Specific amino acid sequences required for O6-methylguanine-DNA methyltransferase activity: Analyses of three residues at or near the methyl acceptor site

Chueh Ling-ling, Takanori Nakamura, Yoshimichi Nakatsu, Kunihiko Sakumi, Hiroshi Hayakawa, Mutsuo Sekiguchi

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Abstract

O6-Methylguanine-DNA methyltransferase plays an important role in preventing tumor induction. To elucidate the significance of a highly conserved amino acid sequence of methyltransferase protein, amino acid substitutions were introduced by site-directed mutagenesis of cloned cDNA for human methyltransferase and the activity and stability of mutant forms of enzyme were examined. When cysteine-145, to which the methyl transfer occurs, was replaced by other amino acids, all of the mutants isolated showed the methyl-transferase-negative phenotype. From one of the negative mutants, methyltransferase-positive revertants were isolated, all of which carried codons for cysteine. Thus the cysteine residue is essential for acceptance of the methyl group and cannot be replaced by other amino acids. Using this negative and positive selectionprocedure, analyses were extended to other residues near the acceptor site. At the histidine-146 site, four substitutions (phenyialanine, methionine, asparagine and glutamine) exhibited the positive phenotype but the levels of methyltransferaseactivity in these mutants were low. With valine-148 substitutions there were six types of positive revertants, among which mutants carrying isoleucine, cysteine and alanine showed significantly high levels of methyltransferase activity. Some mutant forms of cDNA were expressed in methyltransferase-defkient human cells, and the results obtained with Escherichia coli cells were confirmed.

Original languageEnglish
Pages (from-to)837-843
Number of pages7
JournalCarcinogenesis
Volume13
Issue number5
DOIs
Publication statusPublished - May 1 1992

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All Science Journal Classification (ASJC) codes

  • Cancer Research

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