous DME gene and activate maternal MEA:GFP transcription. However, transgenes expressing the D1304N mutant DME do not rescue seed abortion or activate maternal MEA:GFP transcription. Whereas ectopic expression of the wild-type DME polypeptide in pollen is sufficient to activate ectopic paternal MEA and MEA:GUS expression, equivalent expression of the D1304N mutant DME in pollen failed to do so. These results show that the conserved aspartic acid residue is necessary for DME to function in vivo and suggest that an active DNA glycosylase domain, normally associated with DNA repair, promotes gene transcription that is essential for gene imprinting.
Choi
Yeonhee
Y
Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA.
Harada
John J
JJ
Goldberg
Robert B
RB
Fischer
Robert L
RL
D029681:Arabidopsis Proteins / Q000737:chemistry / Q000378:metabolism*; D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D045647:DNA Glycosylases / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers; D009699:N-Glycosyl Hydrolases / Q000737:chemistry / Q000378:metabolism*; D018749:RNA, Plant; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic; D019076:Transgenes
D029681:Arabidopsis Proteins; D017931:DNA Primers; D018749:RNA, Plant; D015534:Trans-Activators; D001224:Aspartic Acid; C463261:DEMETER protein, Arabidopsis; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases