title: Cooperative DNA binding and protein/DNA fiber formation increases the activity of the Dnmt3a DNA methyltransferase.
abstract: The Dnmt3a DNA methyltransferase has been shown to bind cooperatively to DNA and to form large multimeric protein/DNA fibers. However, it has also been reported to methylate DNA in a processive manner, a property that is incompatible with protein/DNA fiber formation. We show here that the DNA methylation rate of Dnmt3a increases more than linearly with increasing enzyme concentration on a long DNA substrate, but not on a short 30-mer oligonucleotide substrate. We also show that addition of a catalytically inactive Dnmt3a mutant, which carries an amino acid exchange in the catalytic center, increases the DNA methylation rate by wild type Dnmt3a on the long substrate but not on the short one. In agreement with this finding, preincubation experiments indicate that stable protein/DNA fibers are formed on the long, but not on the short substrate. In addition, methylation experiments with substrates containing one or two CpG sites did not provide evidence for a processive mechanism over a wide range of enzyme concentrations. These data clearly indicate that Dnmt3a binds to DNA in a cooperative reaction and that the formation of stable protein/DNA fibers increases the DNA methylation rate. Fiber formation occurs at low μm concentrations of Dnmt3a, which are in the range of Dnmt3a concentrations in the nucleus of embryonic stem cells. Understanding the mechanism of Dnmt3a is of vital importance because Dnmt3a is a hotspot of somatic cancer mutations one of which has been implicated in changing Dnmt3a processivity.
authors.affiliation: From the Institute of Biochemistry, Stuttgart University, D-70569 Stuttgart, Germany and.
authors.forename: Max
authors.initials: M
authors.lastname: Emperle
authors.affiliation: From the Institute of Biochemistry, Stuttgart University, D-70569 Stuttgart, Germany and.
authors.forename: Arumugam
authors.initials: A
authors.lastname: Rajavelu
authors.affiliation: Max-Planck-Genomzentrum Köln, D-50829 Köln, Germany.
authors.forename: Richard
authors.initials: R
authors.lastname: Reinhardt
authors.affiliation: From the Institute of Biochemistry, Stuttgart University, D-70569 Stuttgart, Germany and renata.jurkowska@ibc.uni-stuttgart.de.
authors.forename: Renata Z
authors.initials: RZ
authors.lastname: Jurkowska
authors.affiliation: From the Institute of Biochemistry, Stuttgart University, D-70569 Stuttgart, Germany and albert.jeltsch@ibc.uni-stuttgart.de.
authors.forename: Albert
authors.initials: A
authors.lastname: Jeltsch
mesh_terms: D000818:Animals; D001483:Base Sequence; D055162:Biocatalysis; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism*; D019175:DNA Methylation; D000090205:DNA Methyltransferase 3A; D053595:Embryonic Stem Cells / Q000201:enzymology; D005453:Fluorescence; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D059372:Nucleotide Motifs / Q000235:genetics; D011485:Protein Binding; D055503:Protein Multimerization; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
chemical_list: C000715535:Dnmt3a protein, mouse; D050505:Mutant Proteins; D004247:DNA; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A
keywords: Cooperativity; DNA Enzyme; DNA Methylation; DNA Methyltransferase; Enzyme Kinetics; Enzyme Mechanism