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Abstract
The bacteriophage T4 dam gene, encoding the Dam DNA [N6-adenine]methyltransferase (MTase), has been subcloned into the plasmid expression vector, pJW2. In this construct, designated pINT4dam, transcription is from the regulatable phage lambda pR and pL promoters, arranged in tandem. A two-step purification scheme using DEAE-cellulose and phosphocellulose columns in series, followed by hydroxyapatite chromatography, was developed to purify the enzyme to near homogeneity. The yield of purified protein was 2 mg/g of cell paste. The MTase has an s20,w of 3.0 S and a Stokes radius of 23 A and exists in solution as a monomer. The Km for the methyl donor, S-adenosylmethionine, is 0.1 x 10(-6) M, and the Km for substrate nonglucosylated, unmethylated T4 gt- dam DNA is 1.1 x 10(-12) M. The products of DNA methylation, S-adenosyl-L-homocysteine and methylated DNA, are competitive inhibitors of the reaction; Ki values of 2.4 x 10(-6) M and 4.6 x 10(-12) M, respectively, were observed. T4 Dam methylates the palindromic tetranucleotide, GATC, designated the canonical sequence. However, at high MTase:DNA ratios, T4 Dam can methylate some noncanonical sequences belonging to GAY (where Y represents cytosine or thymine).
Highlights
At high MTase:DNA ratios, T4 encodes a DNA [~-adenine]MTase (T4 Dam) can methylate some noncanonical sequences belonging to GAY
Bacteriophage T4 encodes a DNA [~-adenine]MTase (T4 Dam) that methylates adenine in the sequence GATC in cytosine, 5-methylcytosine, or 5-hydroxymethylcytosine-containing DNA [2]
We describe the overexpression, purification, and characterization of kinetic parameters for T4 Dam
Summary
Vol 270, No 24, Issue of June 16, pp. 14389-14393, 1995 Printed in U.S.A. (Received for publication, February 15, 1995, and in revised form, April 11, 1995). DNA [~-adenine]methyltransferase (MTase), has been subcloned into the plasmid expression vector, pJW2. At high MTase:DNA ratios, T4 Dam can methylate some noncanonical sequences belonging to GAY (where Y represents cytosine or thymine). DNA methyltransferases (MTases)l are ubiquitous in living cells and present an interesting example of sequence-specific. DNA MTases recognize specific sequences in DNA and transfer methyl groups from the methyl donor, AdoMet, to adenine or cytosine residues in the recognition sequence. Bacteriophage T4 encodes a DNA [~-adenine]MTase (T4 Dam) that methylates adenine in the sequence GATC in cytosine, 5-methylcytosine, or 5-hydroxymethylcytosine-containing DNA [2]. Gion(s) of T4 Dam responsible for sequence-specific DNA recognition as well as the functional roles of conserved motifs present in all DNA [~-adenine]MTases [6]. We describe the overexpression, purification, and characterization of kinetic parameters for T4 Dam
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