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

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

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    32 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

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Biology
    • Cell Biology

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