Enzymatic properties of recombinant Dnmt3a DNA methyltransferase from mouse: the enzyme modifies DNA in a non-processive manner and also methylates non-CpA sites

Gowher, Humaira; Jeltsch, Albert

doi:10.1006/jmbi.2001.4710

Cited by 219 publications

(154 citation statements)

References 28 publications

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“…Both of these effects are consistent with the known preferences of the DNMT3a methylase for certain flanking sequences in CpGs (see SI Fig. 7) (26) and its ability to methylate cytosines in non-CpG dinucleotides, albeit much less effectively (27)(28)(29). Additionally, the overall relative proportions of methylation found in the dinucleotides CpG, CpA, CpT, and CpC correlate well with the known propensities of the DNMT3a methylase for these sites.…”

Section: Sequence-specific In Vivo Methylation Of Mtdna By a Chimericsupporting

confidence: 81%

Sequence-specific modification of mitochondrial DNA using a chimeric zinc finger methylase

Minczuk

Papworth

Kolasinska

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

141

112

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We used engineered zinc finger peptides (ZFPs) to bind selectively to predetermined sequences in human mtDNA. Surprisingly, we found that engineered ZFPs cannot be reliably routed to mitochondria by using only conventional mitochondrial targeting sequences. We here show that addition of a nuclear export signal allows zinc finger chimeric enzymes to be imported into human mitochondria. The selective binding of mitochondria-specific ZFPs to mtDNA was exemplified by targeting the T8993G mutation, which causes two mitochondrial diseases, neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP) and also maternally inherited Leigh's syndrome. To develop a system that allows the monitoring of site-specific alteration of mtDNA we combined a ZFP with the easily assayed DNA-modifying activity of hDNMT3a methylase. Expression of the mutation-specific chimeric methylase resulted in the selective methylation of cytosines adjacent to the mutation site. This is a proof of principle that it is possible to target and alter mtDNA in a sequence-specific manner by using zinc finger technology.gene therapy ͉ mitochondria ͉ mitochondrial diseases ͉ synthetic zinc finger peptides ͉ nuclear export signal

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Section: Sequence-specific In Vivo Methylation Of Mtdna By a Chimericsupporting

confidence: 81%

Sequence-specific modification of mitochondrial DNA using a chimeric zinc finger methylase

Minczuk

Papworth

Kolasinska

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

141

112

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“…It is noteworthy that Dnmt1 was reported to be post-translationally modified in vivo (29,55). The enzymatic activity of recombinant Dnmt3a expressed in Escherichia coli has recently been reported by other groups (26,56). Their Dnmt3a demonstrated properties different from our baculovirus-expressed Dnmt3a.…”

Section: Substrate Specificity Of Dnmt3amentioning

confidence: 60%

“…Establishment of a Novel Quantitative DNMT Assay System-The low level of methyltransferase activity for Dnmt3a reported by others (15,25,26), coupled with our data, necessitated the development of a reproducible assay with a higher signal-to-noise ratio than currently available methods. A flow chart of the DNMT magnetic beads assay is shown in Fig.…”

Section: Construction Expression and Purification Of Mammalianmentioning

confidence: 94%

“…Their Dnmt3a demonstrated properties different from our baculovirus-expressed Dnmt3a. In particular, one group concluded that their Dnmt3a showed no preference for unmethylated dsDNA (26). Furthermore, the Dnmt3a expressed by these groups possessed relatively strong methylation activity toward non-CpG cytosines, particularly CpA, which was methylated only 2-(26) or 10-fold (56) slower than CpG sites.…”

Section: Substrate Specificity Of Dnmt3amentioning

confidence: 99%

“…Unlike the maintenance methyltransferase DNMT1, mam-malian Dnmt3a has been poorly characterized biochemically, because the enzymatic activity of Dnmt3a is considerably lower than that of DNMT1 (15,25,26). To understand how cellular DNA methylation patterns are regulated enzymatically, we have cloned and purified both enzymes using a baculovirus expression system.…”

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confidence: 99%

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Preferential Methylation of Unmethylated DNA by Mammalian de Novo DNA Methyltransferase Dnmt3a

Yokochi

Robertson

2002

Journal of Biological Chemistry

141

109

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DNA methylation is an epigenetic modification of DNA. There are currently three catalytically active mammalian DNA methyltransferases, DNMT1, -3a, and -3b. DNMT1 has been shown to have a preference for hemimethylated DNA and has therefore been termed the maintenance methyltransferase. Although previous studies on DNMT3a and -3b revealed that they act as functional enzymes during development, there is little biochemical evidence about how new methylation patterns are established and maintained. To study this mechanism we have cloned and expressed Dnmt3a using a baculovirus expression system. The substrate specificity of Dnmt3a and molecular mechanism of its methylation reaction were then analyzed using a novel and highly reproducible assay. We report here that Dnmt3a is a true de novo methyltransferase that prefers unmethylated DNA substrates more than 3-fold to hemimethylated DNA. Furthermore, Dnmt3a binds DNA nonspecifically, regardless of the presence of CpG dinucleotides in the DNA substrate. Kinetic analysis supports an Ordered Bi Bi mechanism for Dnmt3a, where DNA binds first, followed by S-adenosyl-L-methionine.

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Beyond Watson and Crick: DNA Methylation and Molecular Enzymology of DNA Methyltransferases

2002

Self Cite

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DNA methyltransferases catalyze the transfer of a methyl group from S-adenosyl-L-methionine to cytosine or adenine bases in DNA. These enzymes challenge the Watson/Crick dogma in two instances: 1) They attach inheritable information to the DNA that is not encoded in the nucleotide sequence. This so-called epigenetic information has many important biological functions. In prokaryotes, DNA methylation is used to coordinate DNA replication and the cell cycle, to direct postreplicative mismatch repair, and to distinguish self and nonself DNA. In eukaryotes, DNA methylation contributes to the control of gene expression, the protection of the genome against selfish DNA, maintenance of genome integrity, parental imprinting, X-chromosome inactivation in mammals, and regulation of development. 2) The enzymatic mechanism of DNA methyltransferases is unusual, because these enzymes flip their target base out of the DNA helix and, thereby, locally disrupt the B-DNA helix. This review describes the biological functions of DNA methylation in bacteria, fungi, plants, and mammals. In addition, the structures and mechanisms of the DNA methyltransferases, which enable them to specifically recognize their DNA targets and to induce such large conformational changes of the DNA, are discussed.

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Enzymatic properties of recombinant Dnmt3a DNA methyltransferase from mouse: the enzyme modifies DNA in a non-processive manner and also methylates non-CpA sites

Cited by 219 publications

References 28 publications

Sequence-specific modification of mitochondrial DNA using a chimeric zinc finger methylase

Sequence-specific modification of mitochondrial DNA using a chimeric zinc finger methylase

Preferential Methylation of Unmethylated DNA by Mammalian de Novo DNA Methyltransferase Dnmt3a

Beyond Watson and Crick: DNA Methylation and Molecular Enzymology of DNA Methyltransferases

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