PHAGE-T4 DNA [N-6-ADENINE]METHLYLTRANSFERASE - OVEREXPRESSION, PURIFICATION, AND CHARACTERIZATION

The bacteriophage T4 dam gene, encoding the Dam DNA [N-6-adenine]methyltransferase (MTase), has been subcloned into the plasmid expression vector, pJW2, In this construct, designated pINT4dam, transcription is from the regulatable phage lambda p(R) and p(L) 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 s(20,w) of 3.0 S and a Stokes radius of 23 Angstrom and exists in solution as a monomer. The K-m for the methyl donor, S-adenosylmethionine, is 0.1 x 10(-6) M, and the K-m 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; K-i 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).