PMID:3536912
| Citation |
Sakumi, K, Nakabeppu, Y, Yamamoto, Y, Kawabata, S, Iwanaga, S and Sekiguchi, M (1986) Purification and structure of 3-methyladenine-DNA glycosylase I of Escherichia coli. J. Biol. Chem. 261:15761-6 |
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| Abstract |
We constructed a recombinant plasmid carrying a gene that suppresses tag mutation. To overproduce its gene product, a 0.8-kilobase DNA fragment which carries the gene was placed under the control of the lac promoter in pUC8. 3-Methyladenine-DNA glycosylase activity in cells carrying such plasmids (pCY5) was 450-fold higher than that of wild type strain, on exposure to isopropyl-beta-D-thiogalactopyranoside. From an extract of such cells, the enzyme was purified to apparent physical homogeneity, and the amino acid composition and the amino-terminal amino acid sequence of the enzyme were determined. The data were in accord with nucleotide sequence of the gene, determined by the dideoxy method. It was deduced that 3-methyladenine-DNA glycosylase I comprises 187 amino acids and its molecular weight is 21,100, consistent with the value estimated from the sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified protein. Only 3-methyladenine was excised from methylated DNA by the purified glycosylase. These results show that the tag is the structural gene for 3-methyladenine-DNA glycosylase I. |
| Links | |
| Keywords |
Amino Acid Sequence; Amino Acids/analysis; Base Sequence; DNA Glycosylases; DNA, Recombinant; Escherichia coli/enzymology; Molecular Weight; Mutation; N-Glycosyl Hydrolases/genetics; N-Glycosyl Hydrolases/isolation & purification; Plasmids |
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