PubMed · 11367366
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1999. Expanded access.. https://pubmed.ncbi.nlm.nih.gov/11367366/
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DNA N6-methyladenine (6mA) is a newly recognized transcription-associated epigenetic mark in eukaryotes. While its methylation pathway has been well established, the identity of eukaryotic 6mA demethylase(s) responsible for its removal and dynamic regulation has remained elusive. Here, we identify and characterize DMT3 (TtALKBH5), an AlkB family dioxygenase in Tetrahymena thermophila, as a 6mA demethylase in ciliates and potentially several other unicellular eukaryotes with abundant 6mA and a defined AMT1 methyltransferase (MTase) complex, supported by genetic and molecular evidence. DMT3 acts on both fully and hemimethylated ApT dinucleotides, an activity partially facilitated by a naturally occurring cysteine-to-serine substitution. Genome profiling shows that DMT3 is enriched at transcription start sites (TSSs) of 6mA-enriched genes, complementary to the occupancy pattern of the AMT1 complex, where it selectively removes spurious 6mA deposited by AMT1. Genetic disruption of DMT3-mediated demethylation, either by knockout or catalytic inactivation, leads to aberrant 6mA accumulation at TSS regions, transcriptional dysregulation, altered chromatin accessibility, and impaired initiation of sexual reproduction. Notably, simultaneous removal of DMT3 and AMT1 eliminates these defects, indicating that spurious TSS 6mA underlies transcriptional and developmental impairment.
A strategy coupling high-activity nucleases with dimeric TadA-8e optimizes plant Cas12-based adenine base editors, boosts editing efficiency, and provides precise editors for crop breeding and genomics research.
The repair of DNA damage, caused by both endogenous and exogenous sources, is necessary to remove lesions that either miscode or block DNA or RNA polymerases. We propose that damage also must be repaired to maintain sequence-specific DNA-protein interactions. In this paper, we have systematically studied two lesions that interfere with one important DNA landmark, the thymine methyl group. Oxidation of the thymine methyl group in DNA generates 5-hydroxymethyluracil (HmU) whereas the misincorporation of dUMP into DNA generates uracil (U), replacing the methyl group with a hydrogen. Both substitutions are shown to inhibit binding of the AP-1 (c-Jun) transcription factor. The energy cost of the perturbation, approximately 0.4 kcal/mol, is similar in magnitude for both U and HmU substitutions and is additive when multiple substitutions are present. A third lesion, substitution of the central C:G base pair of the AP-1 DNA binding domain with the pro-mutagenic U:G mispair, unexpectedly increases AP-1 binding, allowing the transcription factor to interfere with uracil DNA glycosylase activity. Our results support the hypothesis that an additional role for DNA repair systems is to maintain the integrity of sequence-specific DNA-protein interactions, a role of particular importance in long-lived organisms.