Murine DNA Cytosine-C<sup>5</sup> Methyltransferase:  Pre-Steady- and Steady-State Kinetic Analysis with Regulatory DNA Sequences

Flynn, James E.; Glickman, J. Fraser; Reich, Norbert O.

doi:10.1021/bi9600512

Cited by 75 publications

(120 citation statements)

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“…Mammalian DNMT1 was reported to methylate hemimethylated DNA to a greater extent (2-5-fold) than unmethylated DNA (32)(33)(34)38), an observation confirmed here with a new recombinant enzyme that displays an ϳ30-fold higher activity than previously obtained (34 This effect could be achieved in two ways as follows: (a) m 5 CG "exposes" the enzyme active site, which is otherwise less accessible and/or, (b) m 5 CG binding modifies the active site conformation, improving its fit for AdoMet and/or the DNA. The data with (CGG⅐Cm 5 CG) 12 , which showed saturation at 2 M AdoMet, suggest an active role for m 5 CG in shaping the AdoMet binding pocket.…”

Section: Discussionmentioning

confidence: 99%

“…This region shares sequence homologies with all prokaryotic type II cytosine-5 methyltransferases (39,40), including a PC dipeptide motif that is part of the catalytic center in the crystal structures of M.HhaI (41) and M.HaeIII (42), and the binding site for S-adenosylmethionine (AdoMet), the methyl donor for methyltransferases (43,44). The remaining ϳ1000 N-terminal amino acids, which are not present in the prokaryotic enzymes, contain a nuclear localization signal, a replication foci targeting sequence (15), and are important in the discrimination between unmethylated and hemimethylated substrates (33).…”

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confidence: 99%

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Recombinant Human DNA (Cytosine-5) Methyltransferase

Bacolla¹,

Pradhan²,

Roberts³

et al. 1999

Journal of Biological Chemistry

115

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Initial velocity determinations were conducted with human DNA (cytosine-5) methyltransferase (DNMT1) on unmethylated and hemimethylated DNA templates in order to assess the mechanism of the reaction. Initial velocity data with DNA and S-adenosylmethionine (AdoMet) as variable substrates and product inhibition studies with methylated DNA and S-adenosylhomocysteine (AdoHcy) were obtained and evaluated as double-reciprocal plots. These relationships were linear for plasmid DNA, exon-1 from the imprinted small nuclear ribonucleoprotein-associated polypeptide N, (CGG⅐CCG) 12 , (m 5 CGG⅐CCG) 12 , and (CGG⅐CCG) 73 but were not linear for (CGG⅐Cm 5 CG) 12 . Inhibition by AdoHcy was apparently competitive versus AdoMet and uncompetitive/noncompetitive versus DNA at <20 M AdoMet. Addition of the product (methylated DNA) to unmethylated plasmid DNA increased V max(app) resulting in mixed stimulation and inhibition. Velocity equations indicated a two-step mechanism as follows: first, activation of DNMT1 by methylated DNA that bound to an allosteric site, and second, the addition of AdoMet and DNA to the catalytic site. The preference of DNMT1 for hemimethylated DNA may be the result of positive cooperativity of AdoMet binding mediated by allosteric activation by the methylated CG steps. We propose that this activation plays a role in vivo in the regulation of maintenance methylation.The genome of most organisms contains modified nucleotides including N 6 -methyladenine, N 4 -methylcytosine, and C 5 -methylcytosine (m 5 C) 1 (1-3). However, both the biological significance and the types of DNA methylation differ greatly between prokaryotes and eukaryotes. In prokaryotes, most modified bases participate in restriction-modification, a defense mechanism that protects the host from heterologous phage infection (4). In addition, N 6 -methyladenine plays a role in the initiation of DNA replication (5) and in post-replicative methyl-directed mismatch repair (6).In higher eukaryotes, DNA methylation is confined to m 5 C and is implicated in the regulation of development, genomic imprinting (7, 8), X chromosome inactivation, gene expression (9), and retrotransposon inactivation (10 -12). In mammals, the patterns of methylation are inherited from both parental genomes but are erased and reconstructed (de novo methylation) in somatic cells following implantation (13,14). Such patterns are then copied and maintained by hemimethylation of the daughter strands during semiconservative DNA synthesis in S phase (maintenance methylation) (15, 16). However, the mechanisms by which both de novo and maintenance methylation occur remain to be elucidated.Several DNA methyltransferases have been isolated from human and mouse (17-20), but it is not clear whether the de novo and maintenance methylations are carried out by separate proteins in vivo or whether both activities are shared by one or more enzymes (21-26). Nevertheless, a role for in vivo methylation has been established for isoforms of the human DNMT1 gene (27, 28) whose product, DNMT1, me...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Recombinant Human DNA (Cytosine-5) Methyltransferase

