Phage T4 DNA [N6-adenine]methyltransferase: Overexpression, purification, and characterization

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 λ 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 Å 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).