DNA methylation is recognized as an important DNA modification, which aids in structural integrity and proper regulation of the genome for many species. DNA methylation is catalyzed by a family of DNA methyltransferases (Dnmts) that transfer a methyl group from S-adenyl methionine (AdoMet) to the fifth carbon of a cytosine residue to form canonical m5C (5-methylcytosine). All m5C specific DNA methyltransferases, either eukaryotic or bacterial, contain evolutionarily conserved clusters of amino acid sequence motifs that were used to identify respective coding DNA sequences in a variety of sequenced genomes. In higher eukaryotes, Dnmts methylate on cytosines of CpG dinucleotides, which are initially established by de novo DNA methyltransferase 3 (Dnmt3) and maintained by the maintenance DNA methyltransferase 1 (Dnmt1), which copy methylation marks from methylated CPG dinucleotides on the parental DNA strand to complementary CPG dinucleotides on the daughter strand. DNA methyltransferase 2 (Dnmt2), which is also known as tRNA aspartic acid methyltransferase 1 (Trdmt1) is the second member of the DNA methyltransferase family that exhibits strong sequence conservation to the catalytic motifs of established DNA methyltransferases.
While the role of other Dnmt proteins has been extensively characterized, comparably little is understood about the biological and physiological function of Dnmt2. This is very surprising because Dnmt2 is the most widely conserved protein from the Dnmt family that has homologs in protists, plants, fungi, and animals. Interestingly, a large, diverse group of animal species have retained Dnmt2 as their only candidate form of DNA MTase. The Dnmt2-only organisms include several highly relevant model systems such as Schizosaccharomyces pombe, Dictyostelium discoideum, Entamoeba histolytica, Schistosoma mansoni, and Drosophila melanogaster. D.discoideum serves as a well-suited model organism for characterizing the cellular function of this enzyme, largely because D. discoideum has a haploid genome and also contains a single Dnmt2-like methyltransferase candidate gene, DnmA (DDB_G0288047).
Previous studies have reported that mutation or knockout of Dnmt2 in Dnmt2-only organisms does not result in an obvious phenotype. In this project, we have investigated the effects of DnmAKO on vegetative and multicellular D. discoideum cells. We show that a DnmAKO features relatively mild penetrance on the parental cell line, where mutant axenic cell culture featured defects in proliferation, cytokinesis, karyokinesis, nuclear morphology, centrosome, spindle formation, and localization. These observations indicate that DnmAKO exhibits pleiotropic effects on genome stability, nuclear architecture and microtubule organization. This large disparity of effects from complete knock-out of Dnmt2, indicates its relevancy to either defects in repetitive element suppression in the genome or more likely to defects in amino-acid incorporation, followed by stutter or misincorporation during translation at the ribosome complex which is so crucial to the physiology of this soil-dwelling amoebae.
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|Advisor:||Larochelle, Denis, Drewell, Robert|
|Department:||Biochemistry & Molecular Biology|
|School Location:||United States -- Massachusetts|
|Source:||MAI 82/1(E), Masters Abstracts International|
|Subjects:||Biochemistry, Microbiology, Genetics|
|Keywords:||Dictyostelium discoideum, DnmA, Methylation, Multicellular development, Phenotypic characterization, Vegetative proliferation|
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