Vg. Kossykh et al., PHAGE-T4 DNA [N-6-ADENINE]METHLYLTRANSFERASE - OVEREXPRESSION, PURIFICATION, AND CHARACTERIZATION, The Journal of biological chemistry, 270(24), 1995, pp. 14389-14393
The bacteriophage T4 dam gene, encoding the Dam DNA [N-6-adenine]methy
ltransferase (MTase), has been subcloned into the plasmid expression v
ector, 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 phosp
hocellulose columns in series, followed by hydroxyapatite chromatograp
hy, 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 T
4 gt(-)dam(-) DNA is 1.1 x 10(-12) M. The products of DNA methylation,
S-adenosyl-L-homocysteine and methylated DNA, are competitive inhibit
ors 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 tetran
ucleotide, GATC, designated the canonical sequence, However, at high M
Tase:DNA ratios, T4 Dam can methylate some noncanonical sequences belo
nging to GAY (where Y represents cytosine or thymine).