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 ...
Inter-bacterial toxin DddA-derived cytosine base editors (DdCBEs) enable targeted C-to-T conversions in nuclear and organellar DNA. DddAtox, the deaminase catalytic domain derived from Burkholderia cenocepacia, is split into two inactive halves to avoid its cytotoxicity in eukaryotic cells, when fused to transcription activator-like effector (TALE) DNA-binding proteins to make DdCBEs. As a result, DdCBEs function as pairs, which hampers gene delivery via viral vectors with a small cargo size. Here, we present non-toxic, full-length DddAtox variants to make monomeric DdCBEs (mDdCBEs), enabling mitochondrial DNA editing with high efficiencies of up to 50%, when transiently expressed in human cells. We demonstrate that mDdCBEs expressed via AAV in cultured human cells can achieve nearly homoplasmic C-to-T editing in mitochondrial DNA. Interestingly, mDdCBEs often produce mutation patterns different from those obtained with conventional dimeric DdCBEs. Furthermore, mDdCBEs allow base editing at sites for which only one TALE protein can be designed. We also show that transfection of mDdCBE-encoding mRNA, rather than plasmid, can reduce off-target editing in human mitochondrial DNA.
DNA methylation is a primary epigenetic modification regulating gene expression and chromatin structure in many eukaryotes. Plants have a unique DNA demethylation system in that 5-methylcytosine (5mC) is directly removed by DNA demethylases, such as DME/ROS1 family proteins, but little is known about the downstream events. During 5mC excision, DME produces 3′-phosphor-α, β-unsaturated aldehyde and 3′-phosphate by successive β- and δ-eliminations, respectively. The kinetic studies revealed that these 3′-blocking lesions persist for a significant amount of time and at least two different enzyme activities are required to immediately process them. We demonstrate that Arabidopsis AP endonucleases APE1L, APE2 and ARP have distinct functions to process such harmful lesions to allow nucleotide extension. DME expression is toxic to E. coli due to excessive 5mC excision, but expression of APE1L or ARP significantly reduces DME-induced cytotoxicity. Finally, we propose a model of base excision repair and DNA demethylation pathway unique to plants.
MSN1 is a putative yeast transcriptional activator involved in chromium (Cr) accumulation. Here we show that overexpression of MSN1 enhances Cr and sulfur accumulation and Cr tolerance in transgenic tobacco. In addition, we found that expression of NtST1 (Nicotiana tabacum sulfate transporter 1) was elevated in MSN1-expressing transgenic tobacco, suggesting that chromate and sulfate are taken up via the sulfate transporter in plants. Supporting this, expression of NtST1 increased levels of Cr and S in Saccharomyces cerevisiae. Our findings suggest that yeast transcriptional activators can be used for developing effective metal remediators, and for improving the nutritional status of plants.
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