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 helixhairpin-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.DNA repair | helix-hairpin-helix protein | mutagenesis | mixed charge cluster T he modified base 5-methylcytosine (5mC) is a stable epigenetic mark that silences gene expression and plays an important role in many developmental processes such as gene imprinting, X-chromosome inactivation, and transposon silencing (1-4). DNA methylation primarily occurs at symmetric CG sequences in animals, whereas DNA methylation in plants occurs in all sequence contexts: CG, CHG, and CHH (where H ¼ A, T, or C) (5). The overall CG DNA methylation pattern in the genome is faithfully inherited to daughter cells by maintenance DNA methyltransferases, which convert hemimethylated sites generated by DNA replication to fully methylated sites. When maintenance methyltransferases are absent or down-regulated, DNA methylation is progressively lost during replication, which is referred to as passive DNA demethylation. By contrast, active DNA demethylation that is independent of DNA replication requires alternative pathways.Base excision repair (BER), which normally functions to repair damaged and mispaired bases, is also required for active DNA demethylation and epigenetic reprogramming in eukaryotes (3, 6-9). BER is initiated by DNA glycosylase enzymes that catalyze the hydrolysis of the N-glycosidic (base-ribose) bond. In plants, the DEMETER (DME) family of DNA glycosylases functions to remove 5mC, which is then replaced by unmethylated cytosine (10, 11), resulting in transcriptional activation of target genes (10,12). Arabidopsis has three other DME-like (DML) genes-ROS1, DML2, and DML3 (12-14) (Fig. S1). DME is essential for plant reproduction and influences the endosperm DNA methylation profile (15, 16), whereas DMLs function in vegetative tissues to prevent inappropriate gene silencing and maintain ...

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These results suggest that a DC for increased ICP can reduce the CIB of the brain after severe TBI. We suggest that DC be considered early in a patient's clinical course, particularly when the TIL and ICP are increased.