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Arabidopsis DEMETER DNA glycosylase requires non‐catalytic domains for 5‐methylcytosine excision
Author(s) -
HUH JIN HOE,
Weiner Gregory,
Uzawa Rie,
Eichman Brandt,
Fischer Robert
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.lb38
Subject(s) - dna glycosylase , arabidopsis , 5 methylcytosine , chemistry , biochemistry , dna , mutant , gene , biology , microbiology and biotechnology , dna repair , dna methylation , gene expression
Arabidopsis DEMETER (DME) encodes a 5‐methylcytosine DNA glycosylase and its maternal expression is necessary for establishment of gene imprinting, transcription activation and endosperm development. DME is a bifunctional DNA glycosylase/AP‐lyase which specifically removes 5‐methylcytosine in the genome regardless of the sequence context. DME is an exceptionally large glycosylase (1,729 aa) with the conserved aspartic acid (1304) and lysine (1286) catalytic residues. DME was shown to require an iron‐sulfur cluster following the helix‐hairpin‐helix motif for 5‐methylcytosine excision. A series of terminal deletions revealed two additional conserved domains necessary for the DME activity, which is unique to the DME family genes in Arabidopsis. Expression of active DME in E. coli showed cytotoxicity and this property was exploited to identify critical residues important for DME function. The entire DME region was subjected to random mutagenesis and the resulting mutant library was screened for loss of DME activity as a consequence of amino acid substitutions. We found that the conserved domains were highly susceptible to amino acid substitutions, which implies the functional/structural importance of these uncharacterized regions for 5‐methylcytosine glycosylase activity of DME. This study was supported by the NIH (GM069415).