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Cooperative DNA binding and protein/DNA fiber formation increases the activity of the Dnmt3a DNA methyltransferase

Emperle, Max, Rajavelu, Arumugam, Reinhardt, Richard, Jurkowska, Renata Z. and Jeltsch, Albert 2014. Cooperative DNA binding and protein/DNA fiber formation increases the activity of the Dnmt3a DNA methyltransferase. Journal of Biological Chemistry 289 (43) , pp. 29602-29613. 10.1074/jbc.M114.572032

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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/DNAfiber formation. We show here that the DNA methylation rate of Dnmt3a increases more than linearly with increasing enzymeconcentration on a long DNA substrate, but not on a short 30-mer oligonucleotide substrate. We also show that addition ofa catalytically inactive Dnmt3a mutant, which carries an amino acid exchange in the catalytic center, increases the DNA methylationrate by wild type Dnmt3a on the long substrate but not on the short one. In agreement with this finding, preincubation experimentsindicate that stable protein/DNA fibers are formed on the long, but not on the short substrate. In addition, methylation experimentswith substrates containing one or two CpG sites did not provide evidence for a processive mechanism over a wide range of enzymeconcentrations. These data clearly indicate that Dnmt3a binds to DNA in a cooperative reaction and that the formation of stableprotein/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. Understandingthe mechanism of Dnmt3a is of vital importance because Dnmt3a is a hotspot of somatic cancer mutations one of which has beenimplicated in changing Dnmt3a processivity.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Biosciences
Publisher: American Society for Biochemistry and Molecular Biology
ISSN: 0021-9258
Date of First Compliant Deposit: 31 October 2019
Date of Acceptance: 20 August 2014
Last Modified: 03 Jun 2020 10:45
URI: http://orca.cf.ac.uk/id/eprint/126452

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