TY  - JOUR
A1  - Müller, Sara
A1  - Windhof, Indra M.
A1  - Maximov, Vladimir
A1  - Jurkowski, Tomasz
A1  - Jeltsch, Albert
A1  - Förstner, Konrad U.
A1  - Sharma, Cynthia M.
A1  - Gräf, Ralph
A1  - Nellen, Wolfgang
T1  - Target recognition, RNA methylation activity and transcriptional regulation of the dictyostelium discoideum Dnmt2-homologue (DnmA)
JF  - Nucleic acids research
N2  - Although the DNA methyltransferase 2 family is highly conserved during evolution and recent reports suggested a dual specificity with stronger activity on transfer RNA (tRNA) than DNA substrates, the biological function is still obscure. We show that the Dictyostelium discoideum Dnmt2-homologue DnmA is an active tRNA methyltransferase that modifies C38 in tRNA(Asp(GUC)) in vitro and in vivo. By an ultraviolet-crosslinking and immunoprecipitation approach, we identified further DnmA targets. This revealed specific tRNA fragments bound by the enzyme and identified tRNA(Glu(CUC/UUC)) and tRNA(Gly(GCC)) as new but weaker substrates for both human Dnmt2 and DnmA in vitro but apparently not in vivo. Dnmt2 enzymes form transient covalent complexes with their substrates. The dynamics of complex formation and complex resolution reflect methylation efficiency in vitro. Quantitative PCR analyses revealed alterations in dnmA expression during development, cell cycle and in response to temperature stress. However, dnmA expression only partially correlated with tRNA methylation in vivo. Strikingly, dnmA expression in the laboratory strain AX2 was significantly lower than in the NC4 parent strain. As expression levels and binding of DnmA to a target in vivo are apparently not necessarily accompanied by methylation, we propose an additional biological function of DnmA apart from methylation.
Y1  - 2013
U6  - https://doi.org/10.1093/nar/gkt634
SN  - 0305-1048
SN  - 1362-4962
VL  - 41
IS  - 18
SP  - 8615
EP  - 8627
PB  - Oxford Univ. Press
CY  - Oxford
ER  - 
TY  - JOUR
A1  - Dubin, Manu
A1  - Fuchs, Joerg
A1  - Gräf, Ralph
A1  - Schubert, Ingo
A1  - Nellen, Wolfgang
T1  - Dynamics of a novel centromeric histone variant CenH3 reveals the evolutionary ancestral timing of centromere biogenesis
N2  - The centromeric histone H3 variant (CenH3) serves to target the kinetochore to the centromeres and thus ensures correct chromosome segregation during mitosis and meiosis. The Dictyostelium H3-like variant H3v1 was identified as the CenH3 ortholog. Dictyostelium CenH3 has an extended N-terminal domain with no similarity to any other known proteins and a histone fold domain at its C-terminus. Within the histone fold, alpha-helix 2 (alpha 2) and an extended loop 1 (L1) have been shown to be required for targeting CenH3 to centromeres. Compared to other known and putative CenH3 histones, Dictyostelium CenH3 has a shorter L1, suggesting that the extension is not an obligatory feature. Through ChIP analysis and fluorescence microscopy of live and fixed cells, we provide here the first survey of centromere structure in amoebozoa. The six telocentric centromeres were found to mostly consist of all the DIRS-1 elements and to associate with H3K9me3. During interphase, the centromeres remain attached to the centrosome forming a single CenH3-containing cluster. Loading of Dictyostelium CenH3 onto centromeres occurs at the G2/prophase transition, in contrast to the anaphase/ telophase loading of CenH3 observed in metazoans. This suggests that loading during G2/prophase is the ancestral eukaryotic mechanism and that anaphase/telophase loading of CenH3 has evolved more recently after the amoebozoa diverged from the animal linage.
Y1  - 2010
UR  - http://nar.oxfordjournals.org/
U6  - https://doi.org/10.1093/Nar/Gkq664
SN  - 0305-1048
ER  -