title: Conserved motif VIII of murine DNA methyltransferase Dnmt3a is essential for methylation activity.
abstract: BACKGROUND
  Dnmt3a is a DNA methyltransferase that establishes de novo DNA methylation in mammals. The structure of the Dnmt3a C-terminal domain is similar to the bacterial M. HhaI enzyme, a well-studied prokaryotic DNA methyltransferase. No X-ray structure is available for the complex of Dnmt3a with DNA and the mechanistic details of DNA recognition and catalysis by mammalian Dnmts are not completely understood.
  
  
  RESULTS
  Mutant variants of the catalytic domain of the murine Dnmt3a carrying substitutions of highly conserved N167, R200, and R202 have been generated by site directed mutagenesis and purified. Their methylation activity, DNA binding affinity, ability to flip the target cytosine out of the DNA double helix and covalent complex formation with DNA have been examined. Substitutions of N167 lead to reduced catalytic activity and reduced base flipping. Catalytic activity, base flipping, and covalent conjugate formation were almost completely abolished for the mutant enzymes with substitutions of R200 or R202.
  
  
  CONCLUSIONS
  We conclude that R202 plays a similar role in catalysis in Dnmt3a-CD as R232 in M.SssI and R165 in M.HhaI, which could be positioning of the cytosine for nucleophilic attack by a conserved Cys. R200 of Dnmt3a-CD is important in both catalysis and cytosine flipping. Both conserved R200 and R202 are involved in creating and stabilizing of the transient covalent intermediate of the methylation reaction. N167 might contribute to the positioning of the residues from the motif VI, but does not play a direct role in catalysis.
authors.affiliation: Department of Chemistry, Moscow State University, 119991, Moscow, Russia.
authors.forename: Olga V
authors.initials: OV
authors.lastname: Lukashevich
authors.affiliation: Department of Chemistry, Moscow State University, 119991, Moscow, Russia.
authors.forename: Natalia A
authors.initials: NA
authors.lastname: Cherepanova
authors.affiliation: BioMedX Innovation Center, Im Neuenheimer Feld 583, 69120, Heidelberg, Germany.
authors.forename: Renata Z
authors.initials: RZ
authors.lastname: Jurkovska
authors.affiliation: Institute of Biochemistry, Faculty of Chemistry, University Stuttgart, Pfaffenwaldring 55, 70569, Stuttgart, Germany.
authors.forename: Albert
authors.initials: A
authors.lastname: Jeltsch
authors.affiliation: Department of Chemistry, Moscow State University, 119991, Moscow, Russia. gromova@genebee.msu.ru.
authors.forename: Elizaveta S
authors.initials: ES
authors.lastname: Gromova
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics / Q000378:metabolism*; D019175:DNA Methylation; D000090205:DNA Methyltransferase 3A; D019793:Fluorescein / Q000737:chemistry; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D016415:Sequence Alignment
chemical_list: C000715535:Dnmt3a protein, mouse; D011994:Recombinant Proteins; D004247:DNA; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; D019793:Fluorescein
keywords: Catalytic domain; DNA binding; DNA methylation; Dnmt3a DNA methyltransferase; Dnmt3a mutants