title: Function and disruption of DNA methyltransferase 3a cooperative DNA binding and nucleoprotein filament formation.
abstract: The catalytic domain of Dnmt3a cooperatively multimerizes on DNA forming nucleoprotein filaments. Based on modeling, we identified the interface of Dnmt3a complexes binding next to each other on the DNA and disrupted it by charge reversal of critical residues. This prevented cooperative DNA binding and multimerization of Dnmt3a on the DNA, as shown by the loss of cooperative complex formation in electrophoretic mobility shift assay, the loss of cooperativity in DNA binding in solution, the loss of a characteristic 8- to 10-bp periodicity in DNA methylation and direct imaging of protein-DNA complexes by scanning force microscopy. Non-cooperative Dnmt3a-C variants bound DNA well and retained methylation activity, indicating that cooperative DNA binding and multimerization of Dnmt3a on the DNA are not required for activity. However, one non-cooperative variant showed reduced heterochromatic localization in mammalian cells. We propose two roles of Dnmt3a cooperative DNA binding in the cell: (i) either nucleofilament formation could be required for periodic DNA methylation or (ii) favorable interactions between Dnmt3a complexes may be needed for the tight packing of Dnmt3a at heterochromatic regions. The complex interface optimized for tight packing would then promote the cooperative binding of Dnmt3a to naked DNA in vitro.
authors.affiliation: Biochemistry Laboratory, School of Engineering and Science, Jacobs University Bremen, Campus Ring 1, 28759 Bremen, Germany.
authors.forename: Arumugam
authors.initials: A
authors.lastname: Rajavelu
authors.forename: Renata Z
authors.initials: RZ
authors.lastname: Jurkowska
authors.forename: Jürgen
authors.initials: J
authors.lastname: Fritz
authors.forename: Albert
authors.initials: A
authors.lastname: Jeltsch
mesh_terms: D000818:Animals; D055162:Biocatalysis; D004247:DNA / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019175:DNA Methylation; D000090205:DNA Methyltransferase 3A; D024202:Electrophoretic Mobility Shift Assay; D006570:Heterochromatin / Q000201:enzymology; D051379:Mice; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D041681:NIH 3T3 Cells; D009698:Nucleoproteins / Q000648:ultrastructure; D011485:Protein Binding; D055503:Protein Multimerization
chemical_list: C000715535:Dnmt3a protein, mouse; D006570:Heterochromatin; D009698:Nucleoproteins; D004247:DNA; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A