Purification and biochemical characterization of the Ecal DNA methyltransferase

Abstract

The EcaI GGTNACC-specific DNA-adenine modification methyltransferase has been purified to apparent homogeneity. The active form of the DNA methyltransferase is a single polypeptide. The enzyme has a pH optimum at pH 8.0 and a temperature optimum at 25°C. EcaI DNA methyltransferase transfers one methyl group to the adenine of the recognition site in a single binding event. The K, was 170 nM for DNA and 1.8 pM for the methyl donor S-adenosylmethionine. Methylated DNA is a competitive inhibitor with respect to DNA (Ki = 3.5 nM). The other product of the DNA-methylation reaction, S-adenosylhomocysteine was found to be a competitive inhibitor with respect to S-adenosylmethionine (Ki = 2.7 pM). The Sadenosylmethionine analog sinefungin was shown to be a very strong inhibitor (Ki = 3.5 nM) of the DNA methyltransferase reaction.Type-I1 modification enzymes are sequence-specific DNA methyltransferases that recognize 4 -8-bp sequences in DNA and transfer one methyl group from the methyl donor Sadenosyl-L-methionine (AdoMet) to an adenine or cytosine of the recognition sequence in each strand [l]. These enzymes fall into three groups according to the methylated base generated: 5-methylcytosine, N4-methylcytosine and N6-methyladenine DNA methyltransferases [2]. The other product of the DNA-methylation reaction is S-adenosyl-L-homocysteine (AdoHcy). Most of the known sequence-specific DNA methyltransferases are part of restriction-modification systems, but there are enzymes which exist in the cell without a cognate restriction endonuclease [3]. Type-I1 DNA methyltransferases constitute an interesting example of sequence-specific DNA/protein interaction, especially if we consider the multitude of specificities discovered and the existence of different DNA methyltransferases recognizing the same sequence [4]. Type-I1 DNA methyltransferases have gained practical importance in the analysis of large genomes because of their use in increasing the specificity of certain restriction endonucleases [5]. Understanding the molecular basis of sequence specificity requires the detailed structural and enzymological characterization of type-I1 DNA methyltransferases. The genes of several DNA methyltransferases have been cloned and sequenced [3], but relatively few enzymes have been purified to homogeneity and characterized enzymologically [6 -161.The EcaI DNA methyltransferase is part of the EcaI restriction-modification system that recognizes the sequence 5'-(GGTNACC)-3' [17, 181. We cloned the gene coding for Era1 DNA methyltransferase in Escherichia coli and determined its nucleotide sequence. The nucleotide sequence of the ecaIM gene was used to derive the amino acid sequence of E d DNA methyltransferase [18]. EcaI DNA methyltransferase methylates the adenine of the 5'-GGTNACC-3' recognition sequence to yield p-methyladenine [18]. In this paper, we report the purification and initial enzymological characterization of EcaI DNA methyltransferase. MATERIALS AND METHODS Strains, plasmids and growth conditionsE. coli ER1398 [19] was used as ...

Dam methylase fromEscherichia coli: kinetic studies using modified DNA oligomers: hemimethylated substrates

“…The initial rate of methylation of duplexes (duplex concentrations were 200 nM) measured in the presence of 3 aM AdoMet was linear with respect to enzyme concentration in the range from 0 to 40 nM M-Dam. Although the extent ofmethylation differed widely for the various duplexes linearity was observed in all cases (45 (14).…”

“…It seems that in the case of Dam Mtase, and may be in the case of other Mtases (14,21), DNA-protein interactions are not very tight, the Km values being all in the 10-100 nM range. There are no reasons to propose pronounced conformational differences between the duplexes as was suggested for M-EcoRI by Reich and Danzitz (10,11 ) upon introduction of dIl into their tetradecamer.…”

“…Biochemical studies on EcoRI Mtase (M-EcoRI) (9-13), M*EcaI (14,15), Dam Mtase (16)(17)(18)(19)(20) and M-MvaI (21) have been reported. A mechanism for methylation by M*EcoRI has been proposed (12,22,23).…”

Kinetic characterization of the EcaI methyltransferase

“…EcaI methylase was prepared in our laboratory (Szilik et al, 1992). S-Adenosyl-~-['H]methionine was purchased from Amersham Corp. and diluted to a specific activity of 81.5 kBq/nmol (2.2Ci/mmol) with unlabeled AdoMet obtained from Sigma.…”

Purification, Characterization, and Cloning of an S-Adenosylmethionine-dependent 3-Amino-3-carboxypropyltransferase in Nocardicin Biosynthesis

“…The transferase activity eluted as a single peak at 40.5 kDa, showing that the transferase is active as a monomer. Many small molecule (65)(66)(67)(68) and DNA methyltransferases (69,70) are monomers, especially those isolated from nonmammalian sources. Spermine and spermidine synthase, on the other hand, appear to be dimers of identical subunits (59,61,62).…”