Bacolla¹,

Pradhan²,

Roberts³

et al. 1999

Journal of Biological Chemistry

115

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“…However, it is puzzling that the Δ369 protein loses the ability to methylate non-CpG site because recognition of methylating target is thought to be mediated entirely by the Cterminal MTase domain. As a positive control, Dnmt1 has ~10-fold higher activity on hemimethylated than unmethylated CpG-containing DNA 5,6,32 . The methyl transfer activity of Dnmt3b2 observed here is equivalent to that of Dnmt1 on unmethylated DNA -that is, ~10% of Dnmt1 activity on hemimethylated DNA -comparable to previous reports 8 .…”

Section: The Pwwp Domain Of Dnmt3b Binds To Dnamentioning

confidence: 99%

The PWWP domain of mammalian DNA methyltransferase Dnmt3b defines a new family of DNA-binding folds

et al. 2002

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The PWWP domain is a weakly conserved sequence motif found in >60 eukaryotic proteins, including the mammalian DNA methyltransferases Dnmt3a and Dnmt3b. These proteins often contain other chromatin-association domains. A 135-residue PWWP domain from mouse Dnmt3b (amino acids 223-357) has been structurally characterized at 1.8 Å resolution. The N-terminal half of this domain resembles a barrel-like five-stranded structure, whereas the C-terminal half contains a five-helix bundle. The two halves are packed against each other to form a single structural module that exhibits a prominent positive electrostatic potential. The PWWP domain alone binds DNA in vitro, probably through its basic surface. We also show that recombinant Dnmt3b2 protein (a splice variant of Dnmt3b) and two N-terminal deletion mutants (Δ218 and Δ369) have approximately equal methyl transfer activity on unmethylated and hemimethylated CpG-containing oligonucleotides. The Δ218 protein, which includes the PWWP domain, binds DNA more strongly than Δ369, which lacks the PWWP domain.Genomic patterns of cytosine methylation at the C5 position play key roles in the organization and control of mammalian chromatin (reviewed in refs 1-3). Mammalian DNA methyltransferases (MTases) all contain a C-terminal catalytic region that is structurally and functionally homologous to bacterial MTases. The eukaryotic DNA MTases have been grouped into three families based on (i) distinctive properties of their N-terminal regions and (ii) intriguing differences in DNA substrate preferences. The first eukaryotic DNA MTase, Dnmt1, contains a large N-terminal regulatory region of ~1,000 amino acids and shows a preference for hemimethylated DNA in vitro [4][5][6] . Dnmt2, a relatively small protein, contains only the MTase domain, and its function is still unclear 7 . Recombinant Dnmt3 acts on unmethylated and hemimethylated DNA at equal rates in vitro 8,9 .The task of dissecting the functional roles of the N-terminal region of the different MTase families is just beginning. These regions vary widely in size and are probably responsible for the diverse biological functions of eukaryotic DNA MTases. These functions include the © 2002 Nature Publishing Group Correspondence should be addressed to X.C. xcheng@emory.edu. Competing interests statementThe authors declare that they have no competing financial interests. HHS Public Access Dnmt3 contains a PWWP domainThe sequences of Dnmt3 orthologs have been determined from human and mouse. They all contain an N-terminal variable region (~280 amino acids in Dnmt3a and ~220 amino acids in Dnmt3b), followed by three stretches of conserved regions: the PWWP motif, six repeats of a CXXC motif and a set of 10 motifs conserved among DNA MTase catalytic regions (Fig. 1a). The PWWP motif 28 was first identified in a gene family related to the hepatomaderived growth factor (HDGF) 29 and WHSC1 genes (Wolf-Hirschhorn Syndrome Candidate) 30 . The corresponding sequence in Dnmt3 is SWWP (Fig. 1b); only the last two positions of the motif...

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“…Mammalian maintenance DNA methyltransferase DNMT1 has been extensively studied biochemically and enzymatically (14,(27)(28)(29)(30)(31). DNMT1 prefers hemimethylated dsDNA and its kinetic constants have been reported (14,28,29).…”

Section: Construction Expression and Purification Of Mammalianmentioning

confidence: 99%

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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Murine DNA Cytosine-C⁵ Methyltransferase: Pre-Steady- and Steady-State Kinetic Analysis with Regulatory DNA Sequences

Cited by 75 publications

References 44 publications

Recombinant Human DNA (Cytosine-5) Methyltransferase

Recombinant Human DNA (Cytosine-5) Methyltransferase

The PWWP domain of mammalian DNA methyltransferase Dnmt3b defines a new family of DNA-binding folds

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

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Murine DNA Cytosine-C5 Methyltransferase: Pre-Steady- and Steady-State Kinetic Analysis with Regulatory DNA Sequences

Cited by 75 publications

References 44 publications

Recombinant Human DNA (Cytosine-5) Methyltransferase

Recombinant Human DNA (Cytosine-5) Methyltransferase

The PWWP domain of mammalian DNA methyltransferase Dnmt3b defines a new family of DNA-binding folds

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

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Murine DNA Cytosine-C⁵ Methyltransferase: Pre-Steady- and Steady-State Kinetic Analysis with Regulatory DNA Sequences