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PMID:7782299
| Citation |
Kossykh, VG, Schlagman, SL and Hattman, S (1995) Phage T4 DNA [N6-adenine]methyltransferase. Overexpression, purification, and characterization. J. Biol. Chem. 270:14389-93 |
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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). |
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| Keywords |
Chromatography, DEAE-Cellulose; Chromatography, Gel; Cloning, Molecular; Electrophoresis, Polyacrylamide Gel; Escherichia coli/genetics; Escherichia coli Proteins; Kinetics; Methylation; Methyltransferases/genetics; Methyltransferases/isolation & purification; Methyltransferases/metabolism; Molecular Weight; Plasmids; Site-Specific DNA-Methyltransferase (Adenine-Specific); Viral Proteins |
Significance
Annotations
| Gene product | Qualifier | GO Term | Evidence Code | with/from | Aspect | Extension | Notes | Status |
|---|---|---|---|---|---|---|---|---|
| GO:0009008: DNA-methyltransferase activity |
ECO:0000314: |
F |
Bacteriophage T4 dam gene expressed in plasmid by direct assay by authors. Fig 6 |
complete | ||||
Notes
See also
References
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