Purification and structure of 3-methyladenine-DNA glycosylase I of Escherichia coli

K. Sakumi, Y. Nakabeppu, Y. Yamamoto, S. Kawabata, S. Iwanaga, M. Sekiguchi

Research output: Contribution to journalArticle

30 Citations (Scopus)

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-β-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.

Original languageEnglish
Pages (from-to)15761-15766
Number of pages6
JournalJournal of Biological Chemistry
Volume261
Issue number33
Publication statusPublished - 1986
Externally publishedYes

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3-methyladenine-DNA glycosylase
Escherichia coli
Purification
Genes
Amino Acids
Plasmids
Thiogalactosides
DNA Glycosylases
DNA
Enzymes
Cell Extracts
Electrophoresis
Sodium Dodecyl Sulfate
Polyacrylamide Gel Electrophoresis
Amino Acid Sequence
Nucleotides
Molecular Weight
Molecular weight
Mutation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Sakumi, K., Nakabeppu, Y., Yamamoto, Y., Kawabata, S., Iwanaga, S., & Sekiguchi, M. (1986). Purification and structure of 3-methyladenine-DNA glycosylase I of Escherichia coli. Journal of Biological Chemistry, 261(33), 15761-15766.

Purification and structure of 3-methyladenine-DNA glycosylase I of Escherichia coli. / Sakumi, K.; Nakabeppu, Y.; Yamamoto, Y.; Kawabata, S.; Iwanaga, S.; Sekiguchi, M.

In: Journal of Biological Chemistry, Vol. 261, No. 33, 1986, p. 15761-15766.

Research output: Contribution to journalArticle

Sakumi, K, Nakabeppu, Y, Yamamoto, Y, Kawabata, S, Iwanaga, S & Sekiguchi, M 1986, 'Purification and structure of 3-methyladenine-DNA glycosylase I of Escherichia coli', Journal of Biological Chemistry, vol. 261, no. 33, pp. 15761-15766.
Sakumi K, Nakabeppu Y, Yamamoto Y, Kawabata S, Iwanaga S, Sekiguchi M. Purification and structure of 3-methyladenine-DNA glycosylase I of Escherichia coli. Journal of Biological Chemistry. 1986;261(33):15761-15766.
Sakumi, K. ; Nakabeppu, Y. ; Yamamoto, Y. ; Kawabata, S. ; Iwanaga, S. ; Sekiguchi, M. / Purification and structure of 3-methyladenine-DNA glycosylase I of Escherichia coli. In: Journal of Biological Chemistry. 1986 ; Vol. 261, No. 33. pp. 15761-15766.
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