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

31 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 - Dec 1 1986

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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