Cloning and characterization of the alkA gene of Escherichia coli that encodes 3-methyladenine DNA glycosylase II

Yusaku Nakabeppu, H. Kondo, M. Sekiguchi

Research output: Contribution to journalArticle

72 Citations (Scopus)

Abstract

By in vitro recombination we have constructed hybrid plasmids which can suppress the increased methylmethane sulfonate sensitivity caused the alkA1 mutation in Escherichia coli. Since the cloned DNA fragment was mapped at 44 to 45 min of E. coli K12 genetic map, an area where the alkA gene is located, we conclude that the cloned DNA fragment contains the alkA gene itself but not other gene(s) that suppresses the alkA mutation. Specific labeling of plasmid-encoded proteins by the maxicell method revealed that the alkA codes for a polypeptide whose molecular weight is about 30,000. When cells harboring the alkA+ plasmids were grown in the presence of low doses of a simple alkylating agent (adapted condition), the activity of 3-methyladenine DNA glycosylase II was increased. The enzyme activity was copurified with the M(r) 30,000 polypeptide. These results indicate that the alkA gene codes for 3-methyladenine DNA glycosylase II. Taking advantage of overproduction of the alkA protein in adapted cells that harbor multicopy plasmids carrying the alkA+ gene, 3-methyladenine DNA glycosylase II has been purified to apparent physical homogeneity.

Original languageEnglish
Pages (from-to)13723-13729
Number of pages7
JournalJournal of Biological Chemistry
Volume259
Issue number22
Publication statusPublished - Dec 1 1984

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Cloning
Escherichia coli
Organism Cloning
Genes
Plasmids
Methyl Methanesulfonate
Escherichia coli K12
Peptides
Mutation
Alkylating Agents
DNA
Enzyme activity
Ports and harbors
Labeling
Genetic Recombination
Proteins
Molecular Weight
Molecular weight
DNA-3-methyladenine glycosidase II
Enzymes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Cloning and characterization of the alkA gene of Escherichia coli that encodes 3-methyladenine DNA glycosylase II. / Nakabeppu, Yusaku; Kondo, H.; Sekiguchi, M.

In: Journal of Biological Chemistry, Vol. 259, No. 22, 01.12.1984, p. 13723-13729.

Research output: Contribution to journalArticle

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