Purification, structure, and biochemical properties of human O6-methylguanine-DNA methyltransferase.
The level of O6-methylguanine-DNA methyltransferase activity in a human cell line carrying a 1.1-kilobase cDNA fragment was about 50 times higher than that found in ordinary methyltransferase-proficient (Mer+) cell lines (Hayakawa, H., Koike, G., and Sekiguchi, M. (1990) J. Mol. Biol. 213, 739-747). Taking advantage of this overproduction, the enzyme was purified to apparent physical homogeneity and the physical and biochemical properties investigated. A single polypeptide with a molecular weight of approximately 25,000 was detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the most highly purified preparation. The Stokes radius of 22.5 A and the sedimentation coefficient of 2.0 S were obtained, from which the molecular weight of the native form of the enzyme was calculated to be 19,000. After digestion with lysyl endopeptidase, peptide fragments of the protein were isolated and sequenced. The amino acid sequences of these peptides and the amino acid composition of the protein were in good agreement with those deduced from the nucleotide sequence of the cloned cDNA. The purified enzyme catalyzed transfer of methyl groups from O6-methylguanine and O4-methylthymine, but not from methylphosphotriesters, of methylated DNA to the enzyme molecule.
Koike
G
G
Department of Biochemistry, Faculty of Medicine, Kyushu University, Fukuoka, Japan.
Maki
H
H
Takeya
H
H
Hayakawa
H
H
Sekiguchi
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006801:Humans; D007700:Kinetics; D008780:Methyltransferases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D019853:O(6)-Methylguanine-DNA Methyltransferase; D010446:Peptide Fragments / Q000302:isolation & purification; D011487:Protein Conformation
D010446:Peptide Fragments; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase