Bacteriophage T4 Dam DNA-[N6-adenine]Methyltransferase

Evdokimov, Alexey A.; Zinoviev, Victor V.; Malygin, E. G.; Schlagman, Samuel L.; Hattman, Stanley

doi:10.1074/jbc.m108864200

Cited by 31 publications

(29 citation statements)

References 55 publications

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“…4), which are characteristic of a ternary complex mechanism. Consistent with this notion, there is previous structural and mechanistic data demonstrating that DNMTs form a ternary complex (40,47,58). To determine the order of substrate binding and product release, product inhibition studies were pursued.…”

Section: Molecular Mechanism Of Dnmt3amentioning

confidence: 65%

“…This analysis, also known as substrate inhibition, has been successfully utilized to identify the binding order of substrates for many enzymes (for example, Refs. 37-39) including T4 Dam adenine methyltransferase (40). In a compulsory ordered mechanism, if the initial concentration of the second substrate to bind were sufficiently high, an inhibitory effect on the enzyme would be observed due to the formation of nonproductive binary and/or dead-end ternary complexes.…”

Section: Construction Expression and Purification Of Mammalianmentioning

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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Section: Molecular Mechanism Of Dnmt3amentioning

confidence: 65%

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

View full text Add to dashboard Cite

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“…It should be noted that our assumption about unidirectional movement is not implausible. The transfer of the methyl group from AdoMet to DNA can be considered irreversible for the T4 Dam MTase (22), because the reverse reaction was estimated to be at least 500-fold slower than the forward one. Hence, it follows, that DNA methylation is accompanied by the liberation of significant energy (⌬G 0 ϭ Ϫ3.7 kcal/mole).…”

Section: Resultsmentioning

confidence: 99%

“…Hence, it follows, that DNA methylation is accompanied by the liberation of significant energy (⌬G 0 ϭ Ϫ3.7 kcal/mole). This energy can be used in part for T4 Dam isomerization (22) and for unidirectional 5Ј 3 3Ј movement of T4 Dam along the DNA.…”

Section: Resultsmentioning

confidence: 99%

Bacteriophage T4 Dam DNA-(N 6-adenine)-methyltransferase

Zinoviev

Evdokimov

Malygin

et al. 2003

Journal of Biological Chemistry

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“…If dimeric; however, both enzymatic forms were catalytically active (7), but they had different kinetic characteristics. Since the results summarized above were based primarily on steady state and single turnover methylation studies (5), the present work was undertaken to determine whether methylation under pre-steady state conditions would yield results consistent with the kinetic model we proposed previously (8).…”

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confidence: 99%

Bacteriophage T4Dam DNA-(Adenine-N6)-methyltransferase

Malygin

Sclavi

Zinoviev

et al. 2004

Journal of Biological Chemistry

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We analyzed pre-steady state and single turnover kinetics of bacteriophage T4Dam DNA-(adenine-N 6 )-methyltransferase-mediated methyl group transfer from S-adenosyl-L-methionine (AdoMet) to 40-mer duplexes containing native recognition sites (5-GATC/5-GATC) or some modified variant(s). The results extend a model from studies with single-site 20-mer duplexes. Under pre-steady state conditions, monomeric T4Dam methyltransferase-AdoMet complexes were capable of rapid methylation of adenine residues in 40-mer duplexes containing two sites. During processive movement of T4Dam to the next site, the rate-limiting step was the exchange of the product S-adenosyl-L-homocysteine (AdoHcy) for AdoMet without T4Dam dissociating from the duplex. Consequently, instead of a single exponential rate dependence, complex methylation curves were obtained with at least two pre-steady state steps. With 40-mer duplexes containing a single target site, the kinetics were simpler, fitting a single exponential followed by a linear steady state phase. Single turnover methylation of 40-mer duplexes also proceeded in two stages. First, two dimeric T4Dam-AdoMet molecules bound, and each catalyzed a twostep methylation. Instead of processive movement of T4Dam, a conformational adaptation occurred. We propose that following methyl transfer to one strand, dimeric (T4Dam-AdoMet)-(T4Dam-AdoHcy) was capable of rapidly reorienting itself and catalyzing methyl transfer to the target adenine on the complementary, unmethylated strand. This second stage methyl transfer occurred at a rate about 25-fold slower than in the first step; it was rate-limited by Dam-AdoHcy dissociation or its clearance from the methylated complementary strand. Under single turnover conditions, there was complete methylation of all target adenine residues with each of the two-site 40-mer duplexes.DNA methylation is extremely important for the control of such processes as transcription, genomic imprinting, regulation of development, restoration of the correct structure of DNA, and chromatin organization (1). In contrast to most prokaryotic DNA methyltransferases (MTases), 1 the Dam MTase is not a component of a restriction-modification system; rather, Dam methylation has other important cellular functions such as methyl-directed mismatch repair (reviewed in Ref.2) and bacterial virulence (3). In the latter instance, despite the presence of a large number of virulence factors for intestinal bacteria (Ͼ20), it was sufficient to switch off the gene for the Dam DNA MTase to obtain avirulent live vaccine. Like all natural MTases, Dam MTases use the universal donor, Sadenosyl-L-methionine (AdoMet), transferring methyl groups to the Ade residues in the palindromic sequence 5Ј-GATC-3Ј (1).Earlier, we carried out kinetic analyses of T4Dam-mediated methyl group transfer to oligodeoxynucleotide duplexes containing one or two specific GATC sites with different combinations of (un)methylated targets (reviewed in Ref.

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Bacteriophage T4 Dam DNA-[N6-adenine]Methyltransferase

Cited by 31 publications

References 55 publications

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

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

Bacteriophage T4 Dam DNA-(N 6-adenine)-methyltransferase

Bacteriophage T4Dam DNA-(Adenine-N6)-methyltransferase

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